WO2025015195A1

WO2025015195A1 - Treating alphaherpesvirus infections using gene drive technology

Info

Publication number: WO2025015195A1
Application number: PCT/US2024/037644
Authority: WO
Inventors: Marius WALTER; Keith R. Jerome
Original assignee: Fred Hutchinson Cancer Center
Priority date: 2023-07-13
Filing date: 2024-07-11
Publication date: 2025-01-16

Abstract

Provided are genetically modified viruses and methods of suppressing and/or preventing infection or a recurrence of an infection caused by a wild-type alphaherpesvirus. In certain embodiments, the methods involve co-infecting a cell comprising the wild-type alphaherpesvirus with at least one modified alphaherpesvirus containing a gene drive construct; wherein the modified alphaherpesvirus is effective in genetically modifying/altering the wild-type alphaherpesvirus by integrating/inserting the gene drive construct into the genome of the wild-type virus at a target site, and wherein the integration/insertion of the gene drive construct disrupts at least one viral gene at the target site in the genome of the wild-type virus. In some embodiments, the cell comprises a cell of a latent reservoir. In some embodiments, the wild-type alphaherpesvirus and the modified alphaherpesvirus are selected from HSV-1 and HSV-2. In certain embodiments, the at least one viral gene is a gene involved in anterograde transport of the virus.

Description

TREATING ALPHAHERPESVIRUS INFECTIONS USING GENE DRIVE TECHNOLOGY CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/513467, filed July 13, 2023. STATEMENT REGARDING SEQUENCE LISTING [0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 1896-P92WO_Seq_List_20240709.xml. The XML file is 992,538 bytes; was created on July 9, 2024; and is being submitted electronically via Patent Center with the filing of the specification. STATEMENT OF GOVERNMENT LICENSE RIGHTS [0003] This invention was made with government support under AI132599 and R21AI178255 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND [0004] Herpesviruses are ubiquitous DNA viruses that establish lifelong infections. The lifelong nature of herpesvirus infection creates a burden for individual health as well as for public health policy. They also have a global impact through their effects on veterinary species involved in food production and those kept as companion animals. Within the Herpesviridae family, the alphaherpesvirinae subfamily is characterized by common genomic features, and epithelial and mucosal sites of active replication. Most alphaherpesviruses establish lifelong latency in neurons, with a few non- neuronal exceptions such as the Mardivirus genus (e.g., Marek's disease virus (MDV) or gallid alphaherpesvirus 2). The human alphaherpesviruses consist of herpes simplex virus 1 and 2 (HSV-1,2 or human herpesvirus 1,2, HHV-1,2) and varicella zoster virus (VZV or HHV-3). [0005] HSV-1 and 2 persistently infect close to 70% and 15% of the human population, respectively. Oral and genital herpes infections are very common and can be highly painful and stigmatizing. Infection can be fatal in newborns and immunocompromised hosts, and HSV-2 is a key risk factor for HIV infection. After primary infection, they enter latency and occasionally reactivate, causing recurrent disease. ^{1896-P92WO AP} -1- Herpes simplex viruses (HSV) 1 and 2 first infect mucosal surfaces before spreading to the nervous system via axons. They remain latent in neurons in sensory and autonomic ganglia and reactivation causes lesions in the facial or genital area. HSV-1 and 2 lack vaccines and eradication strategies. Current antiviral drugs, such as acyclovir, cannot eliminate latent HSV and are only suppressive, rather than curative. Thus, new therapies and/or curative treatments and new therapeutic strategies to cure or prevent HSV infection are critically needed. SUMMARY [0006] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. [0007] Gene drive describes genetic sequences that have a high probability of being passed down from one generation to the next and that have the power to disperse a certain trait throughout a population. During sexual reproduction, the majority of engineered gene drive systems replicate a synthetic sequence from one modified chromosome to its wild-type counterpart using CRISPR-Cas9 editing. However, existing gene drive techniques are only effective in sexually reproducing creatures like mammals; they are inapplicable to asexual populations like viruses and bacteria. [0008] The inventors have successfully transferred a gene drive sequence across different human alphaherpesvirus strains to target and replace wild-type populations. The present disclosure describes gene drives and gene drive mechanisms able to propagate a genetically modified feature throughout populations of DNA viruses, specifically alphaherpesviruses. Thus, the present disclosure pertains to viral gene drives and use of the same to offer a novel treatment approach against alphaherpesviruses by suppressing and/or significantly preventing viral infection. [0009] In an aspect, the present disclosure provides a method of suppressing and/or preventing infection, or a recurrence of an infection caused by a wild-type alphaherpesvirus. In certain embodiments, the method comprises genetically modifying/altering a genome of the wild-type alphaherpesvirus. In some embodiments, the method comprises co-infecting at least one cell of a latent reservoir comprising the wild- type alphaherpesvirus with at least one modified alphaherpesvirus. In some embodiments, the at least one modified alphaherpesvirus comprises a gene-drive construct integrated into ^{1896-P92WO AP} -2- the genome of the modified alphaherpesvirus. In some embodiments, the at least one modified alphaherpesvirus is effective in genetically modifying/altering the wild-type alphaherpesvirus by integrating/inserting the gene drive construct into the genome of the alphaherpesvirus at a target site. In an embodiment, the integration/insertion of the gene drive construct disrupts at least one viral gene at the target site in the genome of the wild-type alphaherpesvirus. [0010] In certain embodiments, the gene-drive construct comprises a first nucleic acid sequence operably linked to a first promoter and encoding a functional targeted endonuclease that induces a double stranded break in or near at least one target site in a genome of a wild-type alphaherpesvirus. In some embodiments, the gene-drive construct further comprises flanking sequences homologous to sequences adjacent to the at least one target site that permit insertion of the gene drive construct at the at least one target site in the genome of the wild-type alphaherpesvirus. In some embodiments, the flanking sequences homologous to sequences adjacent to the at least one target site range in length from about 50 bp to about 5 kb. [0011] The functional targeted endonuclease may comprise an endonuclease selected from the group consisting of a class 2 CRISPR/Cas endonuclease, a TALEN, a zinc finger nuclease, and a homing endonuclease. In some embodiments, the functional targeted endonuclease comprises a class 2 CRISPR/Cas endonuclease. In an embodiment, the class 2 CRISPR/Cas endonuclease is a type II CRISPR/Cas endonuclease. In a related embodiment, the class 2 CRISPR/Cas endonuclease comprises a Cas9 protein. The Cas9 protein may be selected from the group consisting of a Streptococcus pyogenes Cas9 protein (spCas9) or a functional portion thereof, a Staphylococcus aureus Cas9 protein (saCas9) or a functional portion thereof, a Streptococcus thermophilus Cas9 protein (stCas9) or a functional portion thereof, a Neisseria meningitides Cas9 protein (nmCas9) or a functional portion thereof, and a Treponema denticola Cas9 protein (tdCas9) or a functional portion thereof. [0012] In a related embodiment, the class 2 CRISPR/Cas endonuclease is a type V or type VI CRISPR/Cas endonuclease. The type V or type VI CRISPR/Cas endonuclease may be selected from the group consisting of a Cpf1 polypeptide or a functional portion thereof, a C2c1 polypeptide or a functional portion thereof, a C2c3 polypeptide or a functional portion thereof, and a C2c2 polypeptide or a functional portion thereof. ^{1896-P92WO AP} -3- [0013] In some embodiments, the gene drive construct further comprises a second nucleic acid sequence encoding at least one guide RNA, and wherein the second nucleic acid sequence is operably linked to a second promoter. In an embodiment, the at least one guide RNA directs the functional targeted endonuclease to a site in the genome of the wild- type alphaherpesvirus where cleavage permits integration/insertion of the gene drive construct into the genome of the wild-type alphaherpesvirus by homologous recombination. [0014] In some embodiments, the first nucleotide sequence encoding the functional targeted endonuclease and the second nucleotide sequence encoding the at least one guide RNA are located between a pair of flanking sequences in the gene drive construct. In some embodiments, the first promoter and/or the second promoter comprises a viral promoter. [0015] In a further embodiment, the disruption of the at least one gene at the target site impairs anterograde transport of the wild-type alphaherpesvirus from a latent reservoir to a site of active infection, and wherein the wild-type alphaherpesvirus remains latent. The latent reservoir may comprise sensory and autonomic ganglia neurons and the site of active infection is mucosal epithelium. In some embodiments, the wild-type and the modified alphaherpesvirus are not impaired in retrograde axonal transport. In an embodiment, the method is effective in preventing viral shedding and recurring symptoms. [0016] The wild-type and/or modified alphaherpesvirus may be selected from Ateline alphaherpesvirus 1; Bovine alphaherpesvirus 2; Bovine mammillitis virus; Cercopithecine alphaherpesvirus 2; Human alphaherpesvirus 1 (HSV-1); Human alphaherpesvirus 2 (HSV-2); Leporid alphaherpesvirus 4; Macacine alphaherpesvirus 1; Macacine alphaherpesvirus 2; Macacine alphaherpesvirus 3; Macropodid alphaherpesvirus 1; Macropodid alphaherpesvirus 2; Panine alphaherpesvirus 3; Papiine alphaherpesvirus 2; Pteropodid alphaherpesvirus 1; Saimiriine alphaherpesvirus 1; Bovine alphaherpesvirus 1; Bovine alphaherpesvirus 5; Bovine encephalitis herpesvirus; Bubaline alphaherpesvirus 1; Canid alphaherpesvirus 1; Caprine alphaherpesvirus 1; Cercopithecine alphaherpesvirus 9; Cervid alphaherpesvirus 1; Cervid alphaherpesvirus 2; Cervid alphaherpesvirus 3; Equid alphaherpesvirus 1; Equid alphaherpesvirus 3; Equid alphaherpesvirus 4; Equid alphaherpesvirus 8; Equid alphaherpesvirus 9; Felid alphaherpesvirus 1; Human alphaherpesvirus 3; Monodontid alphaherpesvirus 1; Phocid alphaherpesvirus 1; and Suid alphaherpesvirus 1. In some embodiment, the wild-type ^{1896-P92WO AP} -4- and/or modified alphaherpesvirus is a Human alphaherpesvirus 1 (Herpes Simplex virus 1). In some embodiments, the wild-type and/or modified alphaherpesvirus is a Human alphaherpesvirus 2 (Herpes simplex virus 2). [0017] The at least one gene at the target site is selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding membrane protein US9) or a homolog thereof. In some embodiments, the at least one gene at the target site is US9 or a homolog thereof. [0018] In some embodiments, the at least one cell comprises a latent reservoir for the wild-type Herpes simplex virus. In an embodiment, the at least one cell is a sensory and/or autonomic ganglia neuron. [0019] In another aspect, the present disclosure pertains to a method of preventing and/or suppressing anterograde transport of a wild-type alphaherpesvirus from a latent reservoir to a site of active infection. In some embodiments, the method comprises co- infecting a subject harboring a wild-type alphaherpesvirus in a latent reservoir with a modified alphaherpesvirus containing a gene-drive construct integrated into the genome of the modified alphaherpesvirus. In an embodiment, the modified alphaherpesvirus is effective in genetically modifying/altering the wild-type alphaherpesvirus by integrating/inserting the gene drive construct at a target site in the genome of the wild-type alphaherpesvirus. In some embodiments, the integration/insertion of the gene drive construct at the target site disrupts at least one viral gene at the target site in the genome of the wild-type alphaherpesvirus. In an embodiment, the at least one viral gene is a gene involved in anterograde transport of the virus. [0020] In some embodiments, the method is effective in preventing viral shedding and recurring symptoms. In an embodiment, the subject is human and the wild- type and/or modified alphaherpesvirus is selected from Human alphaherpesvirus 1 (Herpes Simplex virus1), Human alphaherpesvirus 2 (Herpes Simplex virus2), and Human alphaherpesvirus 3 (Varicella zoster virus). In some embodiments, the subject is human and the wild-type and/or modified alphaherpesvirus is Herpes Simplex virus 1. In yet another embodiment, the subject is human and the wild-type and/or modified alphaherpesvirus is Herpes Simplex virus 2 (HSV-2). In some embodiments, the latent reservoir comprises sensory and autonomic ganglia neurons, and wherein the site of active infection is a mucosal epithelium. ^{1896-P92WO AP} -5- [0021] The at least one gene at the target site may be selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding membrane protein US9) or a homolog thereof. In some embodiments, the at least one gene is US9 (encoding membrane protein US9) or a homolog thereof. In some embodiments, the wild-type and the modified virus are not impaired in retrograde axonal transport. [0022] In yet another aspect, the present disclosure provides a method of suppressing and/or preventing recurrence of an infection in a subject caused by a wild-type Herpes simplex virus. The method may comprise co-infecting the subject with an active and/or a recurrent infection with a therapeutically effective amount of a composition comprising a modified Herpes simplex virus containing a gene-drive construct. In some embodiments, the gene-drive construct is integrated into the genome of the modified Herpes simplex virus. In an embodiment, the composition is effective in impairing anterograde transport of the wild-type Herpes simplex virus from a latent reservoir to a site of active infection. [0023] In some embodiments, the gene-drive construct comprises: (i) a first nucleotide sequence encoding for a functional targeted endonuclease that induces a double stranded break in or near at least one target site in a genome of a wild-type Herpes simplex virus; (ii) a second nucleotide sequence encoding at least one guide RNA sequence complementary to the at least one target site in the genome of a wild-type Herpes simplex virus; and (iii) a pair of flanking sequences homologous to sequences adjacent to the at least one target site. In some embodiments, the first and second nucleotide sequences of the gene-drive construct are located between the pair of flanking sequences in the construct. In some embodiments, the gene-drive construct is effective in integrating/inserting into the genome of the wild-type Herpes simplex virus and disrupting at least one viral gene in the genome of the wild-type Herpes simplex virus. [0024] In an embodiment, the gene-drive construct comprises a first promoter operably linked to the first nucleotide sequence. In a related embodiment, the gene-drive construct comprises a second promoter operably linked to the second nucleotide sequence. In some embodiments, the first promoter linked to the first nucleotide sequence and/or the second promoter linked to the second nucleotide sequence comprises a viral promoter. [0025] In some embodiments, the guide RNA targets the targeted endonuclease to the at least one target site in the wild-type Herpes simplex virus genome where cleavage ^{1896-P92WO AP} -6- permits integration/insertion of the gene drive construct into the genome of the wild-type Herpes simplex virus by homologous recombination. [0026] In some embodiments, the at least one viral gene is selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding membrane protein US9) or a homolog thereof. In an embodiment, the at least one viral gene is US9 or a homolog thereof. [0027] The wild-type and/or the modified Herpes simplex virus may be selected from HSV-1, HSV-2, and VZV. In some embodiments, the wild-type and/or the modified Herpes simplex virus are selected from HSV-1 and HSV-2. In some embodiments, the wild-type and the modified virus are not impaired in retrograde axonal transport. [0028] In yet another aspect, the present disclosure provides a method for prophylactically treating a subject to protect against a disease caused by a wild-type herpes simplex virus. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising a modified Herpes simplex virus containing a gene drive construct integrated into the genome of the modified Herpes simplex virus. In some embodiments, the modified Herpes simplex virus is impaired in anterograde axonal transport of the virus from a latent reservoir to a site of active infection. In some embodiments, the modified Herpes simplex virus is not impaired in retrograde transport of the virus from the site of infection to the latent reservoir. In an embodiment, the subject has had no prior exposure to a wild-type Herpes simplex virus and/or infection caused by a wild-type Herpes simplex virus. In some embodiments, the step of administering comprises infecting the subject with the modified Herpes simplex virus. In some embodiments, the method is effective in keeping the modified Herpes simplex virus latent in the subject. [0029] The compositions and methods disclosed herein are effective in protecting the subject from developing symptoms upon infection with a wild-type Herpes simplex virus. The compositions and methods disclosed herein are effective in genetically modifying a wild-type Herpes simplex virus. The compositions and methods of the present disclosure are effective in impairing anterograde axonal transport of the wild-type virus from a latent reservoir to the site of active infection. [0030] In some embodiments, the gene-drive construct is effective in integrating/inserting into the genome of a wild-type Herpes simplex virus and disrupting at least one viral gene in the genome of the wild-type Herpes simplex virus. The at least one ^{1896-P92WO AP} -7- gene may be selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding tegument protein) or a homolog thereof. In some embodiments, the at least one viral gene is US9 or a homolog thereof. [0031] In some embodiments, the Herpes simplex virus is selected from HSV-1, HSV-2, and VZV. In some embodiments, the Herpes simplex virus is selected from HSV- 1 and HSV-2. DESCRIPTION OF THE DRAWINGS [0032] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: [0033] FIGS. 1A-1E depict design of an exemplary viral gene drive targeting HSV-1 UL37-38 region. Gene drive viruses carry Cas9 and a gRNA targeting the same location in a wild-type genome. After co-infection of cells by wild-type (WT) and gene drive (GD) viruses, Cas9 cleaves the wild-type sequence and homology-directed repair – using the gene drive sequence as a repair template– causes the conversion of the wild-type locus into a new gene drive sequence and the formation of new recombinant gene drive viruses (FIG. 1A). FIG. 1B shows the modified and unmodified UL37-38 region. The gene drive cassette was inserted between the UL37 and UL38 viral genes and is composed of spCas9 under the control of the CBH promoter followed by the SV40 polyA signal, the CMV promoter driving an mCherry reporter, followed by the beta-globin polyA signal, and a U6-driven gRNA (FIG. 1B). FIG. 1C shows localizations of the gene drive sequence and YFP/CFP reporters on HSV-1 genomes. GD represents a functional gene drive virus, GD-ns carries a non-specific gRNA, and Cas9 is deleted in GD-^Cas9. UL/US: unique long/short genome segments (FIG. 1C). Recombination products and examples of viral plaques after cellular co-infection with HSV1-WT expressing YFP and gene drive viruses expressing both mCherry and CFP (FIGS. 1D-1E). [0034] FIGS. 2A-2G show Gene drive spread in vitro. Viral titers in the supernatant after infection of N2a cells with WT, GD, GD-ns or GD-DCas9. Cells were infected with a single virus at M01=1. n=4 (FIG.2A). Viral titers in the supernatant after co-infection of N2a cells with WT+GD, WT+GD-ns or WT+GD-LiCas9, with a starting proportion of gene drive virus of 20% (FIG. 2B) or 40% (FIG. 2C). M01=1, n=4. ^{1896-P92WO AP} -8- Evolution of the viral population after co-infection with WT+GD, WT+GD-ns or WT+GD- LiCas9, with a starting proportion of gene drive virus of 20% (FIG, 2D, FIG.2E) or 40% (FIG. 2F, FIG. 2G). FIG. 2D and FIG. 2F show the proportion of viruses expressing mCherry, representing gene drive virus. FIG. 2E and FIG. 2G show the proportion of viruses expressing the different fluorophore combinations. Viral titers are expressed in log-transformed PFU (plaque-forming unit) per ml of supernatant. Error bars represent the standard error of the mean (SEM) between biological replicates. n=4. [0035] FIGS.3A-3I show Gene drive spread during herpes simplex encephalitis. FIG. 3A. shows infection routes along optic, oculomotor and trigeminal nerves (cranial nerves II, III, and V, respectively) following ocular inoculation of HSV-1. Male and female Balb/c mice were infected with 10⁶ PFU in the left eye. FIG.3B and FIG.3C show viral titers after four days in the eye, TG and whole brain after (FIG.3B) infection with a single virus, n=5, or (FIG.3C) with a starting proportion of gene drive virus of 15%. n=6. FIG. 3D, FIG.3E show viral population in the eye, TG and whole brain after co-infection with WT+GD or WT+GD-ns, after four days. n=6. FIG.3F shows proportion of viral genomes with a mutated target site in the brain after four days. n=3. FIG. 3G shows viral titers in the spinal cord and brain after inoculation of WT, WT+GD, or WT+GD-ns in the right hind leg footpad, after 5-7 days. n=8 for WT and WT+GD, n=4 for WT+GD-ns. FIG.3H, FIG. 3I show viral population in the spinal cord and whole brain after co-infection with WT+GD or WT+GD-ns, after 5-7 days. n=5 for WT+GD, n=1 for WT+GD-ns. Viral titers are expressed in log-transformed PFU. In FIGS.3B, 3C, 3G, black lines indicate the median. n.d.: non-detected. FIGS. 3D, 3E, 3F, 3H and 3I show the average and SEM between biological replicates. [0036] FIGS.4A-4E show high heterogeneity between brain regions during gene drive spread. FIG. 4A. shows infection routes following ocular inoculation of HSV-1. Male and female Balb/c mice were co-infected with 106 PFU of WT+GD in the left eye, with a starting proportion of gene drive virus of 15%. FIG.4B shows viral titers over time. Black lines indicate the median. n.d.: non-detected. FIG.4C, FIG.4D show proportion of viruses over time. Data show the average and SEM between biological replicates FIG.4E depicts heatmap summarizing data represented in FIG.4B and FIG.4C. n=4 for day 2 and 3, n=6 for day 4. [0037] FIGS. 5A-5F show high levels of co-infection in the TG during HSV-1 infection. FIG.5A shows Balb/c mice were co-infected with equivalent amounts of three ^{1896-P92WO AP} -9- viruses expressing YFP, CFP and RFP, respectively, with a total of 106 PFU in the left eye. FIG. 5B shows YFP and CFP cellular intensity after machine learning-assisted cell segmentation of TG sections. Datapoints represent individual cells and were colored by converting YFP and CFP signals into the CYMK color space. 4035 cells were detected, originating from 53 images and n=4 animals. FIG.5C depicts percentage of infected cells expressing YFP, CFP, or both. n=4. FIG.5D shows percentage of infected cells expressing one or two fluorescent markers. n=4. FIG. 5E, FIG. 5F are representative images of TG sections, highlighting high levels of co-infection. Scale bars: 100 µm. Arrows indicate cells co-expressing YFP, CFP and RFP together. [0038] FIGS. 6A-6H show high levels of co-infection in the brain during HSV- 1 infection. FIG.6A shows images of brain sections were collected in three regions in the thalamus, midbrain and brain stem after ocular infection. FIG. 6B shows YFP, CFP and RFP cellular intensity after machine learning-assisted cell segmentation of brain sections. Datapoints were colored by converting YFP, CFP and RFP signals into the CYMK color space. 10,028 cells were detected, originating from 95 images and n=3 animals. FIG.6C depicts the percentage of infected cells expressing one, two, or three fluorescent markers, both in the whole brain and in specific subregions. n=3. FIG. 6D shows representative images of the brain in the thalamus (sections S1), midbrain (sections S2) and brain stem (section S3). FIGS. 6E-6H show representative images and summary of co-infection patterns in specific subregions. LGN: lateral geniculate nucleus; SC: superior colliculus; EW: Edinger–Westphal nucleus; TGN: trigeminal nerve nuclei. Scale bars: 100 µm. [0039] FIGS. 7A-7F show Gene drive spread during latent infection in Swiss- Webster mice. FIG. 7A shows the experimental outline: Swiss-Webster mice were infected with 10⁵ PFU of HSV1-WT on both eyes after corneal scarification. Four weeks later, mice were superinfected with 10⁷ PFU of GD or GD-ns on both eyes, after corneal scarification. Another four weeks later, latent HSV-1 was reactivated twice with JQ1, two weeks apart. n=14 mice per group. FIG.7B shows titer and number of shedding events in eye swabs on days 1-3 following JQ1 treatment, by qPCR. Shedding events from the same mouse are connected by a line. FIG.7C shows genotyping of positive eye swabs from five mice, using two duplex ddPCR assays. The first assay detected and quantified mCherry levels. The second assay distinguished between YFP and CFP. n=8. FIG. 7D shows the number and proportion of TG and mice with detectable CFP. FIG. 7E shows latent viral load in the TG by duplex ddPCR, detecting mCherry, YFP, CFP markers, or all HSV ^{1896-P92WO AP} -10- sequences. n=28. FIG.7F shows proportion of CFP and mCherry in the TG. n=28. Titers are expressed in log-transformed copies per swab, or per million cells after normalization with mouse RPP30 levels. Black lines indicate the median. n.d.: non-detected. [0040] FIGS.8A-8G show Gene drive spread during latent infection in C57Bl/6 mice. C57Bl/6 mice were infected with 10⁶ PFU of HSV1-WT on both eyes after corneal scarification. Four weeks later, mice were superinfected with 10⁷ PFU of GD or GD-ns on both eyes, after corneal scarification. After another four weeks, latent HSV-1 was reactivated three times, two weeks apart, with JQ1 and Buparlisib. n=26 for GD, n=18 for GD-ns. FIG. 8A shows titer and number of shedding events in eye swabs on days 1-3 following JQ1 treatment, by qPCR. Shedding events from the same mouse are connected by a line. FIG.8B shows genotyping of positive eye swabs, detecting mCherry, YFP and CFP markers. Light red indicates low mCherry levels, less than 5% of the total swab titer. Swab genotypes were assessed by ddPCR and confirmed by qPCR for low-titer swabs. Details are shown in supplementary FIG. S12. FIG. 8C shows number and proportion of TG and mice with detectable CFP marker from GD/GD-ns. FIGS.8D-8E show latent viral load and proportion in the TG. n=52 for GD, n=36 for GD-ns. Black lines indicate the median FIG. 8F shows mCherry as a function of CFP, and best line fit. GD datapoints were significantly higher than the identity line, suggesting gene drive propagation in the TG. Same data as FIG.8D, excluding non-detected samples. n=38 for GD, n=23 for GD- ns. FIG.8G. Log2 fold-change between the proportion of latent mCherry and CFP, using data from FIG.8E. Samples with low levels of less than 0.5% were excluded. Black lines show the average. Asterisks summarize the results of Welch's t-test (p=0.0053). n=33 for GD, n=20 for GD-ns. Titers are expressed in log-transformed copies per swab, or per million cells after normalization with mouse RPP30. n.d.: non-detected. [0041] FIGS.9A-9E show N2a cells sustain high levels of co-infection. N2a and Vero cells were coinfected with WT virus expressing YFP and GD-ns virus expressing CFP and mCherry, at low MOI (from 0.1 to 0.001). Two days later, the proportion of infected and co-infected cells was analyzed by microscopy (FIG. 9A, FIG. 9C) or flow cytometry (FIG.9B, FIG.9D, and FIG.9E). At both high, middle, or low total infection rates, N2a cells showed a high level of co-infection, while Vero cells had very little co- infection. Data represented in FIG.9E, show mean and SEM between 6 (high co-infection rate) or 3 (middle and low infection rates) biological replicates. High, middle, and low infection rates indicate that around 90%, 40%, or 10% of all cells are infected, respectively. ^{1896-P92WO AP} -11- [0042] FIGS. 10A-10E show no correlation between viral titers and gene drive- mediated recombination. No correlation was observed after least square linear regression between viral titers and the level of gene drive-mediated recombinants in the different brain regions. R² indicates the goodness of fit. [0043] FIGS. 11A-11F show machine learning-assisted analysis of co-infection in the TG. FIG.11A depicts a summary of the analysis pipeline: individual color channels were merged to create composite gray- scale images. Infected cells were automatically segmented using machine learning analysis software from biodock.ai, allowing quantification of mono- and co-infected cells. 4035 cells were detected, originating from 53 images and n=4 animals. FIG. 11B shows cell area and eccentricity after cell segmentation. FIG. 11C shows co-infected cells and expression thresholds. FIG. 11D shows the percentage of infected cells expressing YFP, CFP, or both, in the four biological replicates. FIG. 11E shows high consistency between biological replicates. FIG. 11F shows representative images of TG sections. Scale bars: 100 µm. Arrows indicate cells co-expressing YFP, CFP and RFP together. [0044] FIGS. 12A-12F shows infection of the visual system in the brain. FIG. 12A shows coronal sections of the brain collected after ocular infection. FIG.12B shows regions of primary spread in the visual system. HSV-1 infects retinal neurons and travels via the optic nerve to the LGN in the thalamus and to the SC in the midbrain. After infection of the ciliary ganglion, HSV-1 travels to the EW in the midbrain via the oculomotor nerve. Finally, HSV-1 travels via the trigeminal nerve through the TG, reaching the TGN in the brain stem. FIG. 12C-12F show reproducible infection of the visual system, as shown in three biological replicates (brain ID# MB112, MB113 and MB114). SCN: suprachiasmatic nuclei, LGN: lateral geniculate nucleus. OT: optic tract, OPT: olivary pretectal nucleus, VC: visual cortex SC: superior colliculus, EW: Edinger- Westphal nucleus, TGN: trigeminal nerve nuclei Scale bars: 100 µm. [0045] FIGS. 13A-13F show machine learning-assisted analysis of co-infection in the brain. 10,028 cells were detected, originating from 95 images and n=3 animals (brain ID# MB112, MB113 and MB114). FIG.13A shows cell area and eccentricity. FIG.13B shows RFP intensity threshold and discarded cells. FIG. 13C Co-infected cells and expression thresholds. FIG. 13D shows percentage of infected cells expressing one, two, or three fluorescent markers in the three biological replicates. FIGS. 13E-F show ^{1896-P92WO AP} -12- percentage of infected cells expressing the different color combinations, in the whole brain and the three biological replicates (FIG.13E), or across the subregions (FIG.13F). n=3. [0046] FIGS. 14A-14B show low co-infection levels in the lateral geniculate nucleus. Representative images of the lateral geniculate nucleus (LGN) from two different biological replicates. Infected cells form tight foci expressing only one color, with co- infected cells at the boundaries. Scale bars: 100 µm. [0047] FIGS. 15A-15B show low co-infection levels in the superior colliculus. Representative images of the superior colliculus (SC) from two different biological replicates. Infected cells form tight foci expressing only one color, with co-infected cells at the boundaries. Scale bars: 100 µm. [0048] FIGS. 16A-16B show high co-infection levels in the Edinger-Westphal nucleus. Representative images of the Edinger-Westphal nucleus (EW) from two different biological replicates. No evidence of spatial clustering and uniform distribution of co- infected cells. Scale bars: 100 µm. [0049] FIGS. 17A-17C shows high co-infection levels in the trigeminal nerve nuclei. Representative images of the trigeminal nerve nuclei (TGN) from three different biological replicates. No evidence of spatial clustering and uniform distribution of co- infected cells. Scale bars: 100 µm. [0050] FIGS.18A-18G show latent infection in Swiss-Webster mice. FIG.18A shows proportion of mice surviving and showing symptoms after primary infection of Swiss-Webster mice with HSV1-WT. n=60. FIG.18B shows weight changes throughout the experiment. FIG. 18C shows cumulative symptom score measured during primary infection Data show means and SEM. FIG.18D shows final eye scarification score at the end of the primary infection. Mice were separated into equivalent groups before superinfection with GD or GD-ns. FIGS. 18E-18F show final titer of GD/GD-ns in the TG as a function of the eye and symptom scores. FIG. 18G. shows swab genotyping by duplex ddPCR. Swabs expressing YFP only are wild-type. Swabs expressing CFP and mCherry represent the original GD/GD-ns. Swabs expressing YFP and mCherry are recombinants. Titers are expressed in log-transformed copies per swab, or per million cells after normalization with mouse RPP30. n.d.: non-detected. [0051] FIGS.19A-19E show latent infection in C57Bl/6 mice. FIG.19A shows a proportion of mice surviving and showing symptoms after primary infection of C57Bl/6 mice with HSV1-WT. n=50. FIG.19B shows weight changes throughout the experiment. ^{1896-P92WO AP} -13- virus, Cas9 cleaves the wild-type genome. Homology-directed repair using the gene drive sequence as a repair template converts the wild-type virus into a new recombinant gene drive virus. HSV virions travel through nerves from the mucosal periphery to neurons in the ganglia (retrograde transport), where they remail latent. After reactivation, virions travel back to the surface (anterograde transport), causing viral outbreaks (FIG. 22B). During orofacial infection, HSV remains latent primarily in the trigeminal ganglia (FIG. 22C). The gene drive virus inactivating anterograde transport, by knocking out US7, US8, US9 could treat or prevent herpes infection by inactivating wild-type viruses in the ganglia and suppressing viral outbreaks. FIG.22D shows gene drive virus knocking out the viral gene US9. The gene drive sequence is inserted into HSV-1 or HSV-2 genome and knockout US9. Schematic gives the composition of GD-US9 viruses around the integration site of the gene drive sequence. [0055] FIGS. 23A-23D show anterograde transport of GD-US9 and GD- US9/gE-Y463E is highly reduced compared to HSV1-WT. Mice were infected ocularly with 10⁶ plaque forming units (pfu) of virus in the intravitreal space of the left eye (FIG. 23D). In this model, HSV-1 travels from the eye to the brain through the cranial nerve II., III. and V. After 5 days, brain sections of infected mice were stained with an antibody recognizing HSV, highlighting the brain regions infected with HSV. Mice infected with HSV1-WT showed extensive staining all over the brain (FIG. 23A). By contrast, mice infected with GD-US9 (FIG. 23B) and GD-US9/gE-Y463E (FIG. 23C) showed much reduced staining. The only region with detectable HSV in the brain of mice infected with GD-US9/gE-Y463E corresponds to the Edinger–Westphal nucleus in the midbrain (arrow) (FIG.23C). This brain region is by reached by retrograde viral transport, while the rest of the brain is reached by anterograde transport. These results confirmed that GD-US9 and GD-US9/gE-Y463E had inactivated anterograde transport while maintaining functional retrograde transport. [0056] FIGS. 24A-24C show GD-US9 and GD-US9/gE-Y463E establish a latent infection. Mice were infected ocularly after corneal scarification with HSV1-WT (10⁵ pfu/eye), GD-US9 (10⁶ pfu/eye) or GD-US9/gE-Y463E (10⁶ pfu/eye) (FIG.24C). A month later, the latent viral load in the trigeminal ganglia (TG) was measured. TG is the main site of latency after orofacial infection. The data shows that both GD-US9 and GD- US9/gE-Y463E could reach the TG and establish latency, confirming that the GD-US9 and GD-US9/gE-Y463E have functional retrograde transport (FIG. 24A). Latent virus in the ^{1896-P92WO AP} -15- ganglia of mice infected with HSV1-WT or GD-US9 was reactivated using the small molecule drug JQ1. Viral shedding was measured in eye swabs collected 1-3 days JQ1 infection. The data shows that in mice infected with GD-US9, the frequency of viral shedding was reduced by 95% (fisher's exact test, p<0.0001). This confirmed that anterograde transport of GD-US9 was almost completely inactivated. With virus GD- US9/gE-Y463E, viral shedding is expected to be reduced by 100% (FIG.24B). pfu: plaque forming unit. [0057] FIGS.25A-25D show GD-US9 and GD-US9/gE-Y463E prevent HSV- 1-associated mortality. To test if GD-US9/gE-Y463E could be used as preventative treatment against HSV-1, mice were treated with GD-US9/gE-Y463E, either by ocular infection with GD-US9/gE-Y463E (10⁶ pfu/eye), or by intravaginal inoculation (10⁶ pfu) (FIG. 25D). A month after inoculation, mice were challenged with HSV-1 WT, either ocularly or vaginally (10⁷ pfu). In both cases, GD-US9/gE-Y463E treatment prevented mortality caused by HSV-1 challenge (FIGS. 25A-25B). This showed that GD-US9/gE- Y463E could be used as a preventive that protect against HSV-1 infection. [0058] Further, to test if GD-US9 and GD-US9/gE-Y463E could be used as a therapeutic treatment against HSV-1 infection, mice were first infected with HSV-1 WT, by intravaginal inoculation (10⁴ pfu). A month after infection, latently infected mice were treated intravaginally with GD-US9 or GD-US9/gE-Y463E (10⁷ pfu). A month following treatment, mice were treated with the small molecule JQ1 to reactivate latent viruses. The data shows that treatment with GD-US9 or GD-US9/gE-Y463E prevented mortality associated with viral reactivation (FIG. 25C). Pfu: plaque forming unit. DETAILED DESCRIPTION [0059] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. [0060] Herpes simplex viruses (HSV) are part of the alphaherpesvirus subfamily of herpesviruses. There are two types of HSV: type-1 (HSV-1) and type-2 (HSV-2). These viruses are neurotropic capable of infecting the nervous system and causing neurological diseases. Moreover, unlike many herpesviruses, HSV has low species specificity and a wide host range. It has the unparalleled ability to infect human and nonhuman cells alike (see, e.g., Spear & Longnecker (2003) J. Virol.77(19): 10179-10185). Ongoing efforts to eliminate latent Herpes simplex virus (HSV), rely on viral vectors such as lentiviruses or ^{1896-P92WO AP} -16- AAVs to deliver nucleases. These promising strategies face challenges in efficiently and specifically targeting the latent reservoir, and by potential toxicity of the vectors. [0061] Gene-drive refers to genetic sequences that are transmitted from one generation to the next with a high probability, and that can transmit a trait over an entire population (see, for example, Esvelt et al. (2014) Elife, 3: e03401; Champer et al. (2016) Nat. Rev. Genet.17: 146-159). Natural homing endonuclease genes exhibit gene drive by cutting the corresponding locus of chromosomes lacking them. This induces the cell to repair the break by copying the nuclease gene onto the damaged chromosome via homologous recombination (see, e.g., Burt & Koufopanou (2004) Curr. Opin. Genet. Dev., 14:609-615). The copying process is termed 'homing', while the endonuclease- containing cassette that is copied is referred to as a "gene drive construct", a "gene drive", or simply a "drive". In sexually reproducing species, copying causes the fraction of offspring that inherit the cassette to be greater than ½, and these genes can therefore drive through a population even if they reduce the reproductive fitness of the individual organisms that carry them. Over many generations, this self-sustaining process can theoretically allow a gene drive to spread from a small number of individuals until it is present in all members of a population. Thus, gene drives are genetic modifications designed to spread efficiently through a population that rely on endonuclease-mediated recombination and have been designed principally in insects to eradicate diseases such as malaria. [0062] An engineered gene drive system can use a targeted endonuclease gene (e.g., a CRISPR/Cas construct) in place of a homing endonuclease gene. The endonuclease transgene is inserted in place of a natural sequence that it can cut. In typical embodiments, the endonuclease transgene construct is provided appropriate flanking homology sequences so that when the expressed endonuclease cleaves the corresponding site in an unmodified genome (e.g., genomic locus) a copy of the construct comprising the endonuclease transgene is inserted into the corresponding (previously unmodified) locus via homologous recombination. Most engineered gene-drive systems use CRISPR-Cas9 editing to duplicate a synthetic sequence from one modified chromosome to its wild-type counterpart during sexual reproduction. Importantly, current gene-drive strategies were understood to only work in sexually reproducing organisms such as animals and plants and it was believed they could not be applied to asexual populations such as viruses and bacteria. ^{1896-P92WO AP} -17- [0063] The present disclosure describes a novel gene-drive system that allows the spreading of an engineered trait in populations of DNA viruses, and in particular herpesviruses. In certain embodiments the gene drive systems described herein can be used to stop or drastically circumvent the spreading of infectious viruses. Thus, for example, such a strategy could be used as a novel cure against any herpesviruses. The gene drive systems described herein additionally or alternatively be used to drive any desired transgene into a population to produce a modified viral population encoding that transgene. [0064] The viral gene drive of the present disclosure represents a novel class of interfering viruses. Upon co-infection with an engineered virus, the wild-type genome is cut and repaired by homologous recombination, producing new recombinant gene drive viruses that progressively replace the wild-type population. An attenuated gene drive virus spreads efficiently until the wild-type population had been eliminated, ultimately reducing viral levels. Gene drive is demonstrated by natural homing endonuclease genes by cutting the relevant region of chromosomes that lack them. By replicating the nuclease gene onto the broken chromosome by homologous recombination, the cell is prompted to repair the break (see, e.g., Burt & Koufopanou (2004) Curr. Opin. Genet. Dev., 14:609–615). The endonuclease-containing cassette that is copied is referred to as a "gene drive construct," "gene drive," or simply a "drive," while the copying procedure is known as "homing." These genes can spread throughout a population even when they have a negative impact on the reproductive success of the organism that carry them because in sexually reproducing species, copying results in a higher than 50% inheritance rate. Theoretically, this self- sustaining process can enable a gene drive to expand from a select few individuals until it is found in every member of a population. [0065] A gene drive offers a complementary approach that may ideally circumvent issues and limitations of conventional strategies relying on viral vectors for delivery of endonucleases. Modified viruses based on HSV-1 have the same tropism and follow the same infection routes as wild-type HSV-1, potentially reaching the latent reservoir efficiently and with few off-target effects. The gene drive may permanently replace the original virus, offering life-long protection against chronic reactivation. Thus, a modified virus containing a gene drive represents an innovative strategy to inactivate latent viruses and functionally cure HSV disease. [0066] Numerous genome-editing techniques have been recently enabled primarily due to CRISPR-Cas9 technology (for examples, see Jinek & Chylinski (2012) ^{1896-P92WO AP} -18- Science, 337: 816-821; Barrangou & Doudna (2016) Nat. Biotechnol.34: 933–941; Wang et al. (2013) Cell, 153: 910–918). A complementary DNA sequence is targeted and cut by the Cas9 protein when it is in combination with a guide RNA (gRNA). Following this, cells either use non-homologous end joining or homology directed repair (HDR) employing a homologous repair template to fix DNA double-strand breaks. The majority of synthetic gene drives employ CRISPR-Cas9 editing, in which a Cas9 transgene is substituted for a natural sequence (homing endonuclease gene) together with a guide RNA that specifically targets this region. When a non-modified allele is repaired during sexual reproduction by homologous recombination following Cas9 cleavage, the synthetic sequence is duplicated, ensuring its spread throughout the population. In typical embodiments, the endonuclease transgene construct is provided appropriate with flanking homology sequences so that when the expressed endonuclease cleaves the corresponding site in an unmodified genome (e.g., genomic locus) a copy of the construct comprising the endonuclease transgene is inserted into the corresponding (previously unmodified) locus via homologous recombination. [0067] In some embodiments, the modified virus containing the gene drive construct retains its ability to infect a cell. In some embodiments, the modified virus containing the gene-drive construct may be introduced into a cell by infection (using the virus's endogenous cell-entry machinery) or by transfection (e.g., by nucleofection, electroporation, etc.). In some embodiments, the gene drive construct inhibits the viral infectivity of the modified virus containing the gene-drive construct. In some embodiments, the gene drive construct further comprises a nucleotide sequence encoding a protein. In some embodiments, the nucleotide sequence encodes a "rescue protein" to permit infectivity of the modified virus containing the gene drive construct. In certain embodiments, the nucleotide sequence encoding the "rescue protein" is operably linked to an inducible promoter. In some embodiments, the infectivity of the modified virus can be initiated/restored by providing the inducer for that promoter. The present disclosure encompasses novel gene drive constructs that do not involve sexual reproduction to allow the spreading of an engineered trait in populations of DNA viruses, and in particular herpesviruses. The preferred requirements for a viral gene drive are: 1) A dsDNA genome large enough to add the 6-7 kb gene drive sequence which, in certain embodiments represents a minimal viral genome size of ˜50 kb; and 2) the capacity to undergo homologous recombination. This second condition is easily satisfied by any nuclear- ^{1896-P92WO AP} -19- replicating virus, because these viruses use cellular recombination machinery. Herpesviruses are nuclear-replicating DNA viruses that harbor a large dsDNA genome (100-200 kb), encoding 100-200 genes (Louten (2016) Chapter 13, pp. 235-256 in Herpesviruses BT-Essential Human Virology, Academic Press, Boston). These properties enabled the design of a new gene drive strategy that doesn't involve sexual reproduction but relies on coinfection of a given cell by a wild-type and an engineered/modified virus. Upon coinfection, the wild-type genome is cleaved and repaired by homologous recombination, producing a new gene drive virus. Thus, utilizing the gene drive constructs of the present disclosure, the inventors demonstrate the ability to carry a gene drive sequence from the genome of an engineered virus to a wild-type one, thereby limiting the spread of wild-type viruses while creating new copies of the mutated one. [0068] In an illustrative, but non-limiting example, a gene drive construct for use in the methods described herein comprises a targeted endonuclease (e.g., Cas9, TALEN, ZFP, etc.) operably linked to (under control of) a promoter). The construct is flanked by a left and a right homology arm to provide for insertion into a target site and the endonuclease is targeted (e.g., by TAL to cleave a target genome at a site corresponding to the location of the gene drive construct so that after cleavage a gene drive construct is inserted into the cleaved site. Where required by the targeted endonuclease, the construct typically encodes one or more guide RNAs, also operably linked to one or more promoters. In certain embodiments the construct can (optionally) additionally include one or more genes (cDNAs) to be expressed. [0069] In certain embodiments to treat a viral invention (e.g., to inhibit or stop a viral infection), the gene drive construct is designed to knock-out one or more essential viral genes. Thus, for example the gene drive construct can be designed to insert into a gene associated with viral infection, and/or a gene associated with viral replication. The genome of the modified viruses (now containing the gene drive construct) will lack an essential viral gene (replaced by the nucleic acid encoding the endonuclease (e.g., encoding Cas9 and gRNA(s)), thereby preventing the production of infectious virions. However, upon co-infection by a gene-drive and a wild-type virus, new infectious gene-drive virions can be produced using the gene products of the wild-type genome. Concomitantly, expression of the targeted endonuclease (e.g., Cas9) from the gene-drive genome would inactivate the wild-type virus and convert it into new gene-drive genome. In various embodiments this strategy relies on the dynamics of expression of the endonuclease (e.g., ^{1896-P92WO AP} -20- Cas9) from the gene drive genome, and the corresponding wild-type gene. In particular, enough wild-type protein should be produced from the wild-type genome before the endonuclease (e.g., Cas9) is expressed and inactivates it. [0070] In certain embodiments, where the construct inhibits/disrupts a gene, the gene/cDNA insert can be a rescue for the knockout. Alternatively, the gene/cDNA can express a detectable marker or can encode another protein that is to be expressed by the target viral genome. Where the targeted endonuclease does not utilize a guide RNA, the nucleic acid encoding the gRNAs can be omitted. In certain embodiments where the targeted endonuclease utilizes a guide RNA (gRNA) multiple guide RNAs can be provided that target multiple sites. Targeting multiple sites increases the cutting frequency and hinders the evolution of drive resistant alleles. In an embodiment, an exemplary gene drive (SEQ ID NO: 1) of the present disclosure comprises a nucleic acid sequence encoding a short synthetic polyA signal, expected to terminate transcription from upstream HSV region; a Roux Sarcoma Virus (RSV) promoter driving expressing of spCas9; spCas9; a SV40 polyA signal terminating spCas9 transcription; a CMV promoter/mCherry/betaglobin polyA signal reporter cassette; gRNA driven by a human U6 promoter; and a short synthetic polyA signal. The mCherry reporter sequence is used for genetic engineering purposes and can be removed. Illustrative gene drives and methods of construction of gene drives are described in detail in U.S. Patent Publication No, US20210222150A, the disclosure of which is incorporated herein in its entirety by reference. [0071] The foregoing exemplary gene drive sequence is used in the different constructs of the present disclosure. See Table 1. The only difference between constructs is the gRNA sequence, which defines the target gene and target site where the gene drive sequence is integrated in the HSV genome. See exemplary target gene sequences in Table 2. In an embodiment, the gRNA targeting HSV2 US9 is represented by SEQ ID NO: 14 (AACGACTTCCTCGTGCGCAT). In an embodiment, the HSV2 comprises the HSV2 strain MS (MK855052.1) (SEQ ID NO: 7). In some embodiments, the gRNA targeting HSV1 US9 is represented by SEQ ID NO: 15. In some embodiments, the HSV1 comprises the HSV2 strain 17+ (NC_001806.2) (SEQ ID NO: 6). The foregoing sequence can be replaced by any other potential CRISPR target site to direct gene drive towards a particular target site on a target gene. Method and tools for designing gRNAs based on target gene sequences are described in, for example, Concordet, J.-P. & Haeussler, M. CRISPOR: ^{1896-P92WO AP} -21- Intuitive Guide Selection For CRISPR/Cas9 Genome Editing Experiments and Screens. Nucleic Acids Res.46, W242–W245 (2018). TABLE 1: Description/Name Guide RNA Targeting HSV1/HSV2 US9 gene 6) 6) : :

^{1896-P92WO AP} -22- [0072] The present disclosure also provides modified viruses comprising the gene drive constructs disclosed herein. Where the modified virus containing the gene drive construct retains its ability to infect a cell, modified virus can be introduced into the cell by infection (using the viruses' endogenous cell-entry machinery) or by transfection (e.g., by nucleofection, electroporation, etc.). Where the gene drive construct inhibits viral infectivity, the modified virus can be provided with a "rescue" gene to permit infectivity, or the modified virus can simply be transfected into the cell. In certain embodiments, where the modified virus contains a rescue gene, that rescue gene can be under control of an inducible promoter and infectivity of the virus can be initiated/restored by providing the inducer for that promoter. [0073] In some embodiments, the methods of utilizing gene drive constructs in viral systems involves 1) transfecting or infecting cells with a modified DNA virus containing a gene drive construct; and infecting that cells with the target virus (virus to be modified) where the genome of the target DNA virus is modified by insertion of the gene drive construct into the genome of the target DNA virus and a population of modified target viruses is produced. [0074] During an infection, cells are often co-infected by several virions and therapeutic approaches that rely on viral co-infection have great potential. Gene drive spread relies on the frequency of co-infection events in vivo, but prior research in this area is limited. Thus, while co-infections are known to occur, their frequency and importance for disease outcome are unknown. The inventors have characterized the frequency of infection events during primary infection and latency for the development of an innovative therapy which utilizes novel CRISPR-based "viral gene drive" constructs that outcompete the replication of their infectious parent. Further, inventors have successfully exploited the ability of Herpes simplex viruses to undergo homologous recombination, the CRISPR/Cas--9 based viral gene drive constructs, and viral co-infection strategies to demonstrate that gene drive viruses can efficiently target and replace wild-type populations and can be used to treat active and latent viral infections, which represents a novel therapeutic strategy against herpes viruses. [0075] The present disclosure thus represents an innovative approach for treating and/or preventing viral infections utilizing gene drives. The present disclosure also represents a method of preventing reactivation of latent virions in an infected host utilizing modified viruses containing gene drives as disclosed herein. The gene drives of the present ^{1896-P92WO AP} -23- disclosure represent a breakthrough strategy to engineer herpesviruses. The present disclosure pertains to methods and compositions utilizing a gene drive and a modified virus comprising the gene drive integrated into the genome of the modified virus. The modified virus comprising a gene drive integrated into the genome of the modified virus is, amongst other things, effective in inactivating a latent virus in infected hosts. The present disclosure thus is directed to new therapies that solve an unmet medical need. [0076] Accordingly, in an aspect, the present disclosure provides a method of suppressing and/or preventing infection, or a recurrence of an infection caused by a wild- type alphaherpesvirus. The method may comprise genetically modifying/altering a genome of the wild-type alphaherpesvirus. In some embodiments, the method comprises co- infecting at least one cell of a latent reservoir comprising the wild-type alphaherpesvirus with at least one modified alphaherpesvirus. In some embodiments, the at least one modified alphaherpesvirus comprises a gene-drive construct integrated into the genome of the modified alphaherpesvirus. In some embodiments, the at least one modified alphaherpesvirus is effective in genetically modifying/altering the wild-type alphaherpesvirus by integrating/inserting the gene drive construct into the genome of the alphaherpesvirus at a target site. In an embodiment, the integration/insertion of the gene drive construct disrupts at least one viral gene at the target site in the genome of the wild-type alphaherpesvirus. [0077] In another aspect, the present disclosure pertains to a method of preventing and/or suppressing anterograde transport of a wild-type alphaherpesvirus from a latent reservoir to a site of active infection. In some embodiments, the method comprises co- infecting a subject harboring a wild-type alphaherpesvirus in a latent reservoir with a modified alphaherpesvirus containing a gene-drive construct integrated into the genome of the modified alphaherpesvirus. In an embodiment, the modified alphaherpesvirus is effective in genetically modifying/altering the wild-type alphaherpesvirus by integrating/inserting the gene drive construct at a target site in the genome of the wild-type alphaherpesvirus. In some embodiments, the integration/insertion of the gene drive construct at the target site disrupts at least one viral gene at the target site in the genome of the wild-type alphaherpesvirus. In an embodiment, the at least one viral gene is a gene involved in anterograde transport of the virus. In some embodiments, the method is effective in preventing viral shedding and recurring symptoms. ^{1896-P92WO AP} -24- [0078] In yet another aspect, the present disclosure provides a method of suppressing and/or preventing recurrence of an infection in a subject caused by a wild-type Herpes simplex virus. The method may comprise co-infecting the subject with an active and/or a recurrent infection with a therapeutically effective amount of a composition comprising a modified Herpes simplex virus containing a gene-drive construct. In some embodiments, the gene-drive construct is integrated into the genome of the modified Herpes simplex virus. In an embodiment, the composition is effective in impairing anterograde transport of the wild-type Herpes simplex virus from a latent reservoir to a site of active infection. [0079] In yet another aspect, the present disclosure provides a method for prophylactically treating a subject to protect against a disease caused by a wild-type herpes simplex virus. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising a modified Herpes simplex virus containing a gene drive construct integrated into the genome of the modified Herpes simplex virus. In some embodiments, the modified Herpes simplex virus is impaired in anterograde axonal transport of the virus from a latent reservoir to a site of active infection. In some embodiments, the modified Herpes simplex virus is not impaired in retrograde transport of the virus from the site of infection to the latent reservoir. In an embodiment, the subject has had no prior exposure to a wild-type Herpes simplex virus and/or infection caused by a wild-type Herpes simplex virus. In some embodiments, the step of administering comprises infecting the subject with the modified Herpes simplex virus. In some embodiments, the method is effective in keeping the modified Herpes simplex virus latent in the subject. Wild-type and modified alphaherpesvirus [0080] The wild-type and/or modified alphaherpesvirus may be selected from Ateline alphaherpesvirus 1; Bovine alphaherpesvirus 2; Bovine mammillitis virus; Cercopithecine alphaherpesvirus 2; Human alphaherpesvirus 1 (HSV-1); Human alphaherpesvirus 2 (HSV-2); Leporid alphaherpesvirus 4; Macacine alphaherpesvirus 1; Macacine alphaherpesvirus 2; Macacine alphaherpesvirus 3; Macropodid alphaherpesvirus 1; Macropodid alphaherpesvirus 2; Panine alphaherpesvirus 3; Papiine alphaherpesvirus 2; Pteropodid alphaherpesvirus 1; Saimiriine alphaherpesvirus 1; Bovine alphaherpesvirus 1; Bovine alphaherpesvirus 5; Bovine encephalitis herpesvirus; Bubaline alphaherpesvirus 1; Canid alphaherpesvirus 1; Caprine alphaherpesvirus 1; ^{1896-P92WO AP} -25- Cercopithecine alphaherpesvirus 9; Cervid alphaherpesvirus 1; Cervid alphaherpesvirus 2; Cervid alphaherpesvirus 3; Equid alphaherpesvirus 1; Equid alphaherpesvirus 3; Equid alphaherpesvirus 4; Equid alphaherpesvirus 8; Equid alphaherpesvirus 9; Felid alphaherpesvirus 1; Human alphaherpesvirus 3; Monodontid alphaherpesvirus 1; Phocid alphaherpesvirus 1; and Suid alphaherpesvirus 1. In some embodiments, the wild-type and/or modified alphaherpesvirus is a Human alphaherpesvirus 1 (Herpes Simplex virus 1). In some embodiments, the wild-type and/or modified alphaherpesvirus is a Human alphaherpesvirus 2 (Herpes simplex virus 2). Viral genes [0081] The methods and compositions of the present disclosure relate to disrupting at least one viral gene in the genome of a wild-type alphaherpesvirus using the modified alphaherpesvirus containing a gene drive construct. In some embodiments, the at least one viral gene is selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding membrane protein US9) or a homolog thereof. In an embodiment, the at least one viral gene is US9 or a homolog thereof. TABLE 2: Target Genes SEQ ID Description/ NO. Name Nucleotide Sequence ccacggt tctcggg atggga tgtcgcg agaggg tggggat ttcggcc ccctcga aaccac ggtgacc gatgtca aagcggc

catggcccgcctcggagccgagctcaaatcgcatccgagcaccccccccaaatcccggcgccggtcgtcacgcacgccaat gccctccctgacggccatcgccgaagagtcggagcccgctggggcggctgggcttccgacgccccccgtggaccccacgac cctggag tactatg cgagac cggggg agacgga gcaacc aagtctcg aagtctc gaacgtc tcgtgtgc cgccgcg gccgag acaacac cgcagt cggggcc ctggggt gggccca tcccgtgg tctgtata ccaggcc cagtttc tcggtat gatttggg cccacgg ttttcgg ctgggg tgtcgctc

gacgcaccacgcccacagccccgcctatccgaccctggagctgggtctggcgcggcagccgcttctgcgggttcgaacggc aacgcgcgactatgccggtctgtatgtcctgcgcgtatgggtcggcagcgcgacgaacgccagccggtttgttttgggggtg cggcccc cctcccc ttccacg cgccttt accccgg cccccaa agtcgtg cctggaa gggccg cggtcgt gactgta acagcgg cgcccct gttcccc cggggt cgccatt cggatct ggcgtac gaaccgg acctgtgc ggtggtg cgccctc ggcgtgc cgtggcg cggaga cgaggg tccgtcct

^{1896-P92WO AP} -28- 13 HSV-2_US9 atgacctcccggcccgccgaccaggactcggtgcgttccagcgcgtcggtgccgctttaccccgcggcctcgcccgtcccgg (MS strain) cagaagcctactactcggaaagcgaagacgaggccgccaacgacttcctcgtgcgcatgggccgccagcagtcggtccta ttcgggg

[0082] In an illustrative non-limiting embodiment, an exemplary gene drive construct of the present disclosure comprises a targeted endonuclease (e.g., Cas9, TALEN, ZFP, etc.) operably linked to (under control of) a promoter. In certain embodiments the encoded targeted endonuclease is a naturally occurring endonuclease (e.g., a site-specific "homing" endonuclease. In certain embodiments the targeted endonuclease includes, but is not limited to, CRISPR/cas endonucleases, zinc finger endonucleases, Transcription Activator-Like Effector Nuclease(s) (TALENs), and the like. The construct is flanked by a left and a right homology arm to provide for insertion into a target site and the endonuclease is targeted (e.g., by TAL) to cleave a target genome at a site corresponding to the location of the gene drive construct so that after cleavage a gene drive construct is inserted into the cleaved site. Where required by the targeted endonuclease, the construct typically encodes one or more guide RNAs, also operably linked to one or more promoters. [0083] In certain embodiments the construct can (optionally) additionally include one or more nucleotide sequences to be expressed, for e.g., a cDNA encoding a gene. In certain embodiments, where the construct inhibits/disrupts a gene, the gene/cDNA insert can be a rescue for the knockout. Alternatively, the gene/cDNA can express a detectable marker or can encode another protein that is to be expressed by the target or wild-type viral genome. Where the targeted endonuclease does not utilize a guide RNA, the nucleic acid encoding the gRNAs can be omitted. In certain embodiments where the targeted endonuclease utilizes a guide RNA (gRNA) multiple guide RNAs can be provided that target multiple sites. Targeting multiple sites increases the cutting frequency and hinders the evolution of drive resistant alleles. [0084] In certain embodiments the targeted endonuclease can comprise a CRISPR/Cas endonuclease that is typically guided to a target site by one or more guide RNAs (gRNAs). CRISPR-based endonucleases are RNA-guided endonucleases derived from CRISPR/Cas systems. Bacteria and archaea have evolved an RNA-based adaptive immune system that uses CRISPR (clustered regularly interspersed short palindromic ^{1896-P92WO AP} -29- repeat) and Cas (CRISPR-associated) proteins to detect and destroy invading viruses or plasmids. CRISPR/Cas endonucleases can be programmed to introduce targeted site- specific double-strand breaks by providing target-specific synthetic guide RNAs (see, e.g., Jinek et al. (2012) Science, 337: 816-821). [0085] In various embodiments the CRISPR-based endonuclease can be derived from a CRISPR/Cas type I, type II, type III, type V, or type VI system. Non-limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, and the like. [0086] In certain embodiments, the CRISPR-based endonuclease is derived from a type II CRISPR/Cas system. In illustrative, but non-limiting embodiments, the CRISPR- based endonuclease is derived from a Cas9 protein. In certain embodiments the Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina. In one specific illustrative ^{1896-P92WO AP} -30- embodiment, the CRISPR-based nuclease is derived from a Cas9 protein from Streptococcus pyogenes. [0087] In general, CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with the guide RNA such that the CRISPR/Cas protein is directed to a specific genomic or genomic sequence. CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, as well as other domains. [0088] In certain embodiments the CRISPR-based endonuclease used in the constructs and methods described herein can be a wild-type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild-type or modified CRISPR/Cas protein. In certain embodiments the CRISPR/Cas protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein. For example, the CRISPR/Cas protein can be truncated to remove domains that are not essential for the function of the protein. The CRISPR/Cas protein also can be truncated or modified to optimize the activity of the protein, or an effector domain fused with the CRISPR/Cas protein. [0089] In some embodiments, the CRISPR-based endonuclease can be derived from a wild-type Cas9 protein, modified forms, or fragment thereof. In other embodiments, the CRISPR-based endonuclease can be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild-type Cas9 protein. [0090] In general, a Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-strand break in DNA (see, e.g., Jinek et al. (2012) Science, 337: 816-821). In one embodiment, the CRISPR-based endonuclease is derived from a Cas9 protein and comprises two function nuclease domains, which together introduce a double- stranded break into the targeted site. [0091] The target sites recognized by naturally occurring CRISPR/Cas systems typically having lengths of about 14-15 bp (see, e.g., Cong et a. (2013) Science, 339: 819- ^{1896-P92WO AP} -31- 823). The target site has no sequence limitation except that sequence complementary to the 5′ end of the guide RNA (i.e., called a protospacer sequence) is typically immediately followed by (3′ or downstream) a consensus sequence. This consensus sequence is also known as a protospacer adjacent motif (or PAM). Examples of PAM include, but are not limited to, NGG, NGGNG, and NNAGAAW (wherein N is defined as any nucleotide and W is defined as either A or T). At the typical length, only about 5-7% of the target sites would be unique within a target genome, indicating that off target effects could be significant. The length of the target site can be expanded by requiring two binding events. For example, CRISPR-based endonucleases can be modified such that they can only cleave one strand of a double-stranded sequence (i.e., converted to nickases). Thus, the use of a CRISPR-based nickase in combination with two different guide RNAs would essentially double the length of the target site, while still effecting a double stranded break. [0092] The requirement of the crRNA-tracrRNA complex in a CRISPR/Cas system can be avoided by use of an engineered "single-guide RNA" (sgRNA) that comprises the hairpin normally formed by the annealing of the crRNA and the tracrRNA (see Jinek et al. (2012) Science 337:816; Cong et al. (2013) Sciencexpress/10.1126/science.1231143). In S. pyogenes, the engineered tracrRNA:crRNA fusion, or the sgRNA, guides Cas9 to cleave the target DNA when a double strand RNA:DNA heterodimer forms between the Cas associated RNAs and the target DNA. This system comprising the Cas9 protein and an engineered sgRNA containing a PAM sequence has been used for RNA guided genome editing and has been useful for zebrafish embryo genomic editing in vivo (see Hwang et al. (2013) Nat. Biotechnol., 31(3):227) with editing efficiencies similar to ZFNs and TALENs. [0093] Accordingly in certain embodiments, a CRISPR/Cas endonuclease complex used in the constructs and methods described herein comprises a Cas protein and at least one to two ribonucleic acids (e.g., gRNAs) that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. In some embodiments, a CRISPR/Cas endonuclease complex used in the methods described herein comprises a Cas protein and one ribonucleic acid (e.g., gRNA) that is capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. [0094] In some embodiments, a Cas protein comprises a core Cas protein. Illustrative Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9. In some embodiments, a Cas protein comprises a Cas protein ^{1896-P92WO AP} -32- of an E. coli subtype (also known as CASS2). Illustrative Cas proteins of the E. Coli subtype include, but are not limited to Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, a Cas protein comprises a Cas protein of the Ypest subtype (also known as CASS3). Illustrative Cas proteins of the Ypest subtype include, but are not limited to Csy1, Csy2, Csy3, and Csy4. In some embodiments, a Cas protein comprises a Cas protein of the Nmeni subtype (also known as CASS4). Illustrative Cas proteins of the Nmeni subtype include, but are not limited to, Csn1 and Csn2. In some embodiments, a Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Illustrative Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, a Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Illustrative Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, Cas5t. In some embodiments, a Cas protein comprises a Cas protein of the Hmari subtype. Illustrative Cas proteins of the Hmari subtype include, but are not limited to Csh1, Csh2, and Cas5h. In some embodiments, a Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Illustrative Cas proteins of the Apern subtype include, but are not limited to Csa1, Csa2, Csa3, Csa4, Csa5, and Cas8a. In some embodiments, a Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Illustrative Cas proteins of the Mtube subtype include, but are not limited to Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, a Cas protein comprises a RAMP module Cas protein. Illustrative RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6. [0095] In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein (spCas9) or a functional portion thereof (see, e.g., UniProtKB-Q99ZW2 (CAS9_STRP1)). In some embodiments, the Cas protein is a Staphylococcus aureus Cas9 protein (saCas9) or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus thermophilus Cas9 protein (stCas9) or a functional portion thereof. In some embodiments, the Cas protein is a Neisseria meningitides Cas9 protein (nmCas9) or a functional portion thereof. In some embodiments, the Cas protein is a Treponema denticola Cas9 protein (tdCas9) or a functional portion thereof. In some embodiments, the Cas protein is Cas9 protein from any other bacterial species or functional portion thereof. [0096] In certain embodiments the Cas 9 is mutated in one or more residues involved in the formation of non-specific DNA interactions. In certain embodiments such a Cas 9 comprises a mutated Cas9 such as eSpCas9 (see, e.g., Slaymaker, et al. ^{1896-P92WO AP} -33- (2016) Science 351: 84-88), SpCas9-HF1 (see, e.g., Kleinstiver et al. (2016) Nature, 529: 490-495), HypaCas9 (see, e.g., Chen et al. (2017) Nature 550: 407-410), and the like. [0097] In certain embodiments the CRISPR/Cas endonuclease systems used in the constructs and methods contemplated herein include but are not limited to a type V or type VI CRISPR/Cas endonuclease (e.g., the genome editing endonuclease is a type V or type VI CRISPR/Cas endonuclease) (e.g., Cpf1, C2c1, C2c2, C2c3). Type V and type VI CRISPR/Cas endonucleases are a type of class 2 CRISPR/Cas endonuclease. Examples of type V CRISPR/Cas endonucleases include but are not limited to: Cpf1, C2c1, and C2c3. An example of a type VI CRISPR/Cas endonuclease is C2c2. In some embodiments, a subject genome targeting composition includes a type V CRISPR/Cas endonuclease (e.g., Cpf1, C2c1, C2c3). In some embodiments, a Type V CRISPR/Cas endonuclease is a Cpf1 protein. In some embodiments, a subject genome targeting composition includes a type VI CRISPR/Cas endonuclease (e.g., C2c2). [0098] Like type II CRISPR/Cas endonucleases, type V and VI CRISPR/Cas endonucleases forma complex with a corresponding guide RNA The guide RNA provides target specificity to an endonuclease-guide RNA RNP complex by having a nucleotide sequence (a guide sequence) that is complementary to a sequence (the target site) of a target nucleic acid (as described elsewhere herein). The endonuclease of the complex provides the site-specific activity. In other words, the endonuclease is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence (e.g., a chromosomal sequence) by virtue of its association with the protein-binding segment of the guide RNA. [0099] Examples and guidance related to type V and type VI CRISPR/Cas proteins (e.g., cpf1, C2c1, C2c2, and C2c3 guide RNAs) can be found in the art (see, e.g., Zetsche et al. (2015) Cell, 163(3):759-771; Makarova et al. (2015) Nat. Rev. Microbiol.13(11): 722-736, Shmakov et al. (2015) Mol. Cell, 60(3):385-397; and the like). [0100] In some embodiments, the Type V or type VI CRISPR/Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3) is enzymatically active, e.g., the Type V or type VI CRISPR/Cas protein, when bound to a guide RNA, and cleaves a target nucleic acid. In some embodiments, the Type V or type VI CRISPR/Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3) exhibits reduced enzymatic activity relative to a corresponding wild-type a Type V or type VI CRISPR/Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3), and retains DNA binding activity. ^{1896-P92WO AP} -34- [0101] In some embodiments, a type V CRISPR/Cas endonuclease is a Cpf1 protein or a functional portion thereof (see, e.g., UniProtKB-AOQ7Q2 (CPF1_FRATN)). Cpf1 protein is a member of the type V CRISPR system and is a polypeptide comprising about 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Unlike Cas9, Cpf1 cleaves target DNA in a staggered pattern using a single ribonuclease domain. The staggered DNA double-stranded break results in a 4 or 5-nt 5′ overhang. [0102] The CRISPR-Cpf1 system, identified in Francisella spp, is a class 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including in their guide RNAs and substrate specificity (see, e.g., Fagerlund et al. (2015) Genom. Bio.16: 251). A major difference between Cas9 and Cpf1 proteins is that Cpf1 does not utilize tracrRNA, and thus requires only a crRNA. The FnCpf1 crRNAs are 42-44 nucleotides long (19- nucleotide repeat and 23-25-nucleotide spacer) and contain a single stem-loop, which tolerates sequence changes that retain secondary structure. In addition, the Cpf1 crRNAs are significantly shorter than the 100-nucleotide engineered sgRNAs required by Cas9, and the PAM requirements for FnCpf1 are 5′-TTN-3′ and 5′-CTA-3′ on the displaced strand. Although both Cas9 and Cpf1 make double strand breaks in the target DNA, Cas9 uses its RuvC- and HNH-like domains to make blunt-ended cuts within the seed sequence of the guide RNA, whereas Cpf1 uses a RuvC-like domain to produce staggered cuts outside of the seed. Because Cpf1 makes staggered cuts away from the critical seed region, NHEJ will not disrupt the target site, therefore ensuring that Cpf1 can continue to cut the same site until the desired HDR recombination event has taken place. Thus, in the methods and compositions described herein, it is understood that the term "Cas" includes both Cas9 and Cfp1 proteins. Accordingly, as used herein, a "CRISPR/Cas system" refers both CRISPR/Cas and/or CRISPR/Cfp1 systems, including both nuclease and/or transcription factor systems. [0103] Accordingly, in certain embodiments the methods described herein the Cas protein is Cpf1 from any bacterial species or functional portion thereof. In some aspects, Cpf1 is a Francisella novicida U112 protein or a functional portion thereof. In some aspects, Cpf1 is an Acidaminococcus sp. BV3L6 protein or a functional portion thereof. In some aspects, Cpf1 is a Lachnospiraceae bacterium ND2006 protein or a function portion thereof. ^{1896-P92WO AP} -35- [0104] In certain embodiments, Cas protein may be a "functional portion" or "functional derivative" of a naturally occurring Cas protein, or of a modified Cas protein. A "functional derivative" of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have abiological activity (e.g., endonuclease activity) in common with a corresponding native sequence polypeptide. As used herein, "functional portion" refers to a portion of a Cas protein that retains its ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked Cpf1 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of the HNH nuclease domain. In some embodiments, a functional portion of the Cpf1 protein comprises a functional portion of a RuvC-like domain. [0105] In certain embodiments a biological activity contemplated herein is the ability of the functional derivative to introduce a double strand break (DSB) at a desired target site in a genomic DNA. The term "derivative" encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. In some aspects, a functional derivative may comprise a single biological property of a naturally occurring Cas protein. In other aspects, a function derivative may comprise a subset of biological properties of a naturally occurring Cas protein. [0106] In view of the foregoing, the term "Cas protein" as used herein encompasses a full-length Cas protein, an enzymatically active fragment of a Cas protein, and enzymatically active derivatives of a Cas protein or fragment thereof. Suitable derivatives of a Cas protein or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof. Cas protein, which ^{1896-P92WO AP} -36- includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or synthesized chemically, recombinantly expressed, or by a combination of these procedures. The cell may be a cell that naturally produces Cas protein, or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas that is same or different from the endogenous Cas. In some cases, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein. [0107] In some embodiments, a Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprises a conservative amino acid substitution. In some instances, substitutions and/or modifications can prevent or reduce proteolytic degradation and/or extend the half-life of the polypeptide in a cell. In some embodiments, the Cas protein can comprise a peptide bond replacement (e.g., urea, thio urea, carbamate, sulfonyl urea, etc.). In some embodiments, the Cas protein can comprise a naturally occurring amino acid. In some embodiments, the Cas protein can comprise an alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, a Cas protein can comprise a modification to include a moiety (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.). [0108] In certain embodiments to treat a viral invention (e.g., to inhibit or stop a viral infection), and/or prevent re-activation of a latent virus, the gene drive construct is designed to knock-out one or more essential viral genes. Thus, for example the construct can be designed to insert into a gene associated with viral infection, and/or a gene associated with viral replication, and/or viral transport from latent reservoir to site of infection, for e.g., mucosa. The modified viruses comprising the gene drive construct integrated into its genome, will lack an essential viral gene (replaced by the nucleic acid encoding the endonuclease (e.g., encoding Cas9 and gRNA(s)), thereby preventing the production of infectious virions, and/or anterograde transport of virions from latent reservoirs to site of active infection. However, upon co-infection by a gene-drive and a wild-type virus, new infectious gene-drive virions can be produced using the gene products of the wild-type genome. Concomitantly, expression of the targeted endonuclease (e.g., Cas9) from the gene-drive genome would inactivate the wild-type virus and convert it into ^{1896-P92WO AP} -37- new gene-drive genome. In various embodiments this strategy relies on the dynamics of expression of the endonuclease (e.g., Cas9) from the gene drive genome, and the corresponding wild-type gene. In particular, enough wild-type protein should be produced from the wild-type genome before the endonuclease (e.g., Cas9) is expressed and inactivates it. [0109] In certain embodiments the Cas protein used in the constructs described herein may be mutated to alter functionality. Illustrative selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98137186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in WO 02/077227. [0110] In certain embodiments the Cas protein (e.g., Cas9 protein) comprise truncated Cas proteins. In one illustrative, but non-limiting, embodiment, the Cas9 comprises only the domain responsible for interaction with the crRNA or sgRNA and the target DNA. [0111] In certain embodiments the Cas proteins comprising the constructs described herein comprise a Cas protein, or truncation thereof, fused to a different functional domain. In some aspects, the functional domain is an activation or a repression domain. In other aspects, the functional domain is a nuclease domain. In some embodiments, the nuclease domain is a FokI endonuclease domain (see, e.g., Tsai (2014) Nat. Biotechnol. doi:10.1038/nbt.2908). In some embodiments, the FokI domain comprises mutations in the dimerization domain. Guide RNA Guide RNA for Type II CRISPR/Cas Endonucleases (e.g., Cas9 gRNA) [0112] A nucleic acid molecule that binds to a class 2 CRISPR/Cas endonuclease (e.g., a Cas9 protein, a type V or type VI CRISPR/Cas protein, a Cpf1 protein; etc.) and targets the complex to a specific location within a target nucleic acid is referred to herein as a "guide RNA" or "CRISPR/Cas guide nucleic acid" or "CRISPR/Cas guide RNA." In various embodiments the guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which typically comprise a nucleotide sequence that is complementary to a sequence of a target nucleic acid. ^{1896-P92WO AP} -38- [0113] A guide RNA can be referred to by the protein to which it corresponds. For example, when the class 2 CRISPR/Cas endonuclease is a Cas9 protein, the corresponding guide RNA can be referred to as a "Cas9 guide RNA." Likewise, as another example, when the class 2 CRISPR/Cas endonuclease is a Cpf1 protein, the corresponding guide RNA can be referred to as a "Cpf1 guide RNA." [0114] In some embodiments, a guide RNA includes two separate nucleic acid molecules (or two segments within a single molecule): an "activator" and a "targeter" and is referred to herein as a "dual guide RNA", a "double-molecule guide RNA", a "two- molecule guide RNA", or a "dgRNA." In some embodiments, the guide RNA is one molecule (e.g., for some class 2 CRISPR/Cas proteins, the corresponding guide RNA is a single molecule; and in some embodiments, an activator and targeter are covalently linked to one another, e.g., via intervening nucleotides and form different segments within a single RNA), and the guide RNA is referred to as a "single guide RNA", a "single-molecule guide RNA," a "one-molecule guide RNA", or simply "sgRNA." By "segment" it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule. A segment can also mean a region/section of a complex such that a segment may comprise regions of more than one molecule. [0115] In various embodiments the first segment (targeting segment) of a type II CRISPR/Cas endonuclease (e.g., a Cas9) guide RNA typically includes a nucleotide sequence (a guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within a target nucleic acid (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.). The protein-binding segment (or "protein-binding sequence") interacts with (binds to) the endonuclease protein. The protein-binding segment of a subject Cas9 guide RNA typically includes two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex). Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas9 guide RNA (the guide sequence of the Cas9 guide RNA) and the target nucleic acid. [0116] A Cas9 guide RNA and a Cas9 protein form a complex (e.g., bind via non- covalent interactions). The Cas9 guide RNA provides target specificity to the complex by including a targeting segment, which includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid). The Cas9 protein of the complex ^{1896-P92WO AP} -39- provides the site-specific activity (e.g., cleavage activity or an activity provided by the Cas9 protein when the Cas9 protein is a Cas9 fusion polypeptide, i.e., has a fusion partner). In other words, the Cas9 protein is guided to a target nucleic acid sequence (e.g., a target sequence in a chromosomal nucleic acid, e.g., a chromosome; a target sequence in an extrachromosomal nucleic acid, e.g., an episomal nucleic acid, a minicircle, an ssRNA, an ssDNA, etc.; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid; a target sequence in a viral nucleic acid; etc.) by virtue of its association with the Cas9 guide RNA. [0117] The "guide sequence" also referred to as the "targeting sequence" of a type II CRISPR/Cas endonuclease guide RNA (e.g., Cas9 guide RNA) can be modified so that the guide RNA can target a CRISPR endonuclease protein to any desired sequence of any desired target nucleic acid, with the exception that the protospacer adjacent motif (PAM) sequence can be taken into account. Thus, for example, a Cas9 guide RNA can have a targeting segment with a sequence (a guide sequence) that has complementarity with (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like. [0118] In some embodiments, a Cas9 guide RNA includes two separate nucleic acid molecules: an "activator" and a "targeter" and is referred to herein as a "dual Cas9 guide RNA", a "double-molecule Cas9 guide RNA", or a "two-molecule Cas9 guide RNA" a "dual guide RNA", or a "dgRNA." In some embodiments, the activator and targeter are covalently linked to one another (e.g., via intervening nucleotides) and the guide RNA is referred to as a "single guide RNA", a "Cas9 single guide RNA", a "single-molecule Cas9 guide RNA," or a "one-molecule Cas9 guide RNA", or simply "sgRNA." [0119] In various embodiments a Cas9 guide RNA comprises a crRNA-like ("CRISPR RNA"/"targeter"/"crRNA"/"crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA"/"activator"/"tracrRNA") molecule. A crRNA-like molecule (targeter) typically comprises both the targeting segment (single stranded) of the Cas9 guide RNA and a stretch ("duplex-forming segment") of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA. A corresponding tracrRNA-like molecule (activator/tracrRNA) typically comprises a stretch of nucleotides (duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the guide nucleic acid. In other words, a stretch ^{1896-P92WO AP} -40- of nucleotides of a crRNA-like molecule are complementary to and hybridize with a stretch of nucleotides of a tracrRNA-like molecule to form the dsRNA duplex of the protein-binding domain of the Cas9 guide RNA. As such, each targeter molecule can be said to have a corresponding activator molecule (which has a region that hybridizes with the targeter). In various embodiments the targeter molecule additionally provides the targeting segment. Thus, in various embodiments, a targeter and an activator molecule (as a corresponding pair) can hybridize to form a Cas9 guide RNA. The exact sequence of a given crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecules are found. A subject dual Cas9 guide RNA can include any corresponding activator and targeter pair. [0120] The term "activator" or "activator RNA" is used herein to mean a tracrRNA-like molecule (tracrRNA: "trans-acting CRISPR RNA") of a Cas9 dual guide RNA (and therefore of a Cas9 single guide RNA when the "activator" and the "targeter" are linked together by, e.g., intervening nucleotides). Thus, for example, a Cas9 guide RNA (dgRNA or sgRNA) typically comprises an activator sequence (e.g., a tracrRNA sequence). A tracr molecule (a tracrRNA) is a naturally existing molecule that hybridizes with a CRISPR RNA molecule (a crRNA) to form a Cas9 dual guide RNA. The term "activator" is used herein to encompass naturally existing tracrRNAs, but also to encompass tracrRNAs with modifications (e.g., truncations, sequence variations, base modifications, backbone modifications, linkage modifications, etc.) where the activator retains at least one function of a tracrRNA (e.g., contributes to the dsRNA duplex to which Cas9 protein binds). In some embodiments, the activator provides one or more stem loops that can interact with Cas9 protein. An activator can be referred to as having a tracr sequence (tracrRNA sequence) and in some embodiments is a tracrRNA, but the term "activator" is not limited to naturally existing tracrRNAs. [0121] The term "targeter" or "targeter RNA" is used herein to refer to a crRNA- like molecule (crRNA: "CRISPR RNA") of a Cas9 dual guide RNA (and therefore of a Cas9 single guide RNA when the "activator" and the "targeter" are linked together, e.g., by intervening nucleotides). Thus, for example, a Cas9 guide RNA (dgRNA or sgRNA) typically comprises a targeting segment (which includes nucleotides that hybridize with (are complementary to) a target nucleic acid, and a duplex-forming segment (e.g., a duplex forming segment of a crRNA, which can also be referred to as a crRNA repeat). Because the sequence of a targeting segment (the segment that hybridizes with a target sequence of ^{1896-P92WO AP} -41- a target nucleic acid) of a targeter is modified by a user to hybridize with a desired target nucleic acid, the sequence of a targeter will often be a non-naturally occurring sequence. However, in various embodiments, the duplex-forming segment of a targeter (described in more detail below), which hybridizes with the duplex-forming segment of an activator, can include a naturally existing sequence (e.g., can include the sequence of a duplex-forming segment of a naturally existing crRNA, which can also be referred to as a crRNA repeat). Thus, the term targeter is used herein to distinguish from naturally occurring crRNAs, despite the fact that part of a targeter (e.g., the duplex-forming segment) often includes a naturally occurring sequence from a crRNA. However, the term "targeter" encompasses naturally occurring crRNAs. [0122] In various embodiments a Cas9 guide RNA can also be said to include 3 parts: (i) a targeting sequence (a nucleotide sequence that hybridizes with a sequence of the target nucleic acid); (ii) an activator sequence (as described above)(in some cases, referred to as a tracr sequence); and (iii) a sequence that hybridizes to at least a portion of the activator sequence to form a double stranded duplex. A targeter has (i) and (iii); while an activator has (ii). [0123] A Cas9 guide RNA (e.g., a dual guide RNA or a single guide RNA) can be comprised of any corresponding activator and targeter pair. In some embodiments, the duplex forming segments can be swapped between the activator and the targeter. In other words, in some embodiments, the targeter includes a sequence of nucleotides from a duplex forming segment of a tracrRNA (which sequence would normally be part of an activator) while the activator includes a sequence of nucleotides from a duplex forming segment of a crRNA (which sequence would normally be part of a targeter). [0124] As noted above, a targeter typically comprises both the targeting segment (single stranded) of the Cas9 guide RNA and a stretch ("duplex-forming segment") of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA. A corresponding tracrRNA-like molecule (activator) typically comprises a stretch of nucleotides (a duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA. In other words, a stretch of nucleotides of the targeter is complementary to and hybridizes with a stretch of nucleotides of the activator to form the dsRNA duplex of the protein-binding segment of a Cas9 guide RNA. As such, each targeter can be said to have a corresponding activator (which has a region that hybridizes with the targeter). The targeter molecule additionally ^{1896-P92WO AP} -42- provides the targeting segment. Thus, a targeter and an activator (as a corresponding pair) hybridize to form a Cas9 guide RNA. The particular sequence of a given naturally existing crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecules are found. Examples of suitable activator and targeter are well known in the art. [0125] In various embodiments a Cas9 guide RNA (e.g., a dual guide RNA or a single guide RNA) can be comprised of any corresponding activator and targeter pair. Targeting Segment of a Type II CRISPR Endonuclease (e.g., Cas9) Guide RNA [0126] The first segment of a subject guide nucleic acid typically includes a guide sequence (e.g., a targeting sequence)(a nucleotide sequence that is complementary to a sequence (a target site) in a target nucleic acid). In other words, the targeting segment of a subject guide nucleic acid can interact with a target nucleic acid (e.g., double stranded DNA (dsDNA)) in a sequence-specific manner via hybridization (i.e., base pairing). As such, the nucleotide sequence of the targeting segment may vary (depending on the target) and can determine the location within the target nucleic acid that the Cas9 guide RNA and the target nucleic acid will interact. The targeting segment of a Cas9 guide RNA can be modified (e.g., by genetic engineering)/designed to hybridize to any desired sequence (target site) within a target nucleic acid (e.g., a eukaryotic target nucleic acid such as genomic DNA). [0127] In certain embodiments the targeting segment can have a length of 7 or more nucleotides (nt) (e.g., 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more nucleotides). In some embodiments, the targeting segment can have a length of from 7 to 100 nucleotides (nt) (e.g., from 7 to 80 nt, from 7 to 60 nt, from 7 to 40 nt, from 7 to 30 nt, from 7 to 25 nt, from 7 to 22 nt, from 7 to 20 nt, from 7 to 18 nt, from 8 to 80 nt, from 8 to 60 nt, from 8 to 40 nt, from 8 to 30 nt, from 8 to 25 nt, from 8 to 22 nt, from 8 to 20 nt, from 8 to 18 nt, from 10 to 100 nt, from 10 to 80 nt, from 10 to 60 nt, from 10 to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 10 to 18 nt, from 12 to 100 nt, from 12 to 80 nt, from 12 to 60 nt, from 12 to 40 nt, from 12 to 30 nt, from 12 to 25 nt, from 12 to 22 nt, from 12 to 20 nt, from 12 to 18 nt, from 14 to 100 nt, from 14 to 80 nt, from 14 to 60 nt, from 14 to 40 nt, from 14 to 30 nt, from 14 to 25 nt, from 14 to 22 nt, from 14 to 20 nt, from 14 to 18 nt, from 16 to 100 nt, from 16 to 80 nt, from 16 to 60 nt, from 16 to 40 nt, from 16 to 30 nt, from 16 to 25 nt, from 16 to 22 nt, from 16 to 20 nt, from 16 to 18 nt, from 18 to 100 nt, ^{1896-P92WO AP} -43- from 13 to 80 nt, from 18 to 60 nt, from 18 to 40 nt, from 18 to 30 nt, from 18 to 25 nt, from 18 to 22 nt, or from 18 to 20 nt). [0128] The nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid can have a length of 10 nt or more. For example, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid can have a length of 12 nt or more, 15 nt or more, 18 nt or more, 19 nt or more, or 20 nt or more. In some embodiments, the nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid has a length of 12 nt or more. In some embodiments, the nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid has a length of 18 nt or more. [0129] For example, in certain embodiments, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid can have a length of from 10 to 100 nucleotides (nt) (e.g., from 10 to 90 nt, from 10 to 75 nt, from 10 to 60 nt, from 10 to 50 nt, from 10 to 35 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 12 to 100 nt, from 12 to 90 nt, from 12 to 75 nt, from 12 to 60 nt, from 12 to 50 nt, from 12 to 35 nt, from 12 to 30 nt, from 12 to 25 nt, from 12 to 22 nt, from 12 to 20 nt, from 15 to 100 nt, from 15 to 90 nt, from 15 to 75 nt, from 15 to 60 nt, from 15 to 50 nt, from 15 to 35 nt, from 15 to 30 nt, from 15 to 25 nt, from 15 to 22 nt, from 15 to 20 nt, from 17 to 100 nt, from 17 to 90 nt, from 17 to 75 nt, from 17 to 60 nt, from 17 to 50 nt, from 17 to 35 nt, from 17 to 30 nt, from 17 to 25 nt, from 17 to 22 nt, from 17 to 20 nt, from 18 to 100 nt, from 18 to 90 nt, from 18 to 75 nt, from 18 to 60 nt, from 18 to 50 nt, from 18 to 35 nt, from 18 to 30 nt, from 18 to 25 nt, from 18 to 22 nt, or from 18 to 20 nt). In some embodiments, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 15 nt to 30 nt. In some embodiments, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 15 nt to 25 nt. In some embodiments, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 30 nt. In some embodiments, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 25 nt. In some embodiments, the targeting sequence of the ^{1896-P92WO AP} -44- targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 22 nt. In some embodiments, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid is 20 nucleotides in length. In some embodiments, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid is 19 nucleotides in length. [0130] In certain embodiments the percent complementarity between the targeting sequence (guide sequence) of the targeting segment and the target site of the target nucleic acid can be 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the seven contiguous 5′-most nucleotides of the target site of the target nucleic acid. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or more over about 20 contiguous nucleotides. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the fourteen contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 14 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the seven contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 20 nucleotides in length. [0131] In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 7 contiguous 5′-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3′-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 8 contiguous 5′-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3′-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some embodiments, the percent complementarity between the targeting sequence ^{1896-P92WO AP} -45- of the targeting segment and the target site of the target nucleic acid is 100% over the 9 contiguous 5′-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3′-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 10 contiguous 5′-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3′-most nucleotides of the targeting sequence of the Cas9 guide RNA). [0132] In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 17 contiguous 5′-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3′-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 18 contiguous 5′-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3′-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or more (e.g., e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over about 20 contiguous nucleotides. [0133] In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 7 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 7 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 8 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 8 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 9 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such ^{1896-P92WO AP} -46- a case, the targeting sequence can be considered to be 9 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 10 contiguous 5′- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 10 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 11 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 11 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 12 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 12 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 13 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 13 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 14 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 14 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 17 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 17 nucleotides in length. In some embodiments, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 18 contiguous 5′-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 18 nucleotides in length. Protein-Binding Segment of a Type II CRISPR Endonuclease (e.g., Cas9) Guide RNA ^{1896-P92WO AP} -47- [0134] The protein-binding segment of a Cas9 guide RNA typically interacts with a Cas9 protein. The Cas9 guide RNA guides the bound Cas9 protein to a specific nucleotide sequence within target nucleic acid via the above-mentioned targeting segment. The protein-binding segment of a Cas9 guide RNA typically comprises two stretches of nucleotides that are complementary to one another and hybridize to form a double stranded RNA duplex (dsRNA duplex). Thus, the protein-binding segment can include a dsRNA duplex. In some embodiments, the protein-binding segment also includes stem loop 1 (the "nexus") of a Cas9 guide RNA. For example, in some embodiments, the activator of a Cas9 guide RNA (dgRNA or sgRNA) includes (i) a duplex forming segment that contributes to the dsRNA duplex of the protein-binding segment; and (ii) nucleotides 3′ of the duplex forming segment, e.g., that form stem loop 1 (the "nexus"). For example, in some embodiments, the protein-binding segment includes stem loop 1 (the "nexus") of a Cas9 guide RNA. In some embodiments, the protein-binding segment includes 5 or more nucleotides (nt) (e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 75 or more, or 80 or more nt) 3′ of the dsRNA duplex (where 3′ is relative to the duplex-forming segment of the activator sequence). [0135] The dsRNA duplex of the guide RNA (sgRNA or dgRNA) that forms between the activator and targeted is sometimes referred to herein as the "stem loop". In addition, the activator (activator RNA, tracrRNA) of many naturally existing Cas9 guide RNAs (e.g., S. pyogenes guide RNAs) has 3 stem loops (3 hairpins) that are 3′ of the duplex-forming segment of the activator. The closest stem loop to the duplex-forming segment of the activator (3′ of the duplex forming segment) is called "stem loop 1" (and is also referred to herein as the "nexus"); the next stem loop is called "stem loop 2" (and is also referred to herein as the "hairpin 1"); and the next stem loop is called "stem loop 3" (and is also referred to herein as the "hairpin 2"). [0136] In some embodiments, a Cas9 guide RNA (sgRNA or dgRNA) (e.g., a full length Cas9 guide RNA) has stem loops 1, 2, and 3. In some embodiments, an activator (of a Cas9 guide RNA) has stem loop 1 but does not have stem loop 2 and does not have stem loop 3. In some embodiments, an activator (of a Cas9 guide RNA) has stem loop 1 and stem loop 2 but does not have stem loop 3. In some embodiments, an activator (of a Cas9 guide RNA) has stem loops 1, 2, and 3. ^{1896-P92WO AP} -48- [0137] In some embodiments, the activator (e.g., tracr sequence) of a Cas9 guide RNA (dgRNA or sgRNA) includes (i) a duplex forming segment that contributes to the dsRNA duplex of the protein-binding segment; and (ii) a stretch of nucleotides (e.g., referred to herein as a 3′ tail) 3′ of the duplex forming segment. In some embodiments, the additional nucleotides 3′ of the duplex forming segment form stem loop 1. In some embodiments, the activator (e.g., tracr sequence) of a Cas9 guide RNA (dgRNA or sgRNA) includes (i) a duplex forming segment that contributes to the dsRNA duplex of the protein- binding segment; and (ii) 5 or more nucleotides (e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 60 or more, 70 or more, or 75 or more nucleotides) 3′ of the duplex forming segment. In some embodiments, the activator (activator RNA) of a Cas9 guide RNA (dgRNA or sgRNA) includes (i) a duplex forming segment that contributes to the dsRNA duplex of the protein-binding segment; and (ii) 5 or more nucleotides (e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 60 or more, 70 or more, or 75 or more nucleotides) 3′ of the duplex forming segment. [0138] In some embodiments, the activator (e.g., tracr sequence) of a Cas9 guide RNA (dgRNA or sgRNA) includes (i) a duplex forming segment that contributes to the dsRNA duplex of the protein-binding segment; and (ii) a stretch of nucleotides (e.g., referred to herein as a 3′ tail) 3′ of the duplex forming segment. In some embodiments, the stretch of nucleotides 3′ of the duplex forming segment has a length in a range of from 5 to 200 nucleotides (nt) (e.g., from 5 to 150 nt, from 5 to 130 nt, from 5 to 120 nt, from 5 to 100 nt, from 5 to 80 nt, from 10 to 200 nt, from 10 to 150 nt, from 10 to 130 nt, from 10 to 120 nt, from 10 to 100 nt, from 10 to 80 nt, from 12 to 200 nt, from 12 to 150 nt, from 12 to 130 nt, from 12 to 120 nt, from 12 to 100 nt, from 12 to 80 nt, from 15 to 200 nt, from 15 to 150 nt, from 15 to 130 nt, from 15 to 120 nt, from 15 to 100 nt, from 15 to 80 nt, from 20 to 200 nt, from 20 to 150 nt, from 20 to 130 nt, from 20 to 120 nt, from 20 to 100 nt, from 20 to 80 nt, from 30 to 200 nt, from 30 to 150 nt, from 30 to 130 nt, from 30 to 120 nt, from 30 to 100 nt, or from 30 to 80 nt). In some embodiments, the nucleotides of the 3′ tail of an activator RNA are wild-type sequences. It will be recognized that a number of different alternative sequences can be used. ^{1896-P92WO AP} -49- [0139] Examples of various Cas9 proteins and Cas9 guide RNAs (as well as information regarding requirements related to protospacer adjacent motif (PAM) sequences present in targeted nucleic acids) can be found in the art (see, e.g., Jinek et al. (2012) Science, 337(6096): 816-821; Chylinski et al (2013) RNA Biol.10(5):726-737; Ma et al., (2013) Biomed. Res. Int.2013: 270805; Hou et al. (2013) Proc. Nat. Acad Sci. USA, 110(39): 15644-15649; Pattanayak et al. (2013) Nat. Biotechnol.31(9): 839-843; Qi et al. (2013) Cell, 152(5): 1173-1183; Wang et al (2013) Cell, 153(4): 910-918; Chen et. al. (2013) Nucl. Acids Res.41(20): e19; Cheng et. al. (2012) Cell Res.23(10): 1163-1171; Cho et. al. (2013) Genetics, 195(3): 1177-1180; DiCarlo et al. (2013) Nucl. Acids Res.41(7): 4336-4343; Dickinson et. al. (2013) Nat. Meth.10(10): 1028-1034; Ebina et. al. (2013) Sci. Rep.3: 2510; Fujii et. al. (2013) Nucl. Acids Res.41(20): e187; Hu et. al. (2013) Cell Res.23(11): 1322-1325; Jiang et. al. (2013) Nucl. Acids Res.41(20): e188; Larson et. al. (2013) Nat. Protoc.8(11): 2180-2196; Mali et. at. (2013) Nat. Meth.10(10): 957-963; Nakayama et. al. (2013) Genesis, 51(12): 835-843; Ran et. al. (2013) Nat. Protoc.8(11): 2281-2308; Ran et. al. (2013) Cell 154(6): 1380-1389; Walsh et. al. (2013) Proc. Natl. Acad. Sci. USA, 110(39): 15514-15515; Yang et. al. (2013) Cell, 154(6): 1370-1379; Briner et al. (2014) Mol. Cell, 56(2): 333-339; and U. S. Patents and Patent Applications: U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 2014/0068797; 2014/0170753; 2014/0179006; 2014/0179770; 2014/0186843; 2014/0186919; 2014/0186958; 2014/0189896; 2014/0227787; 2014/0234972; 2014/0242664; 2014/0242699; 2014/0242700; 2014/0242702; 2014/0248702; 2014/0256046; 2014/0273037; 2014/0273226; 2014/0273230; 2014/0273231; 2014/0273232; 2014/0273233; 2014/0273234; 2014/0273235; 2014/0287938; 2014/0295556; 2014/0295557; 2014/0298547; 2014/0304853; 2014/0309487; 2014/0310828; 2014/0310830; 2014/0315985; 2014/0335063; 2014/0335620; 2014/0342456; 2014/0342457; 2014/0342458; 2014/0349400; 2014/0349405; 2014/0356867; 2014/0356956; 2014/0356958; 2014/0356959; 2014/0357523; 2014/0357530; 2014/0364333; and 2014/0377868; all of which are incorporated herein by reference in their entirety. [0140] In certain embodiments alternative PAM sequences may also be utilized, where a PAM sequence can be NAG as an alternative to NGG (Hsu (2014) supra.) using an S. pyogenes Cas9. Additional PAM sequences may also include those lacking the initial ^{1896-P92WO AP} -50- G (see, e.g., Sander & Joung (2014) Nature Biotech 32(4):347). In addition to the S. pyogenes encoded Cas9 PAM sequences, other PAM sequences can be used that are specific for Cas9 proteins from other bacterial sources. Guide RNAs for Type V and Type VI CRISPR/Cas Endonucleases (e.g., Cpf1 Guide RNA) [0141] A guide RNA that binds to a type V or type VI CRISPR/Cas protein (e.g., Cpf1, C2c1, C2c2, C2c3), and targets the complex to a specific location within a target nucleic acid is referred to herein generally as a "type V or type VI CRISPR/Cas guide RNA". An example of a more specific term is a "Cpf1 guide RNA." [0142] In various embodiments a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) can have a total length of from 30 nucleotides (nt) to 200 nt, e.g., from 30 nt to 180 nt, from 30 nt to 160 nt, from 30 nt to 150 nt, from 30 nt to 125 nt, from 30 nt to 100 nt, from 30 nt to 90 nt, from 30 nt to 80 nt, from 30 nt to 70 nt, from 30 nt to 60 nt, from 30 nt to 50 nt, from 50 nt to 200 nt, from 50 nt to 180 nt, from 50 nt to 160 nt, from 50 nt to 150 nt, from 50 nt to 125 nt, from 50 nt to 100 nt, from 50 nt to 90 nt, from 50 nt to 80 nt, from 50 nt to 70 nt, from 50 nt to 60 nt, from 70 nt to 200 nt, from 70 nt to 180 nt, from 70 nt to 160 nt, from 70 nt to 150 nt, from 70 nt to 125 nt, from 70 nt to 100 nt, from 70 nt to 90 nt, or from 70 nt to 80 nt). In some embodiments, a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) has a total length of at least 30 nt (e.g., at least 40 nt, at least 50 nt, at least 60 nt, at least 70 nt, at least 80 nt, at least 90 nt, at least 100 nt, or at least 120 nt). [0143] In some embodiments, a Cpf1 guide RNA has a total length of 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt. [0144] Like a Cas9 guide RNA, a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) can include a target nucleic acid-binding segment and a duplex- forming region (e.g., in some embodiments formed from two duplex-forming segments, i.e., two stretches of nucleotides that hybridize to one another to form a duplex). [0145] In various embodiments the target nucleic acid-binding segment of a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) can have a length of from 15 nt to 30 nt, e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt. In some embodiments, the target nucleic acid-binding segment has a length of 23 nt. ^{1896-P92WO AP} -51- [0146] In some embodiments, the target nucleic acid-binding segment has a length of 24 nt. In some embodiments, the target nucleic acid-binding segment has a length of 25 nt. [0147] In certain embodiments the guide sequence of a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) can have a length of from 15 nt to 30 nt (e.g., 15 to 25 nt, 15 to 24 nt, 15 to 23 nt, 15 to 22 nt, 15 to 21 nt, 15 to 20 nt, 15 to 19 nt, 15 to 18 nt, 17 to 30 nt, 17 to 25 nt, 17 to 24 nt, 17 to 23 nt, 17 to 22 nt, 17 to 21 nt, 17 to 20 nt, 17 to 19 nt, 17 to 18t, 18 to 30 nt, 18 to 25 nt, 18 to 24 nt, 18 to 23 nt, 18 to 22 nt, 18 to 21 nt, 18 to 20 nt, 18 to 19 nt, 19 to 30 nt, 19 to 25 nt, 19 to 24 nt, 19 to 23 nt, 19 to 22 nt, 19 to 21 nt, 19 to 20 nt, 20 to 30 nt, 20 to 25 nt, 20 to 24 nt, 20 to 23 nt, 20 to 22 nt, 20 to 21 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt). In some embodiments, the guide sequence has a length of 17 nt. In some embodiments, the guide sequence has a length of 18 nt. In some embodiments, the guide sequence has a length of 19 nt. In some embodiments, the guide sequence has a length of 20 nt. In some embodiments, the guide sequence has a length of 21 nt. In some embodiments, the guide sequence has a length of 22 nt. In some embodiments, the guide sequence has a length of 23 nt. In some embodiments, the guide sequence has a length of 24 nt. [0148] In certain embodiments the guide sequence of a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) can have 100% complementarity with a corresponding length of target nucleic acid sequence. The guide sequence can have less than 100% complementarity with a corresponding length of target nucleic acid sequence. For example, the guide sequence of a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) can have 1, 2, 3, 4, or 5 nucleotides that are not complementary to the target nucleic acid sequence. For example, in some embodiments, where a guide sequence has a length of 25 nucleotides, and the target nucleic acid sequence has a length of 25 nucleotides, in some embodiments, the target nucleic acid-binding segment has 100% complementarity to the target nucleic acid sequence. As another example, in some embodiments, where a guide sequence has a length of 25 nucleotides, and the target nucleic acid sequence has a length of 25 nucleotides, in some embodiments, the target nucleic acid- binding segment has 1 non-complementary nucleotide and 24 complementary nucleotides with the target nucleic acid sequence. As another example, in some embodiments, where a guide sequence has a length of 25 nucleotides, and the target nucleic acid sequence has a ^{1896-P92WO AP} -52- length of 25 nucleotides, in some embodiments, the target nucleic acid-binding segment has 2 non-complementary nucleotides and 23 complementary nucleotides with the target nucleic acid sequence. [0149] In certain embodiments the duplex-forming segment of a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) (e.g., of a targeter RNA or an activator RNA) can have a length of from 15 nt to 25 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt). [0150] The RNA duplex of a type V or type VI CRISPR/Cas guide RNA (e.g., cpf1 guide RNA) can have a length of from 5 base pairs (bp) to 40 bp (e.g., from 5 to 35 bp, 5 to 30 bp, 5 to 25 bp, 5 to 20 bp, 5 to 15 bp, 5-12 bp, 5-10 bp, 5-8 bp, 6 to 40 bp, 6 to 35 bp, 6 to 30 bp, 6 to 25 bp, 6 to 20 bp, 6 to 15 bp, 6 to 12 bp, 6 to 10 bp, 6 to 8 bp, 7 to 40 bp, 7 to 35 bp, 7 to 30 bp, 7 to 25 bp, 7 to 20 bp, 7 to 15 bp, 7 to 12 bp, 7 to 10 bp, 8 to 40 bp, 8 to 35 bp, 8 to 30 bp, 8 to 25 bp, 8 to 20 bp, 8 to 15 bp, 8 to 12 bp, 8 to 10 bp, 9 to 40 bp, 9 to 35 bp, 9 to 30 bp, 9 to 25 bp, 9 to 20 bp, 9 to 15 bp, 9 to 12 bp, 9 to 10 bp, 10 to 40 bp, 10 to 35 bp, 10 to 30 bp, 10 to 25 bp, 10 to 20 bp, 10 to 15 bp, or 10 to 12 bp). [0151] Examples and guidance related to type V or type VI CRISPR/Cas endonucleases and guide RNAs (as well as information regarding requirements related to protospacer adjacent motif (PAM) sequences present in targeted nucleic acids) can be found in the art (see, e.g., Zetsche et al. (2015) Cell, 163(3): 759-771; Makarova et al. (2015) Nat. Rev. Microbiol.13(11): 722-736, Shmakov et al. (2015) Mol. Cell, 60(3): 385- 397, and the like). Zinc Finger Endonucleases. [0152] In certain embodiments the targeted endonuclease comprises a zinc finger nuclease (ZFN). Typically, a zinc finger nuclease comprises a DNA binding domain (e.g., zinc finger) and a cleavage domain (e.g., nuclease), both of which are described below. Zinc Finger Binding Domain. [0153] Zinc finger binding domains may be engineered to recognize and bind to any nucleic acid sequence of choice (see, e.g., Beerli et al. (2002) Nat. Biotechnol.20: 135- 141; Pabo et al. (2001) Ann. Rev. Biochem.70: 313-340; Isalan et al. (2001) Nat. Biotechnol.19: 656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12: 632-637; Choo et al. (2000) Curr. Opin. Struct. Biol.10: 411-416; Zhang et a. (2000) J. Biol. Chem.275(43): 33850-33860; Doyon et al. (2008) Nat.

al. (2008) Proc. Natl. Acad. Sci. USA, 105: 5809-5814). An engineered zinc finger binding ^{1896-P92WO AP} -53- domain can have a novel binding specificity compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence (see, e.g., U.S. Pat. Nos. 6,453,242 and 6,534,261, and the like). As an example, the algorithm described in U.S. Pat. No.6,453,242 may be used to design a zinc finger binding domain to target a preselected sequence. Alternative methods, such as rational design using a nondegenerate recognition code table can also be used to design a zinc finger binding domain to target a specific sequence (see, e.g., Sera et al. (2002) Biochemistry 41: 7074-7081; and the like). Publicly available web-based tools for identifying target sites in DNA sequences and designing zinc finger binding domains are found, inter alia, at www.zinefingertools.org and zifit.partners.org/ZiFiT/ (see also Mandell et al. (2006) Nucl. Acida Res.34: W516-W523; Sander et al. (2007) Nucl. Acida Res.35: W599-W605; and the like). [0154] A zinc finger binding domain may be designed to recognize and bind a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, for example, from about 9 to about 18 nucleotides in length. Each zinc finger recognition region (i.e., zinc finger) typically recognizes and binds three nucleotides. In certain embodiments, the zinc finger binding domains of suitable targeted zinc finger nucleases comprise at least three zinc finger recognition regions (i.e., zinc fingers). The zinc finger binding domain, however, may comprise four, or five, or six, or more zinc finger recognition regions. A zinc finger binding domain may be designed to bind to any suitable target DNA sequence (see, e.g., U.S. Pat. Nos. 6,607,882; 6,534,261, 6,453,242, and the like. [0155] Illustrative methods of selecting a zinc finger recognition region include, but are not limited to, phage display and two-hybrid systems, and are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in WO 02/077227. ^{1896-P92WO AP} -54- [0156] Zinc finger binding domains and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and are described in detail in U.S. Patent Application Publication Nos. 2005/0064474 and 2006/0188987. Zinc finger recognition regions and/or multi-fingered zinc finger proteins may be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length (see, e.g., U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949) for non-limiting examples of linker sequences of six or more amino acids in length. Cleavage Domain. [0157] A zinc finger nuclease also typically includes a cleavage domain. The cleavage domain portion of the zinc finger nuclease may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases (see, e.g., New England Biolabs catalog (www.neb.com); Belfort et al. (1997) Nucleic Acids Res.25:3379-3388; and the like). Additional enzymes that cleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). In certain embodiments one or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains. [0158] In certain embodiments, a cleavage domain also may be derived from an enzyme or portion thereof, as described above, that requires dimerization for cleavage activity. Two zinc finger nucleases may be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease can comprise both monomers to create an active enzyme dimer. As used herein, an "active enzyme dimer" is an enzyme dimer capable of cleaving a nucleic acid molecule. The two cleavage monomers may be derived from the same endonuclease (or functional fragments thereof), or each monomer may be derived from a different endonuclease (or functional fragments thereof). [0159] In various embodiments when two cleavage monomers are used to form an active enzyme dimer, the recognition sites for the two zinc finger nucleases are preferably disposed such that binding of the two zinc finger nucleases to their respective recognition sites places the cleavage monomers in a spatial orientation to each other that allows the cleavage monomers to form an active enzyme dimer, e.g., by dimerizing. As a ^{1896-P92WO AP} -55- result, the near edges of the recognition sites may be separated by about 5 to about 18 nucleotides. For instance, the near edges may be separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides. It will however be understood that any integral number of nucleotides or nucleotide pairs can intervene between two recognition sites (e.g., from about 2 to about 50 nucleotide pairs or more). The near edges of the recognition sites of the zinc finger nucleases, such as for example those described in detail herein, may be separated by 6 nucleotides. In general, the site of cleavage lies between the recognition sites. [0160] Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other (see, e.g., U.S. Pat. Nos. 5,356,802; 5,436,150, and 5,487,994; Li et al. (1992) Proc. Natl. Acad Sci. USA, 89: 4275-4279; Li et al. (1993) Proc. Nat. Acad Sci. USA, 90: 2764-2768. Thus, a zinc finger nuclease can comprise the cleavage domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Illustrative type IIS restriction enzymes are described for example in International Patent Publication No: WO 07/014,275. Additional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the present disclosure (see, e.g., Roberts et al. (2003) Nucleic Acids Res.31:418-420. [0161] An illustrative Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI. This particular enzyme is active as a dimer (Bitinaite et al. (1998) Proc. Nat. Acad. Sci. USA 95: 10, 570-10, 575). Accordingly, for the purposes of the present disclosure, the portion of the FokI enzyme used in a zinc finger nuclease is considered a cleavage monomer. Thus, for targeted double-stranded cleavage using a FokI cleavage domain, two zinc finger nucleases, each comprising a FokI cleavage monomer, may be used to reconstitute an active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage monomers can also be used. ^{1896-P92WO AP} -56- [0162] In certain embodiments the cleavage domain may comprise one or more engineered cleavage monomers that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos.2005/0064474, 2006/0188987, 2008/0131962, and the like. By way of non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are all targets for influencing dimerization of the FokI cleavage half-domains. Illustrative engineered cleavage monomers of FokI that form obligate heterodimers include a pair in which a first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of FokI and a second cleavage monomer that includes mutations at amino-acid residue positions 486 and 499 (see, e.g., Miller et al (2007) Nat. Biotechnol.25: 778-785; Szczpek et al. (2007) Nat. Biotechnol.25: 786-793). For example, the Glu (E) at position 490 may be changed to Lys (K) and the lie (I) at position 538 may be changed to K in one domain (E490K, 1538K), and the Gin (Q) at position 486 may be changed to E and the I at position 499 may be changed to Leu (L) in another cleavage domain (Q486E, 1499L). In other aspects, modified FokI cleavage domains can include three amino acid changes (see, e.g., Doyon et al. (2011) Nat Methods, 8: 74-81). For example, one modified FokI domain (which is termed ELD) can comprise Q486E, 1499L, N496D mutations and the other modified FokI domain (which is termed KKR) can comprise E490K, 1538K, H537R mutations. [0163] In certain embodiments the Zinc finger protein can be modified to have an activator, a repressor, and/or an epigenetically modifying domain (e.g., in a manner similar to modified CRISPR constructs). TALENs [0164] In certain embodiments the targeted endonuclease comprises a Transcription Activator-Like Effector Nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases derived from Xanthomonas bacteria, that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism. The DNA binding domain of the TAL effector contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence. ^{1896-P92WO AP} -57- [0165] TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, FokI The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences (see, e.g., WO 2010/079430; Morbitzer et al. (2010) Proc. Nat. Acad. Sci. USA, 107(50): 21617-21622; Scholze & Boch (2010) Virulence, 1: 428-432; Christian et al (2010) Genetics, 186:757-761; Li et al. (2010) Nucl. Acids Res. (1):359-372; and Miller et al. (2011) Nat. Biotech.29: 143-148). [0166] To produce a TALEN, a TAL protein is fused to a nuclease, which is typically a wild-type or mutated FokI endonuclease. Several mutations to FokI have been made for its use in TALENs. These, for example, improve cleavage specificity or activity (see, e.g., Cermak eta. (2011) Nucl. Acids Res.39: e82; Miller et al. (2011) Nat. Biotech.29: 143-148; Hockemeyer et al. (2011) Nat. Biotech.29: 731-734; Wood et al. (2011) Science, 333: 307; Doyon et al. (2010) Nat. Meth.8: 74-79; Szczepek et al. (2007) Nat. Biotech.25: 786-793; and Guo et al. (2010) J. Mol. Biol.200: 96). [0167] The FokI domain functions as a dimer, typically requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity (see, e.g., Miller et al. (2011) Nat. Biotech., 29: 143-148). [0168] Examples of suitable TAL nucleases, and methods for preparing suitable TAL nucleases, are disclosed, e.g., in US Patent Application Nos. 2011/0239315 A1, 2011/0269234 A1, 2011/0145940 A1, 200310232410 A1, 2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1, 2006/0188987 A1, and 2006/0063231 A1. In various embodiments, TAL effector nucleases are engineered that out in or near a target nucleic acid sequence in, e.g., a genomic locus of interest, where the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector. In various embodiments, the TAL nucleases suitable for use with the various methods and compositions provided herein include those that are specifically designed to bind at or near target nucleic acid sequences to be modified, e.g., by targeting vectors. ^{1896-P92WO AP} -58- [0169] In one illustrative, but non-limiting embodiment, each monomer of the TALEN comprises 10 or more DNA binding repeats, and in some embodiments 15 or more DNA binding repeats (e.g., in certain embodiments, 12-25 TAL repeats), wherein each TAL repeat binds a 1 bp subsite. In one embodiment, the nuclease agent is a chimeric protein comprising a TAL repeat-based DNA binding domain operably linked to an independent nuclease. In one embodiment, the independent nuclease is a Fok1 endonuclease (see e.g., Kim et al. (1996) Proc. Natl. Acad. Sci. USA, 93:1156-1160), however, other useful endonucleases may include, but are not limited to, for example, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. [0170] In some embodiments, the TAL effector domain that binds to a specific nucleotide sequence within the target DNA comprises a plurality of repeat variable- diresidues (RVD) each of which determines recognition of a base pair in the target DNA sequence, where each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence, and wherein the RVD comprises one or more of HD for recognizing C; NG for recognizing T; NI for recognizing A; NN for recognizing G or A; NS for recognizing A or C or G or T; N* for recognizing C or T, where * represents a gap in the second position of the RVD; HG for recognizing T; H* for recognizing T, where * represents a gap in the second position of the RVD; IG for recognizing T; NK for recognizing G; HA for recognizing C; ND for recognizing C; H1 for recognizing C; HN for recognizing G; NA for recognizing G; SN for recognizing G or A; and YG for recognizing T. [0171] If the genome editing endonuclease to be utilized is a TALEN, in some embodiments, optimal target sites may be selected in accordance with the methods described by Sanjana et al. (2012) Nat. Protocol., 7: 171-192, which is hereby incorporated by reference in its entirety. In brief, in various embodiments, TALENs function as dimers, and a pair of TALENs, referred to as the left and right TALENs, target sequences on opposite strands of DNA. TALENs can be engineered as a fusion of the TALE DNA- binding domain and a monomeric FokI catalytic domain. In certain embodiments to facilitate FokI dimerization, the left and right TALEN target sites can be chosen with a spacing of approximately 14-20 bases. Therefore, for a pair of TALENs, each targeting 20-bp sequences, an optimal target site can have the form 5′-TN¹⁹N^14-20N¹⁹A-3′, where the left TALEN targets 5′-TN¹⁹-3′ and the right TALEN targets the antisense strand of 5′- N¹⁹A-3′ (N=A, G, T or C). This is of course illustrative and non-limiting and examples of ^{1896-P92WO AP} -59- TALENs that bind to particular target sites are well known to those of skill in the art. For more information on TALENs, refer to U.S. Pat. No. 8,685,737, which is hereby incorporated by reference in its entirety. [0172] In certain embodiments the TALENs can be modified to have an activator, a repressor, and/or an epigenetically modifying domain (e.g., in a manner similar to modified CRISPR constructs). [0173] In various embodiments the methods described herein involve transfecting or infecting a cell (or cell population) with a modified DNA virus containing a gene drive construct (e.g., as described herein), and infecting cells with the target virus where the genome of the target DNA virus is modified by insertion of the gene drive construct into the genome of the target DNA virus and a population of modified target viruses (containing the gene drive construct) is produced. [0174] In certain embodiments the modified DNA virus (containing gene drive construct) and the target virus (e.g., wild-type virus) are introduced into a cell ex vivo. In certain embodiments the cell is maintained in a cell culture. However, in certain embodiments, the modified DNA virus (containing gene drive construct) and the target virus (e.g., wild-type virus) are introduced into a cell in vivo. In certain embodiments the cell can be a cell, e.g., in a mammal, that is already infected with the target virus (virus to be modified). Thus, for example, where the mammal is infected with a wild-type virus, the gene drive virus can be introduced into the subject, e.g., by infection where the gene drive virus retains infectivity (or has infectivity temporarily restored by, e.g., an inducible or transitional rescue gene). In certain embodiments the gene drive virus can be introduced into cells of the subject by other means (e.g., encapsulated in delivery vehicles, complexed with dendrimers or polymeric delivery particles, and the like). EXAMPLE 1 Cells and viruses [0175] African green monkey epithelial Vero cells and murine neuroblastoma N2a cells were obtained from the ATCC and cultured in DMEM (Corning, Corning, NY, USA) supplemented with 10% FBS (Sigma-Aldrich, St-Louis, MO, USA). Cells were maintained at 37 °C in a 5% CO2 humidified incubator and frequently tested negative for mycoplasma contamination. [0176] Unmodified HSV-1 strain 17+ and HSV1-CFP expressing cyan fluorescent protein mTurquoise2 from the US1/2 locus were provided by Matthew Taylor ^{1896-P92WO AP} -60- (Montana State University, USA). Viruses generated for this study were made by modifying HSV-1 and HSV1-CFP, as described below. To prepare viral stocks for cell culture experiments, Vero cells in 15 cm dishes were infected for one hour at MOl=0.01 and kept in culture for 48hours or until 100% cytopathic effect was observed. Cells and supernatant were scraped out of the plate, sonicated three times at maximum power with a probe sonicator, and debris pelleted away by centrifugation (2000 rpm, 10 minutes, 4 °C). Media containing viruses was collected in single-use aliquots and titers measured by plaque assay. [0177] For high-titer and high-purity viral stocks used for animal experiments, Vero cells in 15 cm dishes were infected for one hour at Mol = 0.01 and kept in culture for 48 hours or until 100% cytopathic effect was observed. Supernatants and cells were collected, and cells were pelleted by centrifugation (2000 rpm, 5 minutes, 4 °C). Supernatants were collected in clean tubes and reserved for later. Cell pellets were resuspended in a small volume of culture media and cell-bound virions were released by two cycles of freeze-thaw in dry ice. Debris were pelleted again and the supernatant containing released virions was combined with the supernatant reserved earlier. Virions were then pelleted by ultracentrifugation (22,000 rpm, 90 min, 4 °C, Beckman-Coulter rotor SW28) on a 5-mL cushion of 30% sucrose. Supernatants were discarded, and virions were resuspended in PBS containing 2% BSA. Single-use aliquots were prepared, and titers were measured by plaque assay. [0178] Co-infection experiments were performed in 12-well plates by co- infecting N2a cells with HSV1-WT and gene drive viruses for 1h, with a total MOl of 1, before replacing inoculum with 1mL of fresh medium. 100uL of supernatant was collected at regular intervals and analyzed by plaque assay. EXAMPLE 2 Cloning and generation of recombinant viruses. [0179] A donor plasmid containing the gene drive cassette against the HSV-1 UL37-38 intergenic region (GD and derivatives) was generated by serial modifications of the GD-mCherry donor plasmid. All modifications were carried out by Gibson assembly (NEB, Ipswich, MA, USA), using PCR products from other plasmids or synthesized DNA fragments (GeneArtTM StringTM fragments, ThermoFisher, USA). The final GD donor plasmid included homology arms for the UL37-38 region, the CBH promoter driving SpCas9 followed by the SV40 polyA terminator, the CMV promoter driving an mCherry ^{1896-P92WO AP} -61- reporter followed by the beta-globin polyA signal, and the U6 promoter controlling gRNA expression. The functional GD plasmid carried a gRNA targeting the UL37-38 region (ACGGGATGCCGGGACTTAAG), while the non-specific GD-ns control targeted a sequence absent in HSV-1 (ACATCGCGGTCGCGCGTCGG). GD-L1Cas9 donor construct was subsequently generated by removing SpCas9 by digestion and ligation. Donor constructs to insert CMV-driven yellow (YFP) or red (RFP) fluorescent protein reporters into the US1/US2 locus were built similarly, by replacing mTurquoise with mCitrine2 or mScarlet2 in a donor plasmid for the US1/US2 region, respectively (pGL002). Of note, the YFP, CFP, and RFP reporters carried a nuclear localization signal. [0180] To build recombinant viruses, 1.5 million Vero cells were co-transfected with linearized donor plasmids and purified HSV-1 strain 17+ or HSV1-CFP viral DNA. Viral DNA was purified from infected cells by HIRT DNA extraction. Transfection was performed by Nucleofection (Lonza, Basel, Switzerland) and cells were plated in a single 6-well. After 2-4 days, mCherry-expressing viral plaques were isolated and purified by several rounds of serial dilutions and plaque purification. Purity and absence of unmodified viruses were assayed by PCR and Sanger sequencing after DNA extraction (DNeasy kit, Qiagen, Germantown, MD, USA). Viral stocks were produced as specified above and tittered by plaque assay. EXAMPLE 3 [0181] Plaque assays were performed either directly from cell culture supernatants, or from frozen mouse tissues. To release infectious virions from tissues, frozen samples were resuspended in cell culture media and disrupted using a gentle tissue homogenizer (Pellet Pestle, Fisher Scientific, USA). Samples were sonicated three times at maximum power with a probe sonicator, and debris pelleted away by centrifugation (2000 rpm, 10 minutes, 4 °C). Volumes were adjusted to a final volume of 1 ml, and titers were measured by plaque assay. [0182] Viral titers and recombination levels were determined by plaque assay with 10-fold serial dilutions. Confluent Vero cells in 24-well plates were incubated for 1 h with 100uL of inoculum and overlaid with 1mL of complete media containing 1% methylcellulose, prepared using DMEM powder (Thermo) and Methylcellulose (sigma). After two or three days, fluorescent plaques expressing YFP, CFP and/or mCherry were manually counted using a Nikon Eclipse Ti2 inverted microscope. Every viral plaque was ^{1896-P92WO AP} -62- analyzed on both YFP, CFP and red channel. 5–100 plaques were counted per well, and each data point was the average of 3–4 technical replicates (i.e., 3–4 different wells). Images of fluorescent viral plaques were acquired with an EVOS automated microscope and adjusted for contrast and exposure with ImageJ (v2.1.0). [0183] The deconvolution of Sanger sequencing in FIG.3F was performed using Synthego ICE online tools (https://ice.synthego.com). EXAMPLE 4 Mouse experiments Acute infection after intravitreal inoculation [0184] Male and female Balb/c between five and eight weeks-old were infected by intravitreal injection in the left eye. Briefly, mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and laid prone under a stereo microscope. The left eye was treated with a thin layer of veterinary ophthalmic ointment and the sclera was exposed using ophthalmic forceps. 2 μl of HSV-1 stock containing 10⁶ pfu was injected slowly in the intravitreal space, using a 5^L Hamilton syringe and a 30- gauge needle. In the days following infection, mice were treated with sustained-release buprenorphine to minimize pain (Ethiqa XR, Fidelis Animal Health, North Brunswick, NJ, USA). Animals were humanely euthanized after two to four days. For plaque assay analysis, tissues were collected and snap-frozen in liquid nitrogen. Latent infection after corneal scarification [0185] Latent infections were performed using female Swiss-Webster or C57bl/6 mice five to six weeks-old purchased from Taconic (Germantown, NY, USA). Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and laid under a stereo microscope. Mice corneas were lightly scarified using a 28-gauge needle, and 4uL of viral inoculum dispensed on both eyes. Swiss-Webster and C57bl/6 mice were infected with 10⁵ and 10⁶ PFU, respectively. Following inoculation, ophthalmic drops of local analgesic (Diclofenac) were deposited on both eyes, and the analgesic Meloxicam was added to the drinking water ad libitum for 1-5 days following infection. From five to fifteen days following primary infection, symptoms of infection were reported and scored using an in-house scoring system. Mice experiencing severe symptoms were humanely euthanized. Once the infection had fully resolved, final eye scarification levels were scored in both eyes and averaged, using the following scores: 0: perfect eye; 1: lightly ^{1896-P92WO AP} -63- damaged and/or cloudy cornea, 2: scar tissue covering a small portion of the eye; 3: scar tissue covering most of the eye; 4: extremely bad looking eye, fully blind. [0186] The second infection with GD and GD-ns was performed the same way four weeks after the primary infection, with 10⁷ PFU per eye. Mice were transiently immunosuppressed with dexamethasone (FIG. 7 in Swiss-Webster) or with dexamethasone and tacrolimus (FIG.8 in C57bl/6). Tacrolimus and Dexamethasone were diluted in the drinking water and administrated ad libitum from one day before to seven days after infection. Drug concentration was calculated according to the average mice weight, considering that mice drink around 5mL per day, in order to reach a dose of 1 mg/kg/day and 2 mg/kg/day for dexamethasone and tacrolimus, respectively. For example, for an average mouse weight of 25g, dexamethasone and tacrolimus were diluted at 5mg/L and 10mg/L, respectively. Of note, drugs were diluted in medidrop sucralose (ClearH20, Westbrook, ME, USA) instead of regular drinking water, to make tacrolimus more palatable to mice. HSV reactivation [0187] HSV reactivation was performed by intraperitoneal injection of JQ1 (MedChemExpress, NJ, USA) at a dose of 50 mg/kg, and, when indicated, Buparlisib (MedChemExpress, NJ, USA) at a dose of 20 mg/kg. JQ1 and Buparlisib were prepared from stock solutions (10x at 50mg/mL and 100x at 200mg/mL, in DMSO, respectively) by dilution in a vehicle solution of 10% w/v 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich, St-Louis, MO, USA) in PBS. In the C57bl/6 experiments (FIG.8), mice were transiently immunosuppressed with dexamethasone and tacrolimus in the drinking water, at the concentrations indicated above, from one day before to three days after JQ1/Buparlisib injection. Mouse eyes were gently swabbed with cotton swabs moistened with PBS, on day one to three following injection. Swabs were collected into vials containing 1 ml of digestion buffer (KCL, Tris HCl pH8.0, EDTA, Igepal CA-630) and stored at 4°C before DNA extraction. EXAMPLE 5 HSV quantification of viral loads in swabs and tissues [0188] DNA was extracted from 200 μl of swab digestion buffer using QiaAmp 96 DNA Blood Kits (Qiagen, Germantown, MD, USA) and eluted into 100 μl AE buffer (Qiagen, Germantown, MD, USA). 10 μl of eluted DNA was used to setup 30 μl real-time Taqman quantitative PCR reactions, using QuantiTect multiplex PCR mix (Qiagen, ^{1896-P92WO AP} -64- Germantown, MD, USA), using the following PCR cycling conditions: 1 cycle at 50°C for 2 minutes, 1 cycle at 95°C for 15 minutes, and 45 cycles of 94°C for 1 minute and 60°C for 1 minute. Exo internal control was spiked into each PCR reaction to monitor inhibition. A negative result was accepted only if the internal control was positive with a cycle threshold (CT) within 3 cycles of the Exo CT of no template controls. Primers and probes have been described previously and are provided in Table 3. Swabs positive for HSV were then further analyzed by ddPCR, as described below. TABLE 3. qPCR/ddPCR primers Forward Reverse probe HSV B CGGC TTG GGCC TGTGC GG GACTC

1^{896-P92WO AP} -65- [0189] Total genomic DNA was isolated from ganglionic tissues using the DNeasy Blood and tissues kit (Qiagen, Germantown, MD, USA) and eluted in 60 μl of EB buffer, per the manufacturer's protocol. [0190] Quantification of the YFP, CFP and mCherry markers, as well as total HSV viral load was measured with two separate duplex ddPCR, using 10uL of eluted DNA. ddPCR was performed using the QX200 Droplet Digital PCR System and ddPCR Supermix for Probes (No dUTP) from Biorad (Hercules, CA, USA), following the manufacturer's instructions. Primers were used at a final concentration of 900nM and probes at 250nM (Table S1). Primers and probes were ordered from IDT (Coralville, IA, USA), using their custom PrimeTime ZEN double-quenched qPCR probes, with FAM and HEX fluorescent dyes. The first duplex assay used two sets of primers/probes to quantify mCherry (HEX probe) and HSV UL38 gene (FAM probe). UL38 primers/probe set was located in the UL38 viral gene and recognized both wild-type and gene drive genomes. The second duplex assay distinguished between YFP and CFP, using one set of common primers amplifying both markers and YFP and CFP-specific probes with FAM and HEX dyes, respectively. Primer specificity and sensitivity were validated on plasmid DNA before use in mouse samples. A limit of detection of three copies per reaction was used throughout the study, except in FIGS.7D-7F, where a cutoff of 10 was applied to mCherry to account for a small PCR contamination. Final titers were normalized and expressed in log-transformed copies per million cells (MCells). Cell numbers in tissue samples were quantified by ddPCR using a mouse-specific RPP30 primer/probe set. [0191] The duplex assays allowed us to determine the proportion of latent mCherry and CFP, using absolute values measured in the same PCR reaction, thus, limiting technical variation. Proportions were calculated as follows: %^^^^^^^ ^ 100 ∗ ^^^^^^^^/^^38^^ %^^^ ^ ^100 ∗ ^^^/^^^^ ^ ^^^^ Swab genotyping [0192] Positive swabs identified by qPCR were analyzed using the duplex ddPCR assays described above. Samples expressing YFP only with no detectable CFP and mCherry were categorized as wild-type. Swabs expressing mCherry at the same level as UL38 were categorized as gene drive. Swabs expressing both CFP and mCherry represented the original GD/GD-ns, while swabs expressing both YFP and mCherry represented recombinants (FIG. 18G and FIGS. 20A-20C). Some swabs expressed ^{1896-P92WO AP} -66- mCherry one to two orders of magnitude lower than HSV and were genotyped as wild-type but with detectable amounts of mCherry. For low-titer swabs, the genotype was further confirmed by duplex qPCR using the same primers. EXAMPLE 6 Brain and TG imaging and image analysis Tissue processing and image collection [0193] Balb/c mice were infected ocularly with equivalent amounts of three viruses expressing either YFP, CFP or RFP from the US1-US2 locus. A total of 10⁶ PFU was inoculated intravitreally in the left eye. Of note, the fluorescent proteins carry nuclear localization signals. They are expressed in infected cells and are not incorporated into virions. Four days after infection, mice were injected intraperitoneally with a terminal dose of euthanasia solution containing Sodium Pentobarbitol (Euthasol). Once unresponsive, mice were subjected to thoracotomy and transcardially perfused with PBS followed by 4% Paraformaldehyde-Lysine-Periodate solution (PLP) through the aorta to fix tissues (43). Brains, TG, and eyes were dissected, fixed in PLP overnight, and transferred to a 20% sucrose solution for 24 hours, and finally to a 30% sucrose solution for at least 24 hours for cryo-protection. All tissues were stored in 30% sucrose before processing. [0194] TG were embedded in OCT and serial sections of 15 µm made using a Cryostat (Zeiss) at -20oC. TG sections were mounted on subbing solution-treated slides and polymerizing mounting media containing DAPI (Vectashield, Vector Labs, Burlingame, CA, USA) was added before coverslipping. Brains were immobilized in 30% sucrose on a freezing microtome stage set to -18˚ C (Physitemp, Clifton, NJ). Serial coronal sections at 30 µm on a horizontal sliding microtome (AO Optical) were collected. Brain sections were binned into six parallel groups. One bin was arranged and mounted on slides before counterstaining with polymerizing mounting media containing DAPI and coverslipping. Epifluorescence imaging was performed on a Nikon Ti-Eclipse (Nikon Instruments, Melville, NY, USA) inverted microscope equipped with a SpectraX LED (Lumencor, Beaverton, OR, USA) excitation module and fast-switching emission filter wheels (Prior Scientific, Rockland, MA, USA). Fluorescence imaging used paired excitation/emission filters and dichroic mirrors for DAPI, CFP, YFP and TRITC (Chroma Technology Corp., Bellow Falls, VT, USA). All images were acquired with an iXon 896 EM-CCD (Andor Technology LTD, Belfast, NI, USA) camera using NIS-Elements software. Image tiles with 4x and 10x Phase objectives were acquired to assess fluorescent ^{1896-P92WO AP} -67- protein expression across sectioned tissues. Specific regions were imaged with the 20x ELWD to acquire detailed localization and fluorescent protein expression images for subsequent data analysis. Image analysis [0195] From the five animals originally infected, one animal was not included as very few infected cells could be detected in the brain and TG, suggesting that the infection had failed. Furthermore, one brain from the remaining four mice was irremediably damaged during processing. Thus, the analysis was conducted on four TG and three brains. [0196] Using ImageJ (v2.14.0/1.54f), Nd2 images acquired with Nikon NIS- Element software were batch converted into tiff files using an ImageJ macro (modified from https://github.com/singingstars/). During the conversion process, brightfield and DAPI channels were discarded, and ImageJ background subtraction was performed on the YFP, CFP and RFP channels. An additional grayscale channel was created, composed of the maximum projection of the YFP, CFP and RFP channels. This composite channel contained every cell irrespective of the original color and was used for segmentation (FIG. 11A). Machine learning-assisted segmentation was performed using an online analysis tool from www.biodock.ai (Biodock, AI Software Platform. Biodock 2023). The software was trained to recognize cells on the gray channel using a few training images, and segmentation was then run on the entire dataset. Around 3-4% of cells with aberrant area or eccentricity were discarded, and average signal intensity was measured in the original YFP, CFP and RFP channels for each detected cell. Of note, for the TG, this analysis was performed using only the YFP and CFP channels. Data was then further processed and plotted using R (RStudio v2023.09.1+494). For YFP and CFP, the intensity was simply log10 converted. Because RFP had a higher background and different intensity ranges across images, RFP intensity was first scaled across images and then log10 converted. Stringent intensity thresholds were applied on the three channels and used to quantify cells infected with one, two, or three viruses, with around 5% of cells below thresholds being discarded (FIG. 13B). Thresholds were chosen stringently to unequivocally identify co- infected cells and are reported in FIG.11C and FIG.13C. For visualization and plotting, signal intensities in the YFP, CFP and RFP channels were converted into CYMK color space. [0197] Representative images shown herein were minimally adjusted for contrast and exposure using ImageJ. Some images were rotated or flipped horizontally to ^{1896-P92WO AP} -68- consistently present the brain in a caudal direction, with the left hemisphere on the right side. EXAMPLE 7 Statistics and reproducibility [0198] Experiments were carried out in multiple replicates. Investigators were blinded when performing plaque assays, collecting swabs, and analyzing DNA samples. No data was excluded, except when indicated in the main text, methods or figure legends. Statistical analyses were performed using GraphPad Prism version 10.1.1 for macOS (GraphPad Software, USA, www.graphpad.com). Statistical tests and their results are described in the text and figure legends. EXAMPLE 8 Design of a gene drive against HSV-1 [0199] The inventors aimed to build a gene drive that would not affect viral infectivity and could spread efficiently into the wild-type population. A gene drive targeting an intergenic sequence between the UL37 and UL38 genes, a region known to tolerate the insertion of transgenes with little or no impact on viral replication in vitro and in vivo, was designed. A donor plasmid containing homology arms, Cas9 (from Streptococcus pyogenes) under the CBH promoter, an mCherry fluorescent reporter under the CMV promoter, and a U6-driven gRNA targeting the intergenic UL37-UL38 region (FIG.1B) was created. Importantly, to prevent self-cleaving, introduction of the gene drive cassette, removed the gRNA target sequence from the construct. To create the gene drive virus (GD), Vero cells were co-transfected with purified HSV-1 viral DNA and the gene drive donor plasmid, and mCherry-expressing viruses created by homologous recombination were isolated by three rounds of plaque purification until a pure population was obtained. To follow or track the recombination events between viral genomes, the gene drive virus also carried a cyan, fluorescent reporter (CFP) inserted into another neutral region between the US1 and US2 viral genes (FIG. 1C). Similarly, two control viruses with non-functional CRISPR systems, one with a non-specific gRNA (GD-ns) that did not target HSV-1, and one without Cas9 (GD-ΔCas9) were also built. Finally, to be used as a wild-type virus with an unmodified UL37-UL38 region, a virus expressing a yellow, fluorescent reporter (YFP) from the same US1-US2 region, hereafter referred to as HSV1- WT or simply WT (FIG. 1C) was generated. All the viruses described here originated from the highly neurovirulent HSV-1 strain 17+. ^{1896-P92WO AP} -69- [0200] Recombination between gene drive and wild-type genomes can result in four different genome configurations expressing the different fluorescent reporters, which can be followed by plaque assay (FIG.1D and FIG. 1E). In highly susceptible cell lines such as Vero cells, viral plaques originate from single virions, which allows reconstituting the recombination history of individual viral genomes. Plaques expressing YFP only, or CFP and mCherry together, represent the original WT and gene drive viruses, respectively, while plaques expressing CFP only, or YFP and mCherry together, represent recombination products that exchanged the gene drive cassette. EXAMPLE 9 Gene drive spread in cell culture [0201] The gene drive was first tested to assess if it could spread in vitro and co- infection experiments were conducted in cell culture. Some cell lines, in particular Vero cells or other epithelial cells, efficiently restrict co-infection by a mechanism known as superinfection exclusion. However, it was observed that neuronal N2a cells could sustain high levels of co-infection (FIGS. 9A-9E). Thus, co-infection experiments were conducted in N2a cells, while plaque assays were performed in Vero cells. Importantly, the WT, GD, GD-ns and GD-ΔCas9 viruses individually replicated with similar dynamics in N2a cells, showing that insertion of the gene drive cassette in the UL37-UL38 region did not affect infectivity in vitro, as expected (FIG.2A). [0202] To test if the gene drive could efficiently spread in the viral population, N2a cells were co-infected with WT+GD, WT+GD-ns, or WT+GD-ΔCas9. Cells were infected at a combined MOI of 1 with an initial proportion of gene drive virus of 20% (FIG. 2B, FIG. 2D and FIG. 2E). Titers and proportion of progeny viruses expressing the different fluorescent markers were measured by plaque assay, from day one to day three post-infection. The proportion of viruses expressing mCherry, which represents gene drive viruses, increased from 20% to 85% when cells were co-infected with WT+GD (FIG.2D). Importantly, YFP-only viruses disappeared and were replaced by recombinant viruses expressing both YFP and mCherry, representing 45% of the final population (FIG. 2E). This indicated that the WT population had been converted to new recombinant gene drive viruses, as anticipated. The proportion of viruses expressing both CFP and mCherry, which represent the original gene drive virus, increased slightly from 20% to 40% (FIG.2E). By contrast, in the control experiments with GD-ns or GD-ΔCas9, the proportion of mCherry- expressing viruses did not change and remained close to its initial value of 20% (FIG.2D). ^{1896-P92WO AP} -70- In these control experiments, around 10% of viruses expressed both YFP and mCherry at day 3, but this population of recombinant viruses was mirrored by viruses that had lost mCherry and expressed CFP only (FIG. 2E). These represented viruses that had exchanged their YFP and CFP regions in a nonspecific manner. Importantly, CFP-only viruses were not observed after co-infection with WT+GD, highlighting that the efficient incorporation of the gene drive cassette into unmodified genomes is a unilateral and targeted process requiring both Cas9 and a specific gRNA. [0203] To confirm these observations, the co-infection experiments were repeated with an initial proportion of gene drive virus of 40% (FIG. 2C, FIG. 2F, and FIG.2G). With this higher starting point, the gene drive achieved almost complete penetrance and the proportion of mCherry-expressing viruses reached 95% after 3 days, with the population of wild-type viruses expressing YFP-only being converted to recombinant gene drive viruses expressing both YFP and mCherry. As observed above, in the WT+GD-ns or WT+GD-ΔCas9 control experiments, the proportion of mCherry- expressing viruses remained constant at around 40%, and approximately 15% of YFP+mCherry and CFP-only viruses symmetrically appeared by CRISPR-independent recombination. This further showed that a gene drive could spread efficiently in the wild- type population and that the spread of the gene drive was mediated by a functional CRISPR system. [0204] Together, these results indicated that a gene drive could be designed against HSV-1 and spread efficiently in vitro. Importantly, both during single and co- infections, viral growth of WT and GD viruses followed similar dynamics and the proportion of viruses expressing CFP remained constant. Thus, the rapid increase of recombinant viruses expressing both mCherry and YFP could not be explained by a higher fitness of the GD virus but resulted from efficient CRISPR-directed homologous recombination. These results show that a viral gene drive could be developed in a second, unrelated, herpesvirus. EXAMPLE 10 Gene drive spread during herpes encephalitis [0205] Next, the ability of the gene drive to spread during acute HSV-1 infection in mice was tested. Previous studies showed that HSV-1 naturally sustains high levels of recombination in the mouse brain during herpes simplex encephalitis. Thus, it was hypothesized that a gene drive could spread efficiently in this context. A well-established ^{1896-P92WO AP} -71- infection model, where HSV-1 is inoculated intravitreally in the eye and infects the retina and other ocular tissues before propagating to the nervous system via cranial nerves was utilized to test this hypothesis (FIG. 3A). Specifically, HSV-1 travels to the brain via the optic, oculomotor and trigeminal nerves (cranial nerve CN II, III, and V, respectively), either directly or indirectly by first infecting ganglionic neurons of the peripheral nervous system. HSV-1 infects the trigeminal ganglia (TG) before reaching the brain stem. In the following experiments, a total of 10⁶ plaque-forming units (PFU) were inoculated intravitreally in the left eye, and tissues were collected, dissociated, and analyzed by plaque assay after four days. Five to eight-week-old male and female Balb/c mice were used and no differences were observed between sexes. Importantly, individual infections with WT, GD or GD-ns reached similar titers in the eye, the TG and the brain, showing that the gene drive cassette in the UL37-UL38 region did not impact infectivity in vivo (FIG.3B). [0206] To test if the gene drive could efficiently spread in vivo, mice were inoculated intravitreally with WT only, WT+GD, or WT+GD-ns, with an initial proportion of gene drive virus of 15% (FIGS.3C-3E). Total viral titers in the eye, TG and brain after four days were indistinguishable between the different conditions, showing that the gene drive did not perturb the overall dynamics of infection (FIG. 3C). The population of gene drive viruses expressing mCherry increased from 15% to 30% in the eye, to 60% in the TG, and 70% in the brain, respectively. A high variation between replicates, with the percentage of gene drive viruses in the brain ranging from 50% to 90%, with a median of 77% was observed (FIG. 3D). Furthermore, and as observed in vitro, wild-type viruses expressing YFP-only were converted to recombinant gene drive viruses expressing YFP and mCherry, representing 40% of the final population. The proportion of original gene drive viruses expressing CFP and mCherry increased slightly, from 15% to 20% in the TG, and 30% in the brain, respectively (FIG. 3E). Critically, in the control experiment with GD-ns, the proportion of gene drive viruses did not change and remained close to its initial value around 15% in all tissues, with a similar proportion of YFP+mCherry and CFP-only viruses appearing by CRISPR-independent recombination (FIG. 3E). Altogether, these result show that the gene drive of the present disclosure can efficiently spread in the viral population as the infection progressed to the brain, with recombinant gene drive viruses increasing from 15% to 70% in four days. [0207] Gene drive propagation relies on efficient homologous recombination after CRISPR cleavage, but DNA double-strand breaks can also be repaired by non- ^{1896-P92WO AP} -72- homologous end joining (NHEJ), resulting in small insertions and deletions that render viruses resistant to the drive. After PCR and Sanger sequencing of infectious viruses isolated from the brain, 20% of the remaining target sites were observed to be mutated by NHEJ (FIG. 3F). Since the gene drive represented 70% of viruses at this point, this result suggests that gene drive-resistant viruses represented around 6% of the total viral population. This indicated that the drive did not achieve full penetrance after four days and indicate that mutagenic repair by NHEJ is infrequent compared to homologous recombination during gene drive spread. [0208] Next, the gene drive's ability to spread in a second infection model, where HSV-1 is inoculated in the hind leg footpad, was tested. In this model, HSV-1 travels to the spinal cord via the sciatic nerve, and finally to the brain. 10⁶ PFU of WT, WT+GD or WT+GD-ns were inoculated in the right hind footpad, with an initial proportion of gene drive virus of 15%. Tissues were collected five to seven days later and analyzed by plaque assay (FIGS.3G-3I). Around half the mice did not show any symptoms of infection and had no detectable virus in the spinal cord and the brain, while others developed severe neurological symptoms with very high titers in both tissues (FIG. 3G). In animals with detectable virus, the average proportion of gene drive viruses reached 60% in the brain, with a range between 30% and 80%, and 50% in the spinal cord (FIG. 3H). Once again, wild-type viruses expressing YFP-only had been converted to recombinant gene drive viruses expressing both YFP and mCherry, while the population of viruses expressing CFP and mCherry remained constant (FIG. 3I). Only one control animal infected with WT+GD-ns developed a detectable infection, with mCherry-expressing viruses reaching 35% in the spinal cord. These results confirmed that a gene drive could spread after inoculation via a second route of HSV-1 infection. [0209] Altogether, these findings showed that a gene drive can propagate efficiently in vivo during acute HSV-1 infection in mice, and that the gene drive spread was mediated by a functional CRISPR system. EXAMPLE 11 High heterogeneity during gene drive spread in the brain [0210] HSV-1 travels to the brain via different neuronal pathways, and we hypothesized that following gene drive propagation in different regions of the brain could bring novel insight into the dynamics of HSV-1 infection and recombination. After intravitreal inoculation, HSV-1 infects retinal neurons and travels via the optic nerve (CN ^{1896-P92WO AP} -73- II) to the hypothalamus and the thalamus –both part of the interbrain– before reaching the cortex through visual pathways. Secondary branches of the optic nerve also connect to the midbrain –the rostral part of the brain stem. After infecting other ocular tissues and specifically the ciliary ganglion, HSV-1 separately reaches the midbrain via the oculomotor nerve (CN III). Finally, HSV-1 travels via the trigeminal nerve (CN V) to the TG and then to the brain stem (FIG.4A). [0211] To investigate tissue-specific differences in gene drive propagation, Balb/c mice were inoculated intravitreally with 10⁶ PFU of WT+GD, with an initial proportion of gene drive virus of 15%. Viral titers and gene drive-directed recombination were measured by plaque assay in the eye, TG, brain stem, interbrain, cortex and cerebellum at days two to four post-infection (FIGS. 4A-4E). Viral titers increased progressively throughout the brain, first reaching the interbrain and brain stem after 2 days and then spreading to the cortex and cerebellum (FIG.4B and summary heatmap in FIG. 4E, upper panel). As described above, the proportion of gene drive viruses remained low in the eye and reached around 60% in the TG (FIGS. 4C-4E). Interestingly, gene drive levels varied greatly between the different brain regions. The gene drive reached 80% in the brain stem after only two days, and progressively increased to 65% in the interbrain and cerebellum, with a penetrance of up to 90% in some animals. By contrast, gene drive levels remained low in the cortex, at 25% on average. As observed above, in the TG, brain stem and interbrain –but not in the eye and cortex, viruses expressing YFP-only had been converted to recombinant gene drive viruses expressing both YFP and mCherry (FIG.4D). Together, this showed that gene drive spread in the brain was highly heterogeneous, with almost complete penetrance in some regions and barely any in adjacent ones. [0212] This heterogeneity gives an interesting insight into the dynamic of HSV- 1 infection. Gene drive propagation relies on cellular co-infection and one intuitive hypothesis is that higher viral levels would increase the probability that cells are co-infected by several virions, and, thus, that recombination levels should positively correlate with viral titers. However, no correlation between viral titers and recombination was observed (FIGS.10A - 10E). For example, HSV-1 levels were the highest in the eye, but gene drive levels remained the lowest in this organ. Similarly, HSV-1 reached similar titers in the brain stem, interbrain and cortex, but almost no recombination occurred in the cortex while high levels were observed in the brain stem and interbrain (FIG. 4D). This shows that ^{1896-P92WO AP} -74- recombination does not simply correlate with viral titers, as could be expected, but is likely explained by other tissue-specific cellular or viral mechanisms. EXAMPLE 12 High levels of cellular co-infection in the TG and the brain [0213] The results described above suggest that cells are frequently co-infected by several virions during HSV-1 infection. Thus, direct measurement of co-infection levels during HSV-1 infection was assessed to determine the basic biology that supports gene drive propagation. [0214] HSV-1 and related viruses expressing fluorescent proteins have long been used to probe neuronal pathways of the visual system. To investigate cellular co-infection, Balb/c mice were infected ocularly with equal amounts of three different viruses, expressing either YFP, CFP or RFP from the same US1/US2 locus (FIG. 5A). The fluorescent reporters carried nuclear localization signals and were not incorporated into virions, and, thus, marked infected nuclei. Mice were injected intravitreally with a total of 10⁶ PFU in the left eye and dissected four days later. Fluorescence was observed directly on frozen sections without staining. Strikingly, we observed very high levels of co- infection in the TG (FIGS. 5A-5E) and different regions of the brain (FIG. 6), with cells often co-expressing two or more fluorophores. [0215] In the TG, YFP and CFP were expressed at high levels, but the RFP signal was weaker and rarely above background. Thus, for the TG, the analysis was restricted to YFP and CFP. A widespread expression distributed over the entire length of the TG on some sections, or more tightly localized clusters in others was observed (FIG.5E). Areas with the strongest signal likely localized to the neuron-rich ophthalmic division of the TG, but the widely disseminated expression indicated that HSV-1 had spread to other areas. Importantly, a majority of infected cells appeared to express both YFP and CFP, indicating extensive cellular co-infection in the TG (FIG. 5E). To quantify co-infection precisely, machine learning was utilized to automatically segment cells and measure YFP and CFP intensity on TG sections (FIGS. 5B-5D, FIGS. 11A-11F). A total of 4035 cells were detected, with a clear separation between YFP and CFP signals and high consistency between replicates. An average of 52% of cells were found to express both YFP and CFP, with a range of 45% to 60% between replicates. Since RFP was excluded from this analysis, this was probably an underestimate of the co-infection frequency. In fact, in the few images with strong RFP signal, numerous cells co-expressing RFP, YFP and/or CFP, ^{1896-P92WO AP} -75- with instances of cells expressing all three markers (FIG. 5F, FIG. 11F) were detected. Together, this showed that more than 50% of cells were co-infected by two or more virions in the TG. [0216] The above analysis was repeated in the brain (FIGS.6A-6H, FIGS.12A- 12F, 13A-13F, and FIGS. 14-17). A strong fluorescence was detected along well- characterized routes through the optic, oculomotor and trigeminal nerves (FIG. 6D, FIG. 12A-12F). In particular, fluorescence was observed: 1) in the lateral geniculate nucleus (LGN) in the thalamus and the superior colliculus (SC) in the midbrain, where most axons of the optic nerves terminate; 2) in the Edinger–Westphal nucleus (EW), one of the two nuclei of the oculomotor nerve in the midbrain; and 3) throughout the hindbrain, likely corresponding to trigeminal nerve nuclei (TGN). Fluorescence was also detected in other areas associated with visual pathways such as the optic tract, olivary pretectal nucleus, suprachiasmatic nuclei or visual cortex (FIG. 12A-12F). Additionally, disseminated fluorescence was detected over wide areas not easily associated with the visual system, with important variation between replicates. This likely corresponded to the secondary or tertiary spread of HSV-1 throughout the brain. Images were collected in three main regions in the thalamus, midbrain, and hindbrain (FIG.6A). This time, RFP had a stronger signal and was included in the analysis. After machine learning-assisted segmentation, a total of 10,028 cells were analyzed over three brains. RFP was observed in 10-15% of infected cells, while YFP and CFP were detected in equivalent proportions in 40-60% of cells (FIGS. 13A-13F). When analyzing all images together, a high level of co-infection was observed. In total, 29% of cells expressed two colors, and 5% of cells had three colors, with results highly consistent between replicates (FIG.6B, FIG.6C, and FIGS.13A-13F). This confirmed the high level of co-infection found in the TG, with more than 34% of cells co-infected by two or more virions in the whole brain. [0217] Next, the brain regions associated with primary HSV-1 spread, namely the LGN, SC, EW and TGN were investigated. Interestingly, the LGN and SC, where the optic nerve terminates, had relatively low co-infection levels, around 20%. By contrast, the EW and TGN, where the oculomotor and trigeminal nerves terminate, respectively, had much higher co-infection levels, around 40% (FIG. 6C). This visually correlated with very different infection patterns. In the LGN and SC, well-separated and tight foci expressing only one color were observed, with co-infected cells at the boundaries (FIG.6E, FIG.6F, closeups and additional examples in FIGS. 14A-14B and FIGS. 15A-15B). This was ^{1896-P92WO AP} -76- reminiscent of viral plaques in cell culture and may suggest clonal spread from a single infected cell. By contrast, in the EW and TGN, infected cells were broadly disseminated, with few infected cells touching each other. The different colors were uniformly distributed and cells with one, two, or three colors were observed without evidence of spatial clustering (FIGS.6G-6H, FIG.16, and FIG.17). These differences in co-infection patterns are intriguing. Together with the high heterogeneity observed during gene drive spread (FIG. 4), these observations suggested that infection dynamics vary significantly depending on the viral propagation route. [0218] Altogether, this analysis revealed that neuronal tissues sustain high levels of co-infection during HSV-1 infection, with more than 50% and 40% of cells infected with two or more viruses in the TG and specific brain regions, respectively. EXAMPLE 13 Gene drive spread during latent infection [0219] The results so far show that a gene drive can spread efficiently during acute infection. Next, the ability of a gene drive to spread in the context of a latent infection was investigated. This would be critical in opening important avenues for therapeutic interventions. After primary orofacial infection, HSV-1 typically establishes latency in the TG and other peripheral ganglia. Following reactivation, HSV-1 travels back to the mucosal surface, causing lesions or shedding asymptomatically. In the following experiments, an ocular model of latent infection and drug-induced reactivation in mice was used to test if a gene drive virus –administered at a later time point, thus "superinfecting"– could target and recombine with latent HSV1-WT (FIGS.7A-7F and 8A-8G). [0220] To establish a latent infection, Swiss-Webster mice were infected ocularly with HSV1-WT, with 10⁵ PFU in both eyes after corneal scarification. Four weeks later, animals were inoculated with GD or control GD-ns after corneal scarification (FIG. 7A, FIGS.18A-18F). Immune responses induced by the primary infection limit the spread of a superinfecting virus, allowing mice to be safely inoculated with 10⁷ PFU per eye of GD or GD-ns while being transiently immunosuppressed with the glucocorticoid dexamethasone. After four more weeks and once the second infection had resolved, latent HSV-1 –originating from the primary, secondary infection, or both– was reactivated twice by treating animals with the bromodomain inhibitor JQ1 (FIG.7B). Mice do not naturally reactivate HSV-1, but JQ1 induction reproducibly induces viral shedding. Eye swabs collected on days 1-3 following JQ1 treatment were screened for viral DNA by qPCR. ^{1896-P92WO AP} -77- Overall low shedding rates were observed. A total of 10 shedding events were detected, with some animals shedding on consecutive days (FIG. 7B). Gene drive-directed recombination was measured in positive swabs using two duplex digital droplet (dd)PCR assays, that distinguish between the YFP, CFP and mCherry markers of WT and gene drive viruses and quantify the proportion of gene drive recombinants. Eight out of ten positive swabs could be successfully genotyped, with the gene drive sequence detected in three of them (FIG. 7C, FIG. 18G). Critically, one of the reactivated viruses (from mouse #24) was a recombinant gene drive virus carrying YFP and mCherry markers, whereas two others carried CFP and mCherry, representingFFF the original gene drive virus (FIG. 18G). Despite the limited number of shedding events, this showed that GD and GD-ns could successfully reach the latent reservoir and later reactivate, with one recombination event detected. [0221] Next, both TG from each animal were collected and latent viral loads were measured by duplex ddPCR. The CFP marker originating from the superinfecting GD or GD-ns viruses could be detected in around 40% of TG, with 60% of mice having detectable CFP in at least one TG (FIG. 7D). When detected, GD and GD-ns viral loads were approximately two orders of magnitude lower than WT, as measured by the respective titers of YFP and CFP/mCherry (FIG.7E). Overall, GD and GD-ns represented between 0 and 60% of the total latent viral load (average around 5%, median at 0%), with most detected samples ranging from 1% to 10% (FIG. 7F). This low proportion contrasted with the relatively high frequency of gene drive sequences detected in reactivated swabs (3/8, or 37%), suggesting that the superinfecting GD/GD-ns viruses could successfully reactivate and shed despite representing only a small proportion of the latent reservoir. [0222] Swiss Webster mice are highly susceptible to HSV-1 infection. During primary infection, mice exhibited moderate to severe symptoms, with often extended facial lesions. As a result, most animals had residual scar tissue on their eyes once the primary infection had resolved. Symptoms and final eye scarification were scored during the primary infection, and mice separated into groups with homogenous symptoms and eye scores before superinfection with GD or GD-ns (FIGS.18C-18D). Importantly, at the end of the experiment, GD and GD-ns were detected almost exclusively in the TG of mice with perfect eyes, with even low levels of scarification preventing a successful superinfection (FIG.18E). HSV-1 typically does not cause long-lasting scars in humans, and this residual scar tissue represented an unfortunate confounding factor in the mouse model. ^{1896-P92WO AP} -78- [0223] To alleviate this effect, the experiment in C57Bl/6 mice were repeated (FIGS. 8A-8G). C57Bl/6 are more resistant to HSV-1 infection and typically experience minimal symptoms. C57Bl/6 mice were infected ocularly with HSV1-WT, with 10⁶ PFU in both eyes after corneal scarification. Compared to Swiss Webster and despite the higher dose, mice exhibited limited symptoms and reduced mortality during primary infection. Around 20% of mice had residual scar tissues, usually on only one eye (FIGS.19A-19D). Four weeks later, mice were superinfected with 10⁷ PFU per eye of GD or GD-ns, while being transiently immunosuppressed with dexamethasone and tacrolimus. Then, another four weeks later, mice were injected with JQ1 and Buparlisib to reactivate latent HSV-1. Buparlisib is a phosphoinositide 3-kinase inhibitor and evidence suggested that it could improve HSV-1 reactivation (FIG.19E). Reactivation rates ranged from 0 to 33% (FIG. 8A). Most events were close to the detection limit (around 100 copies/swab) and viral genotypes from 17 out of 22 reactivation events (FIG. 8B, FIGS. 20A-20C) were successfully obtained. Nine out of 17 swabs (53%) were either recombinant viruses carrying mCherry and YFP, or original gene drive viruses with mCherry and CFP. In addition, two swabs were genotyped as predominantly WT (from mice #25 and #33), but had detectable amounts of mCherry, representing less than 5% of the total titer (FIG. 8B, FIGS. 20A-20C). In one positive swab with a low viral titer (mouse #45), both YFP and CFP markers, originating from the same US1/US2 locus, were detected, suggesting a mix of reactivated viruses. Importantly, we detected recombinants expressing YFP and mCherry in mice superinfected with either GD (1/9) or GD-ns (3/8), showing that both viruses could recombine with HSV1-WT. Because of the overall low number of reactivation events, further statistical analysis could not be conducted. In summary, it was established that gene drive viruses represented more than 50% of reactivation events, with several examples of recombination with HSV1-WT. [0224] Next, TG were dissected, and latent viral loads were measured by duplex ddPCR. GD and GD-ns viruses could be detected in around 79% and 66% of TG, respectively, with more than 90% of mice having detectable GD or GD-ns in at least one TG (FIG.8C). GD and GD-ns titers were one to two orders of magnitude lower than WT (FIG. 8D) and represented a small percentage of the total latent viral load, ranging from 0.5% to 50% for most samples (average at 10% and median around 1%, FIG.8E). [0225] Evidence of gene drive spread in the TG was investigated. In particular, whether mCherry was significantly overrepresented compared to the CFP baseline, was ^{1896-P92WO AP} -79- determined, as the gene drive cassette containing mCherry potentially recombined with HSV1-WT and increased in frequency. Of note, no correlation between the viral loads in the left and right TG collected from the same animals (Pearson correlation, r2=0.11), was observed, which allowed all samples to be treated as independent replicates in these analyses (FIG.21A). When plotting the titers of mCherry versus CFP, GD datapoints were observed to be significantly above the identity line (p<0.0001), while GD-ns datapoints were mostly situated along the line and did not significantly deviate from it (FIG. 8F, statistical analysis in FIG.21B). This suggested that the gene drive had recombined with wild-type viruses and increased in frequency in the TG of GD-superinfected mice. To quantify this enrichment, the fold change between the proportion of mCherry and CFP in the TG was calculated (FIG. 8G). On average, in the GD-superinfected samples, a 70% increase in the relative proportion of mCherry compared to CFP, was observed, which was significantly higher than the control samples with GD-ns where no enrichment was observed (p=0.0053, t-test). In the most extreme cases, the gene drive had spread more than 10-fold over the CFP baseline, for example increasing from 9% to 90% in one sample, or from 1.3% to 10% in another (FIG. 21C). Together, this analysis showed a limited but statistically significant spread of the gene drive in the latent wild-type population. [0226] In summary, these data establish that a superinfecting gene drive virus could reach the latent reservoir and spread in the wild-type population. In both Swiss- Webster and C57Bl/6, GD and GD-ns were detected in more than 50% of shedding events after JQ1 reactivation. This indicated that the superinfecting GD and GD-ns viruses could successfully reactivate and shed despite representing only a small proportion of the latent reservoir. EXAMPLE 14 Design and generation of gene drive virus targeting US9 [0227] In an exemplary embodiment of a modified alphaherpesvirus containing a gene-drive construct of the present disclosure relies on co-infection of cells to replace wild-type viruses with an engineered version. CRISPR-based viral gene drives include Cas9 and a guide RNA targeting the complementary wild-type locus. After co-infection of neurons by wild-type and gene drive virus, Cas9 cleaves the wild-type genome. Homology-directed repair using the gene drive sequence as a repair template converts the wild-type virus into a new recombinant gene drive virus (FIG.22A). HSV virions travel through nerves from the mucosal periphery to neurons in the ganglia (retrograde ^{1896-P92WO AP} -80- transport), where they remail latent. After reactivation, virions travel back to the surface (anterograde transport), causing viral outbreaks (FIG.22B). During orofacial infection, HSV remains latent primarily in the trigeminal ganglia (FIG.22C). [0228] A donor plasmid containing the gene drive cassette against the HSV-1 and HSV-2 US9 coding sequence (HSV1_GD-US9 and HSV2-GD-US9, respectively) were generated by serial modifications of the GD-mCherry donor plasmid (FIG.22D). All modifications were carried out by Gibson assembly (NEB, Ipswich, MA, USA), using PCR products from other plasmids or synthesized DNA fragments (GeneArtTM StringTM fragments, ThermoFisher, USA). The final HSV1_GD-US9 and HSV2_GD- US9 donor plasmid included homology arms for the US9 region, the Roux sarcoma virus (RSV) promoter driving SpCas9 followed by the SV40 polyA terminator, the CMV promoter driving an mCherry reporter followed by the beta-globin polyA signal, and the U6 promoter controlling gRNA expression. HSV1_GD-US9 plasmid carried a guide RNA targeting the HSV-1 US9 coding sequence (TTCGGTCGAAGCCTACTACT) (SEQ ID NO: 15). HSV2_GD-US9 carried a guide RNA targeting the HSV-2 US9 coding sequence (AACGACTTCCTCGTGCGCAT) (SEQ ID NO: 14). [0229] To build recombinant viruses HSV1_GD-US9 (SEQ ID NO: 2) and HSV2_GD-US9 (SEQ ID NO: 4), 1 million Vero cells were infected with HSV-1 (strain 17+) (SEQ ID NO: 6), or HSV-2 (strain MS) (SEQ ID NO: 7),, respectively, and transfected with linearized donor plasmids containing the gene drive donor sequence. Transfection was performed by Nucleofection (Lonza, Basel, Switzerland) and cells were plated in a single 24-well. After 2-4 days, mCherry-expressing viral plaques were isolated and purified by several rounds of serial dilutions and plaque purification. Purity and absence of unmodified viruses were assayed by PCR and Sanger sequencing after DNA extraction (DNeasy kit, Qiagen, Germantown, MD, USA). Viral stocks were produced as specified above and tittered by plaque assay. [0230] To improve safety of HSV1_GD-US9 (SEQ ID NO: 2), a second virus, named HSV1_GD-US9/gE-Y463A (SEQ ID NO: 3) was created, carrying an additional mutation in the US8 viral gene (coding for glycoprotein gE), in order to further attenuate the vector and improve safety. The point mutation result in the Y463A substitution in the gene product of US8 (glycoprotein gE). [0231] In addition to the gene drive sequence introduced in the US9 coding sequence, HSV2_GD-US9 ) carries a mutation in the US8 viral gene (coding for ^{1896-P92WO AP} -81- glycoprotein gE), to further attenuate the vector and improve safety. The point mutation result in the Y458A substitution in the gene product of US8 (glycoprotein gE) (SEQ ID NO: 4). EXAMPLE 15 Characterization of gene drive virus HSV1-GD-US9 and HSV1_GD-US9/gE-Y463A [0232] Gene drive viruses HSV1-GD-US9 (SEQ ID NO: 2) and HSV1_GD- US9/gE-Y463A (SEQ ID NO: 3) knockout viral gene US9. To confirm that these viruses have inactivated anterograde transport as expected, mice were infected ocularly with 10⁶ plaque forming units (pfu) of virus in the intravitreal space of the left eye (FIG. 23D). In this model, HSV-1 travels from the eye to the brain through the cranial nerve II., III. and V. After 5 days, brain sections of infected mice were stained with an antibody recognizing HSV, highlighting the brain regions infected with HSV. Mice infected with HSV1-WT showed extensive staining all over the brain (FIG. 23A). By contrast, mice infected with HSV1_GD-US9 (SEQ ID NO: 2) (FIG. 23B) and HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3) (FIG.23C) showed much reduced staining. The only region with detectable HSV in the brain of mice infected with GD-US9/gE-Y463E corresponds to the Edinger– Westphal nucleus in the midbrain. This brain region is by reached by retrograde viral transport in cranial nerve III, while the rest of the brain is reached by anterograde transport. These results confirmed that HSV1_GD-US9 (SEQ ID NO: 2) and HSV1_GD-US9/gE- Y463E (SEQ ID NO: 3) had inactivated anterograde transport while maintaining functional retrograde transport. [0233] To show that gene drive viruses HSV1_GD-US9 (SEQ ID NO: 2) and HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3) can establish a latent infection but are unable to reactivate and cause disease in the mucosa, mice were infected ocularly after corneal scarification with HSV1-WT (10⁵ pfu/eye), HSV1_GD-US9 (10⁶ pfu/eye) or HSV1_GD- US9/gE-Y463E (10⁶ pfu/eye) (FIG. 24C). A month later, the latent viral load in the trigeminal ganglia (TG) was measured. The TG is the main site of latency after orofacial infection. FIG. 24A shows that both HSV1_GD-US9 and HSV1_GD-US9/gE-Y463E could reach the TG and establish latency, confirming that the HSV1_GD-US9 and HSV1_GD-US9/gE-Y463E have functional retrograde transport (FIG.24A). Latent virus in the ganglia of mice infected with HSV1-WT or HSV1_GD-US9 (SEQ ID NO: 2) was reactivated using the small molecule drug JQ1. Viral shedding was measured by qPCR in eye swabs collected 1-3 days JQ1 infection. The data shows that in mice infected with ^{1896-P92WO AP} -82- HSV1_GD-US9 (SEQ ID NO: 2), the frequency of viral shedding was reduced by 95% (fisher's exact test, p<0.0001). This confirmed that anterograde transport of HSV1_GD- US9 (SEQ ID NO: 2) was almost completely inactivated. With virus HSV1_GD-US9/gE- Y463E (SEQ ID NO: 3), viral shedding is expected to be reduced by 100% (FIG.24B). EXAMPLE 16 GD-US9 and GD-US9/gE-Y463E prevent HSV-1-associated mortality. [0234] To test if HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3) could be used as preventative treatment against HSV-1, mice were treated with HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3), either by FFFocular infection with HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3) (10⁶ pfu/eye) (FIG.25D), or by intravaginal inoculation (10⁶ pfu). A month after inoculation, mice were challenged with HSV1-WT, either ocularly or vaginally (10⁷ pfu). In both cases, HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3) treatment prevented mortality caused by HSV-1 challenge (FIGS. 25A-25B). This showed that HSV1_GD-US9/gE- Y463E (SEQ ID NO: 3) could be used as a preventive treatment that protect against HSV- 1 infection. [0235] To test if HSV1_GD-US9 (SEQ ID NO: 2) and HSV1_GD-US9/gE- Y463E (SEQ ID NO: 3) could be used as a therapeutic treatment against genital HSV-1 infection, mice were first infected with HSV1-WT, by intravaginal inoculation (10⁴ pfu). A month after infection, latently infected mice were treated intravaginally with HSV1_GD- US9 (SEQ ID NO: 2) or HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3) (10⁷ pfu). A month following treatment, mice were treated with the small molecule JQ1 to reactivate latent viruses. The data shows that treatment with HSV1_GD-US9 (SEQ ID NO: 2) or HSV1_GD-US9/gE-Y463E (SEQ ID NO: 3) prevented mortality associated with viral reactivation (FIGS.25C). ^{1896-P92WO AP} -83-

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 1. A method of suppressing and/or preventing infection or a recurrence of an infection caused by a wild-type alphaherpesvirus comprising genetically modifying/altering a genome of the wild-type alphaherpesvirus, the method comprising: co-infecting at least one cell of a latent reservoir comprising the wild-type alphaherpesvirus with at least one modified alphaherpesvirus, wherein the at least one modified alphaherpesvirus comprises a gene-drive construct integrated into the genome of the modified alphaherpesvirus, wherein the modified alphaherpesvirus is effective in genetically modifying/altering the wild-type alphaherpesvirus by integrating/inserting the gene drive construct into the genome of the alphaherpesvirus at a target site, and wherein the integration/insertion of the gene drive construct disrupts at least one viral gene at the target site in the genome of the wild-type alphaherpesvirus.

2. The method of claim 1, wherein the gene-drive construct comprises: a first nucleic acid sequence operably linked to a first promoter and encoding a functional targeted endonuclease that induces a double stranded break in or near at least one target site in a genome of a wild-type alphaherpesvirus.

3. The method of claim 2, wherein the gene-drive construct further comprises flanking sequences homologous to sequences adjacent to the at least one target site that permit insertion of the gene drive construct at the at least one target site in the genome of the wild-type alphaherpesvirus.

4. The method of claim 3, wherein the flanking sequences homologous to sequences adjacent to the at least one target site range in length from about 50 bp to about 5 kb.

5. The method of any one of claims 2 to 4, wherein the functional targeted endonuclease comprises an endonuclease selected from the group consisting of a class 2 CRISPR/Cas endonuclease, a TALEN, a zinc finger nuclease, and a homing endonuclease. ^{1896-P92WO AP} -84-

6. The method of claim 5, wherein the functional targeted endonuclease comprises a class 2 CRISPR/Cas endonuclease.

7. The method of claim 6, wherein the class 2 CRISPR/Cas endonuclease is a type II CRISPR/Cas endonuclease.

8. The method of claim 6 or claim 7, wherein the class 2 CRISPR/Cas endonuclease comprises a Cas9 protein.

9. The method of claim 8, wherein the Cas9 protein is selected from the group consisting of a Streptococcus pyogenes Cas9 protein (spCas9) or a functional portion thereof, a Staphylococcus aureus Cas9 protein (saCas9) or a functional portion thereof, a Streptococcus thermophilus Cas9 protein (stCas9) or a functional portion thereof, a Neisseria meningitides Cas9 protein (nmCas9) or a functional portion thereof, and a Treponema denticola Cas9 protein (tdCas9) or a functional portion thereof.

10. The method of claim 6, wherein the class 2 CRISPR/Cas endonuclease is a type V or type VI CRISPR/Cas endonuclease.

11. The method of claim 10, wherein the type V or type VI CRISPR/Cas endonuclease is selected from the group consisting of a Cpf1 polypeptide or a functional portion thereof, a C2c1 polypeptide or a functional portion thereof, a C2c3 polypeptide or a functional portion thereof, and a C2c2 polypeptide or a functional portion thereof.

12. The method of any one of claims 6-11, wherein the gene drive construct further comprises a second nucleic acid sequence encoding at least one guide RNA, and wherein the second nucleic acid sequence is operably linked to a second promoter.

13. The method of any one of claims 6-12, wherein the first promoter and/or the second promoter comprises a viral promoter.

14. The method of claim 12 or claim 13, wherein the at least one guide RNA directs the functional targeted endonuclease to a site in the genome of the wild-type alphaherpesvirus where cleavage permits integration/insertion of the gene drive construct into the genome of the wild-type alphaherpesvirus by homologous recombination. ^{1896-P92WO AP} -85-

15. The method of any one of claims 6-14, wherein the first nucleotide sequence encoding the functional targeted endonuclease and the second nucleotide sequence encoding the at least one guide RNA are located between a pair of flanking sequences in the gene drive construct.

16. The method of any one of claims 1-15, wherein the disruption of the at least one gene at the target site impairs anterograde transport of the wild-type alphaherpesvirus from a latent reservoir to a site of active infection, and wherein the wild-type alphaherpesvirus remains latent.

17. The method of any one of claims 1-16, wherein the latent reservoir comprises sensory and autonomic ganglia neurons and the site of active infection is a mucosal epithelium.

18. The method of any one of claims 1-16, wherein the wild-type and the modified alphaherpesvirus are not impaired in retrograde axonal transport.

19. The method of claim 1-18, wherein the method is effective in preventing viral shedding and recurring symptoms.

20. The method of any one of claims 1-19, wherein the wild-type and/or modified alphaherpesvirus is selected from Ateline alphaherpesvirus 1; Bovine alphaherpesvirus 2; Bovine mammillitis virus; Cercopithecine alphaherpesvirus 2; Human alphaherpesvirus 1 (HSV-1); Human alphaherpesvirus 2 (HSV-2); Leporid alphaherpesvirus 4; Macacine alphaherpesvirus 1; Macacine alphaherpesvirus 2; Macacine alphaherpesvirus 3; Macropodid alphaherpesvirus 1; Macropodid alphaherpesvirus 2; Panine alphaherpesvirus 3; Papiine alphaherpesvirus 2; Pteropodid alphaherpesvirus 1; Saimiriine alphaherpesvirus 1; Bovine alphaherpesvirus 1; Bovine alphaherpesvirus 5; Bovine encephalitis herpesvirus; Bubaline alphaherpesvirus 1; Canid alphaherpesvirus 1; Caprine alphaherpesvirus 1; Cercopithecine alphaherpesvirus 9; Cervid alphaherpesvirus 1; Cervid alphaherpesvirus 2; Cervid alphaherpesvirus 3; Equid alphaherpesvirus 1; Equid alphaherpesvirus 3; Equid alphaherpesvirus 4; Equid alphaherpesvirus 8; Equid alphaherpesvirus 9; Felid alphaherpesvirus 1; Human alphaherpesvirus 3; Monodontid alphaherpesvirus 1; Phocid alphaherpesvirus 1; and Suid alphaherpesvirus 1. ^{1896-P92WO AP} -86-

21. The method of any one of claims 1-20, wherein the wild-type and/or modified alphaherpesvirus is a Human alphaherpesvirus 1 (Herpes Simplex virus 1).

22. The method of any one of claims 1-20, wherein the wild-type and/or modified alphaherpesvirus is a Human alphaherpesvirus 2 (Herpes simplex virus 2).

23. The method of any one of claims 1-22, wherein the at least one gene at the target site is selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding membrane protein US9) or a homolog thereof.

24. The method of any one of claims 1-23, wherein the at least one gene at the target site is US9 or a homolog thereof.

25. The method of any one of claims 1-24, wherein the at least one cell comprises a latent reservoir for the wild-type Herpes simplex virus.

26. The method of claim 25, wherein the at least one cell is a sensory and/or autonomic ganglia neuron.

27. A method of preventing and/or suppressing anterograde transport of a wild-type alphaherpesvirus from a latent reservoir to a site of active infection, the method comprising: co-infecting a subject harboring a wild-type alphaherpesvirus in a latent reservoir with a modified alphaherpesvirus containing a gene-drive construct integrated into the genome of the modified alphaherpesvirus, wherein the modified alphaherpesvirus is effective in genetically modifying/altering the wild-type alphaherpesvirus by integrating/inserting the gene drive construct at a target site in the genome of the wild-type alphaherpesvirus, wherein the integration/insertion of the gene drive construct at the target site disrupts at least one viral gene at the target site in the genome of the wild-type alphaherpesvirus, and wherein the at least one viral gene is a gene involved in anterograde transport of the virus. ^{1896-P92WO AP} -87-

28. The method of claim 27, wherein the method is effective in preventing viral shedding and recurring symptoms.

29. The method of claim 27 or claim 28, wherein the subject is human and the wild-type and/or modified alphaherpesvirus is selected from Human alphaherpesvirus 1 (Herpes Simplex virus1), Human alphaherpesvirus 2 (Herpes Simplex virus2), and Human alphaherpesvirus 3 (Varicella zoster virus).

30. The method of any one of claims 27-29, wherein the subject is human and the wild-type and/or modified alphaherpesvirus is Herpes Simplex virus 1.

31. The method of any one of claims 27-29, wherein the subject is human and the wild-type and/or modified alphaherpesvirus is Herpes Simplex virus 2 (HSV-2).

32. The method of claim 30 or claim 31, wherein the latent reservoir comprises sensory and autonomic ganglia neurons, and wherein the site of active infection is a mucosal epithelium.

33. The method of any one of claims 27-32, wherein the at least one gene at the target site is selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding membrane protein US9) or a homolog thereof.

34. The method of any one of claims 27-33, wherein the wild-type and the modified virus are not impaired in retrograde axonal transport.

35. A method of suppressing and/or preventing recurrence of an infection in a subject caused by a wild-type Herpes simplex virus comprising: co-infecting the subject with an active and/or a recurrent infection with a therapeutically effective amount of a composition comprising a modified Herpes simplex virus containing a gene-drive construct, wherein the gene-drive construct is integrated into the genome of the modified Herpes simplex virus, and wherein the composition is effective in impairing anterograde transport of the wild-type Herpes simplex virus from a latent reservoir to a site of active infection. ^{1896-P92WO AP} -88-

36. The method of claim 35, wherein the gene-drive construct comprises: (i) a first nucleotide sequence encoding for a functional targeted endonuclease that induces a double stranded break in or near at least one target site in a genome of a wild-type Herpes simplex virus; (ii) a second nucleotide sequence encoding at least one guide RNA sequence complementary to the at least one target site in the genome of a wild-type Herpes simplex virus; and (iii) a pair of flanking sequences homologous to sequences adjacent to the at least one target site, wherein the first and second nucleotide sequences are located between the pair of flanking sequences in the construct, and wherein the gene-drive construct is effective in integrating/inserting into the genome of the wild-type Herpes simplex virus and disrupting at least one viral gene in the genome of the wild-type Herpes simplex virus.

37. The method of claim 36, wherein the gene-drive construct comprises a first promoter operably linked to the first nucleotide sequence.

38. The method of claim 36, wherein the gene-drive construct comprises a second promoter operably linked to the second nucleotide sequence.

39. The method of claim 37 or claim 38, wherein the first promoter linked to the first nucleotide sequence and/or the second promoter linked to the second nucleotide sequence comprises a viral promoter.

40. The method of any one of claims 36-39, wherein the guide RNA targets the targeted endonuclease to the at least one target site in the wild-type Herpes simplex virus genome where cleavage permits integration/insertion of the gene drive construct into the genome of the wild-type Herpes simplex virus by homologous recombination.

41. The method of any one of claims 36-40, wherein the at least one viral gene is selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding membrane protein US9) or a homolog thereof. ^{1896-P92WO AP} -89-

42. The method of any one of claims 36-41, wherein the at least one viral gene is US9 or a homolog thereof.

43. The method of any one of claims 35-42, wherein the wild-type and/or the modified Herpes simplex virus is selected from HSV-1 and HSV-2.

44. The method of any one of claims 35-43, wherein the wild-type and the modified virus are not impaired in retrograde axonal transport.

45. A method for prophylactically treating a subject to protect against disease caused by a wild-type herpes simplex virus, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising a modified Herpes simplex virus containing a gene drive construct integrated into the genome of the modified Herpes simplex virus, wherein the modified Herpes simplex virus is impaired in anterograde axonal transport of the virus from a latent reservoir to a site of active infection, wherein the modified Herpes simplex virus is not impaired in retrograde transport of the virus from the site of infection to the latent reservoir, and wherein the subject has had no prior exposure to a wild-type Herpes simplex virus and/or infection caused by a wild-type Herpes simplex virus.

46. The method of claim 45, wherein the step of administering comprises infecting the subject with the modified Herpes simplex virus.

47. The method of any one of claims 45 or 46, wherein the modified Herpes simplex virus remains latent in the subject.

48. The method of claim 47, wherein the composition is effective in protecting the subject from developing symptoms upon infection with a wild-type Herpes simplex virus.

49. The method of any one of claims 45-48, wherein the composition is effective in genetically modifying a wild-type Herpes simplex virus. ^{1896-P92WO AP} -90-

50. The method of claim 49, wherein the composition is effective in impairing anterograde axonal transport of the wild-type virus from a latent reservoir to the site of active infection.

51. The method of any one of claims 45-50, wherein the gene-drive construct is effective in integrating/inserting into the genome of a wild-type Herpes simplex virus and disrupting at least one viral gene in the genome of the wild-type Herpes simplex virus.

52. The method of claim 51, wherein the at least one gene is selected from US7 (encoding glycoprotein gI) or a homolog thereof, US8 (encoding glycoprotein gE) or a homolog thereof, US9 (encoding tegument protein) or a homolog thereof.

53. The method of any one of claims 45-52, wherein the at least one viral gene is US9 or a homolog thereof.

54. The method of any one of claims 45-53, wherein the Herpes simplex virus is selected from HSV-1 and HSV-2. ^{1896-P92WO AP} -91-