WO2019193205A1

WO2019193205A1 - Chimeric protein having photoactivatable recombinase activity

Info

Publication number: WO2019193205A1
Application number: PCT/EP2019/058829
Authority: WO
Inventors: Gaël YVERT; Hélène DUPLUS; Martin SPICHTY
Original assignee: Centre National De La Recherche Scientifique; Ecole Normale Superieure De Lyon; Universite Claude Bernard Lyon 1
Priority date: 2018-04-06
Filing date: 2019-04-08
Publication date: 2019-10-10
Also published as: FR3079832A1

Abstract

The present invention relates to a chimeric protein, the recombinase activity of which is photo-inducible. It also relates to the use of such a protein for controlling, via exposure to light, recombination events in a host cell or in vitro.

Description

Chimeric protein with photoactivatable recombinase activity

The present invention relates to a chimeric protein whose recombinase activity is photoinducible. It also relates to the use of such a protein for controlling, via light exposure, recombination events in a host cell or in vitro.

BACKGROUND OF THE INVENTION

The rise of biotechnology is largely based on the ability to control and modify the genetic heritage of cells. For this purpose, recombinases are among the most effective tools in genetic engineering, whether for handling transgenes in eukaryotic or prokaryotic cells.

Among the available recombinases, the CRE recombinase is very widely used in various prokaryotic and eukaryotic models as a tool for handling DNA in vitro and in vivo. This recombinase is derived from bacteriophage P1 and catalyzes the recombination reaction between two specific LoxP sites. The LoxP site, 34 base pairs long, comprises at its ends two palindromic regions of 13 base pairs each called RBE (Recombinase Binding Element) which surround an oriented region of 8 base pairs, which determines the direction of the LoxP site. If the two LoxP sites are oriented in the same direction, the recombinase causes irreversible excision of the region between the two sites. On the other hand, if the two LoxP sites are oriented in the opposite direction, the recombinase induces the reversible inversion of the sequence lying between the two sites.

Many techniques for the conditional activation of the CRE-LoxP system have been developed in recent decades.

One of these techniques allows in vivo conditional mutagenesis in mice and thus to study the effect of mutations that would be lethal if they were non-tissue-specific. Briefly, the LoxP sites are first integrated on either side of an allele of the gene of interest by homologous recombination in embryonic stem cells. These cells are then injected into a mouse blastocyst to obtain a cell line that will be crossed with another line of transgenic mice expressing CRE recombinase under the control of a tissue-specific promoter. The Animals from this cross are "doubly" transgenic and present the desired mutation only in the target tissue. These animals are called "mosaics" in the sense that their genetic inheritance is not unitary and differs according to the cells (Babinet and Cohen-Tannoudji, 2000, Med Sci (Paris), 16, 1, 31-42).

The conditional activation of the CRE-LoxP system has also been used in yeast, and in particular S. cerevisae (Sauer, 1987, Mol Cell Biol 7, 2087-2096). The gene coding for the CRE recombinase was placed under the control of the GAL1 promoter whose activity is repressed in the presence of glucose and stimulated in the presence of galactose. It has been demonstrated that the addition of galactose in the culture medium induces the excision of the target gene LEU2 flanked by two LoxP sites of the same orientation and previously integrated into the genome. The kinetics of leucine auxotrophy show that CRE recombinase activity in yeast begins after eight hours of galactose induction to be maximal after 24 hours. This kinetics has been improved by performing a pre-culture in the presence of raffinose.

Time control of CRE-LoxP system activity was also obtained using CRE recombinase whose activity is ligand-dependent (Metzger et al., 1995, Proc Natl Acad Sci 92, 6991). -6995). This enzyme is obtained by fusion of the gene coding for the CRE recombinase with a cDNA of the ligand binding domain (LBD) of the steroid hormone receptors and in particular of the human estrogen receptor (hER). In the absence of ligand (progesterone, estrogen), the CRE recombinase is not or only slightly active. Conversely, in the presence of the ligand, the chimeric protein undergoes a conformational change and exhibits CRE recombinase activity. This system has been improved by introducing different mutations in the LBD domain in order to replace the natural hormones with an artificial steroid, tamoxifen, and / or to increase the specificity and activation efficiency (Indra et al., 1999, Nucleic Acids Res 27, 4324- 4327, Metzger and Chambon, 2001, Methods 24, 71-80).

The activity of the CRE-LoxP system can also be controlled by temperature. Using a fish as a study model, it has indeed been shown that the expression of CRE recombinase can be controlled by a "heat shock element" (HSE) (Kobayashi et al., 2013, Genoa, NY, N, 2000). , 59-67). The HSE-CRE system can thus be activated by thermal shock (39 ° C for two hours). The response obtained, however, varies according to the different cell types and according to the stage of development during which the thermal shock has occurred. Several light-inducible CRE-LoxP systems have also been described.

One of these systems is an adaptation of the CRE-ER system made locally photo-activatable by G using a photo-chemical inductor, cyclofen-OH. This substrate, whose structure is close to tamoxifen-OH, is surrounded by labile photo groups. In response to illumination, the ligand released from these groups can bind to the ER receptor and thereby activate the CRE recombinase. This system has been used in fish (Sinha et al., 2010, Zebrafish, 7, 199-204, Sinha et al., 2010, ChemBioChem 11, 653-663) and in mice (Lu et al., 2012, Bioconjug, Chem 23, 1945-1951). The spatial and temporal control of this activation is, however, particularly complex in that it requires controlling the quality, the quantity and the diffusion of the photoactivatable ligand.

Similarly, the light inducible Light On gene expression system was used to control the expression of CRE recombinase (Wang et al., 2012, Nat Methods 9, 266-269). This system is based on the use of a transactivator which, under the action of light, binds to a transcriptional promoter and activates it. However, this system has the disadvantage of requiring very long illumination periods (of the order of 22 hours).

Another photo-activatable Cre-LoxP system has also been described by Kawano et al. (Nat Chem Biol 2016 Dec 12 (12): 1059-1064). This system uses two fusion proteins, each comprising a fragment of CRE recombinase fused to a switch photo called "Magnet". The illumination of the system causes the dimerization of the "Magnets" and the association of the fragments of the CRE recombinase which thus becomes active.

A similar system of photoactivatable Cre-LoxP has been described by Taslimi et al. (Nat Chem Biol 2016 June, l2 (6): 425-430). This system also uses two fusion proteins, each comprising a fragment of CRE recombinase fused to a photosensitive dimerization domain called CRY2-CIB. The illumination of the system causes the dimerization of the two chimeric proteins and the association of the fragments of the CRE recombinase which thus becomes active.

Another photoactivatable Cre-LoxP system has been described in human cells by Zhang et al. (Nature Methods, 2017 l4 (4): 39l-394). This system uses a protein domain called PhoCl which is cleaved upon photoactivation, as well as a ligand binding domain of steroid hormone receptors (SR). Protein The described chimeric SR-PhoCl-Cre-PhoCl-SR is inactive due to the SR domains that recruit the Hsp90 inhibitory protein from the host cell. After illumination, the double cleavage of the protein releases the SR domains and thus lifts the inhibition imposed by Hsp90.

Bearing in mind the importance of recombinases as tools in genetic engineering, many studies have been and are being conducted to improve their control. The various inducible systems available to date, however, remain complex, expensive and / or imprecise. There remains therefore a real need for a simple and effective technology ensuring the control of recombinase activity in an individual cell or a set of cells at a given time.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a recombinase system whose use would be simple and inexpensive to be adapted for industrial use, while ensuring a precise temporal and spatial control of recombinase activity.

For this purpose, the inventors have developed a photoactivatable recombinase.

Unlike the previously developed systems where the light dependence is indirect (for example via a photo-protected chemical activator or a photoinducible transcriptional activator), the recombinase according to the invention is directly activated by light, thus considerably simplifying the process. .

Thus, according to a first aspect, the present invention relates to a fusion protein comprising a recombinase domain fused to a photoreceptor domain and wherein the recombinase activity is photoactivatable.

Preferably, the recombinase domain is fused at the N-terminal to the photoreceptor domain.

Preferably, the photoreceptor domain comprises, or consists of, a domain

LOV2 of phototropin, or a variant or derivative thereof. In particular, the photoreceptor domain may be a derivative of a LOV2 domain, preferably an iLID (enhanced light inducing dimer) domain, and more particularly preferably an iLID domain derived from the LOV2 domain of phototropin 1 to Avenu sativa. In some particular embodiments, the photoreceptor domain of the fusion protein is a iLID domain having the sequence SEQ ID NO: 13 wherein X is one to three amino acids, preferably X is A, D, K, L, IL, VV, AI or QPG.

The recombinase domain is preferably selected from the group consisting of CRE recombinase (SEQ ID NO: 1), and functional variants thereof. In particular, the recombinase domain may be a functional variant of the CRE recombinase comprising or consisting of the sequence SEQ ID NO: 2, or a sequence having at least 80% sequence identity with SEQ ID NO: 2.

More particularly preferably, the recombinase domain is a functional variant of the CRE recombinase, said variant comprising, or consisting of, a CRE recombinase of SEQ ID NO: 1 in which 18, 19, 26 or 31 amino acids have been deleted. N-terminal, and optionally having a substitution at the residue corresponding to residue E340 of SEQ ID NO: 1 and / or a substitution at the residue corresponding to residue D341 of SEQ ID NO: 1

The present invention also relates to a nucleic acid encoding a fusion protein according to the invention, an expression cassette comprising a nucleic acid according to the invention, an expression vector comprising a nucleic acid according to the invention or a cassette of expression according to the invention, a host cell comprising a nucleic acid, an expression cassette or an expression vector according to the invention.

The present invention also relates to a method for inducing recombinase activity in a cell comprising

the provision of a cell expressing a fusion protein according to the invention or the insertion of a nucleic acid, an expression cassette or an expression vector according to the invention in a cell and

exposing said cell to a light capable of activating the photoreceptor domain of said fusion protein and thus recombinase activity.

The present invention also relates to a method for modifying a nucleic acid, comprising

contacting a fusion protein according to the invention with said nucleic acid of interest comprising one or more recognition sites recognized by the recombinase domain of said fusion protein, and exposing the fusion protein and said nucleic acid to a light capable of activating the photoreceptor domain of said fusion protein and thus the recombinase activity.

This method can in particular be used to modulate the expression of a molecule of interest in the host cell, to induce genetic diversity in the host cell or to modify a biological activity in the context of a gene therapy.

The present invention further relates to a kit comprising a fusion protein, a nucleic acid, an expression cassette, an expression vector and / or a host cell according to the invention, and optionally a light source capable of activating the photoreceptor domain of the fusion protein. It also relates to the use of said kit for inducing recombinase activity in a cell according to the method according to the invention and / or for modifying one or more nucleic acids according to the process according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Reporter system for detecting and quantifying CRE recombinase activity in yeast cells. The left part represents the genetic construction of the system. The right side represents the phenotype of a yeast cell carrying this construct.

Figure 2: Representation of CRE recombinase activity test results. A) The histogram shows data produced by flow cytometry. Abscissa: intensity of the FL1-H signal corresponding to the GFP fluorescence. Ordinate: number of cells with this signal strength. The data is analyzed to determine a GFP signal threshold for separating the OFF (low signal) cells of the ON (high signal) cells. B) The recombination efficiency is the proportion of ON cells in a population of cells having expressed the tested protein.

Figure 3: CRE C-Ter mutant activity test results. Error bars: standard deviation, n = 3.

Figure 4: CRE N-Ter mutant activity test results. Error bars: standard deviation, n = 3. Figure 5: Diagram of the construction of iLID-CRE fusions by homologous recombination. Cre / AA: coding sequence of the or a portion of the Cre protein or Cre_A340A341 variant. TRP1: S. cerevisiae gene allowing auxotrophic selection of transformed yeasts. Black arrows: oligonucleotides (primers) used for amplification. Star: mutation possibly carried on the upstream oligonucleotide. PromGAL1: promoter of the S. cerevisiae GAL1 gene for inducing or repressing the expression of the protein of interest.

Figure 6: The iLID-Crel9, iLID-Cre27, iLID-Cre32 and iLID-CreAA20 fusions have increased CRE activity after light stimulation. Black (darkness): without illumination. Blue (light): after illumination of cells at 460 nm of 36.3 mW / cm ² for 30 minutes. Error bars: standard deviation, n = 5.

Figure 7: Activity of iLID-xn-Cre32 mergers. Sequence of the trunk: 24A and 24B: iLID-QIA-Cre32, 24F: iLID-QIK-Cre32, 241: iLID-QIL-Cre32, 25C: iLID-QIIL-Cre32, 25F: iLID-QIVV-Cre32, 25X: iLID -QIAI-Cre32, 26R: iLID-QIQPG-Cre32. Black (darkness): without illumination. Blue (light): including illumination at 460 nm of 2.2 mW / cm2 for 90 minutes. Error bars: standard deviation, n = 3.

Figure 8: Effects of different conditions of illumination of the fusion protein iLID_Cre32 in the context of an expression controlled by the promoter GAL1 (A) or by the promoter MET17 (B). Black: without illumination. Blue: including an illumination at 460nm during the indicated time (30, 60 or 90) minutes and different intensities: 10, 20, 50, 100 respectively corresponding to 2.2, 5.1, 17.9 and 36.3 mW / cm2. Error bars: standard deviation, n = 3.

Figure 9: Effects of different conditions of illumination of the fusion protein iLID_CreAA20 in the context of an expression controlled by the promoter MET17. A: illumination at 460nm for 30 minutes with different intensities. B: illumination at 460nm during different times and with an intensity of 36.3 mW / cm ² .

Figure 10: Comparison of iLID-CreAA20 fusion activity alone or with NLS. Black (darkness): without illumination. Blue (10%): including illumination at 460 nm of 2.2 mW / cm2 for 180 minutes. Error bars: standard deviation, n = 3.

Figure 11: Comparison of the system according to the invention with the system nMag / pMag-Cre. Black (darkness): without illumination. Blue (light): including illumination at 460 nm of 2.2 mW / cm2 for 180 minutes. Error bars: standard deviation, n = 3. DETAILED DESCRIPTION OF THE INVENTION

The inventors have designed a chimeric protein endowed with a directly photoactivatable recombinase activity. To do this, they fused a recombinase domain to a photoreceptor domain and demonstrated that activation by light of the photoreceptor domain made it possible to activate the recombinase domain. The recombinase activity of this protein is therefore directly dependent on exposure to light. In the absence of light, the fusion protein according to the invention has little or no recombinase activity. On the other hand, the inventors have demonstrated that a short exposure to light of this protein makes it possible to strongly induce this activity.

The fusion protein developed by the inventors thus constitutes a simple and effective genetic tool for carrying out DNA modifications in vivo or in vitro under the impulse of a simple light stimulation.

Thus, according to a first aspect, the present invention relates to a fusion protein comprising a recombinase domain fused to a photoreceptor domain.

In this document, the terms "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of residues of amino acid constituting this chain.

The amino acids are here represented by their one-letter or three-letter code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine (Tyr). The amino acids constituting the fusion protein according to the invention may be of L or D configuration, preferably of L configuration.

As used herein, the term "fusion protein" refers to an artificial protein comprising at least two fused protein domains in a single molecule, it being understood that these two domains are never associated in a natural protein. Each of the domains of this protein may be a natural domain, i.e., a natural protein or a fragment thereof, or a synthetic domain, i.e. a Protein domain does not exist in nature or in a natural protein. The fusion protein is most often the product of a fusion gene in which the coding sequences of the different domains are in phase.

The fusion protein according to the invention may have one or more post-translational modifications and / or one or more chemical modifications such as glycosylations, amidations, acylations, acetylations, ubiquitinations, phosphorylations or methylations. In order to increase the resistance of the fusion protein according to the invention to proteases, especially in the case of in vitro use, protective groups may be added to the C- and / or N-terminal ends. For example, the protecting group at the N-terminus may be acylation or acetylation and the protective group at the C-terminus may be amidation or esterification. The protein according to the invention may also comprise pseudo-peptide bonds replacing the "conventional" CONH peptide bonds and conferring increased resistance to proteases, such as CHOH-CH 2, NHCO, CH 2 -O, CH 2 CH 2, CO-CH 2, NN, CH = CH, CH2NH, and CH2-S.

According to some embodiments, the fusion protein may comprise, in addition to the recombinase domain fused to a photoreceptor domain, an additional N-terminal or C-terminal, preferably C-terminal, peptide domain. This additional domain is preferably fused to the recombinase domain of the fusion protein. The additional domain may be a tag (or tag) useful for purification or immobilization of the fusion protein. Such tags are well known to those skilled in the art and include, for example, the tags histidine (His ₆ ), FLAG, HA (epitope derived from the hemagglutinin of the influenza virus), MYC (epitope derived from the proto-oncoprotein MYC) or GST (glutathione-S-transferase). Optionally, the fusion protein may comprise a protease cleavage site or a chemical agent to suppress this additional domain.

According to preferred embodiments, the fusion protein does not comprise any additional N-terminal sequence or domain of the photoreceptor or C-terminal domain of the recombinase domain.

The recombinase domain of the fusion protein according to the invention may be any protein domain exhibiting recombinase activity. It is understood that the recombinase activity does not require any hetero-dimerization with another domain or another protein. It is intrinsic to the recombinase domain and can easily be tested by any technique known to those skilled in the art (see, for example, one of the techniques described below) applied to the recombinase domain alone (not fused to the photoreceptor domain).

As used herein, the term "recombinase" refers to a site-specific recombinase DNA, i.e. a recombinase DNA that recognizes and binds to specific target regions, or recognition sites, of the DNA and catalyzes a recombination reaction between the nucleic acids of these sites. The recognition sites are generally short (about 30 to 40 nucleotides) sequences that are specific for each recombinase. These enzymes exhibit both an endonuclease activity and a ligase activity and catalyze (i) the deletion of a DNA fragment flanked by two compatible recognition sites and in the same orientation and / or (ii) the inversion of a DNA fragment flanked by two compatible recognition sites and in opposite orientations and / or (iii) the intra- or intermolecular translocation of a DNA fragment flanked from one recognition site to another compatible recognition site . The recombinase activity of a protein can be evaluated by any method known to those skilled in the art, in particular by the method described in the experimental part of this document or by one of the many tests described in the literature such as, for example the appearance or the disappearance of a signal relating the presence or the integrity of a nucleic acid targeted by the recombinase, such as a DNA molecule comprising on the one hand a coding sequence for a reporter protein such as beta -galactosidase, GLP (Green Lluorescent Protein), luciferase or mCherry fluorescent protein, and secondly one or more recombinase recognition sites, and whose recombinase rearrangement affects the expression of the reporter protein ( disappearance or appearance of the signal).

The recombinase domain may be a recombinase protein or a functional variant thereof.

Many recombinases have been described in the literature and are useful in the present invention. Examples of recombinases include, but are not limited to, phage P1 CRE recombinase, Saccharomyces cerevisiae PLP recombinase, Zygosaccharomyces rouxii R recombinase, Salmonella HIN recombinase, Kluyveromyces drosophilarium recombinase A or Kluyveromyces waltii, int integrase, the Mu phage GIN recombinase, the PhiC3l integrase, the Tn3 resolvase, TRE recombinase, DRE recombinase (Anastassiadis et al., Disease Models & Mechanisms 2009 2: 508-515), and prokaryotic beta-recombinase.

Preferably, the recombinase protein is selected from the group consisting of CRE recombinase and FLP recombinase.

The FLP recombinase is an enzyme derived from the Saccharomyces cerevisiae 2-micron plasmid and catalyzes the recombination reaction between two specific FRT sites. Like the LoxP sites, the FRT sites consist of an asymmetric 8 bp sequence framed by two 13 bp palindromic arms. There are many variations / mutants of the FRT and LoxP sites.

More preferably, the recombinase protein is CRE recombinase. The recombinase CRE (Uniprot: P06956, NCBI GenelD: 2777477) is a tyrosine recombinase derived from the bacteriophage P1 whose sequence is presented in SEQ ID NO: 1. As mentioned above, this enzyme catalyzes the recombination reaction between two specific LoxP sites.

The term "functional variant" refers to a protein whose sequence differs from the parent protein by at least one substitution, insertion or deletion but which retains recombinase activity.

Numerous recombinase protein variants have been described in the literature, in particular variants of CRE and FLP recombinases, and are useful in the present invention. These variants may be natural or synthetic and have recombinase activity as defined above. They may, for example, recognize other recognition sites than the wild-type enzyme (see, for example, the article by Santoro and Schultz, Proc Natl Acad Sci US A, 2002 Apr 2, 99 (7): 41 85-90 describing variants of the CRE recombinase), have improved characteristics such as increased thermostability (see, for example, the thermostable variants of the FLP recombinase described in the articles by Buchholz et al., 1998, Nat Biotechnol 16, 657-662 and Raymond and Soriano, 2007, PLoS ONE 2, el62), increased precision (see, for example, the variants of the CRE recombinase described in application WO 2014/158593), improved expression in mammalian cells (see, for example, the variants of CRE recombinase described in US Patent 6,734,295).

Preferably, the functional variants have at least 80% sequence identity with the parent recombinase protein, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the parent recombinase protein.

As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) at the positions arising from an alignment of two polypeptide sequences. Sequence identity is determined by comparing sequences when aligned to maximize overlap and identity while minimizing sequence interruptions. In particular, the sequence identity can be determined using any of the many global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (eg Needleman & Wunsch, J. Mol Biol 48: 443, 1970) that optimally align the sequences over the entire length, while sequences substantially different lengths are preferably aligned using a local alignment algorithm, for example the Smith and Waterman algorithm (Smith and Waterman, Adv Appl Math 2: 482, 1981) or the Altschul algorithm. (Altschul et al (1997) Nucleic Acids Res 25: 3389-3402, Altschul et al (2005) FEBS J. 272: 5101-5109). The alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved by any method known to those skilled in the art, for example by using software available on Internet sites such as http: // blast.ncbi.nlm. nih.gov / or http://www.ebi.ac.uk/Tools/emboss/. Those skilled in the art can readily determine the appropriate parameters for measuring alignment. For purposes of the present invention, amino acid sequence percent identity values refer to values generated using the pairwise sequence alignment program EMBOSS Needle which creates an optimal overall alignment of two sequences using the Needleman-Wunsch algorithm in which the parameters are the default parameters: Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5.

Alternatively, the functional variants of a recombinase differ from the latter by 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 substitutions, insertions and / or deletions of amino acids, preferably 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 substitutions and / or deletions of amino acids. In particular, the functional variants of a recombinase may differ from the latter by 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 at 10 or 1-5 amino acid substitutions, preferably with 1 or 2 amino acid substitutions.

The term "substitution" as used herein with respect to a position or an amino acid means that the amino acid in the particular position has been replaced by another amino acid or amino acid different from that of the wild-type protein is present. The term "substitution" may refer to the replacement of an amino acid residue by another one of the 20 natural standard amino acid residues, the rare natural amino acid residues (eg hydroxyproline, hydroxylysine, allohydroxylysine, 6 N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, omithine) and unnatural amino acids (eg norleucine, norvaline and cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of one amino acid residue with another selected from the 20 standard natural amino acid residues (G, P, A, V, L, I, M, C , F, Y, W, H, K, R, Q, N, E, D, S and T). In this document, the following terminology is used to denote a substitution: D341A indicates that the amino acid residue at position 341 of SEQ ID NO: 1 (aspartic acid, D) is changed to alanine (A).

The substitution (s) may be conservative or non-conservative substitutions. The term "conservative substitution" as used herein refers to a substitution of one amino acid residue for another that has similar chemical or physical properties (size, charge, or polarity). Examples of conservative substitutions are among (i) basic amino acids (arginine, lysine and histidine), (ii) acidic amino acids (glutamic acid and aspartic acid), (iii) polar amino acids (glutamine and asparagine or serine, threonine and tyrosine), (iv) hydrophobic amino acids (methionine, leucine, isoleucine and valine), (v) aromatic amino acids (phenylalanine, tryptophan) and (vi) small amino acids (glycine, alanine).

According to some embodiments, the functional variant is obtained by deleting N-ter and / or C-ter residues of a recombinase to obtain a fragment comprising at least 100, 150, 200, 250 or 300 contiguous amino acids. of said recombinase, while retaining recombinase activity. Preferably, the fragment is obtained by deletion of 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 amino acids in N-terminal and / or C-terminal of a recombinase protein. . The number of amino acids that can be deleted without affecting recombinase activity depends on the nature of the protein and can be readily determined by those skilled in the art using routine techniques.

In a particular embodiment, the recombinase domain is the CRE recombinase or a functional variant thereof.

The functional variant may be any variant of the CRE recombinase known to those skilled in the art and described in the literature such as, for example, the variants described in the article by Santoro and Schultz (Proc Natl Acad Sci US A, 2002 Apr 2; 99 (7): 4185-90), in WO 2014/158593, in US 6,734,295, and in the article by Frank Buchholz and A. Francis Stewart (Nature Biotechnology 2001 19: 1047-1052).

According to one embodiment, the functional variant of the CRE recombinase has a sequence having at least 80% sequence identity with SEQ ID NO: 1, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1.

The inventors have here demonstrated that the CRE recombinase retains its activity after deletion of 31 residues in N-ter and / or 3 residues in C-ter.

Thus, according to a particular embodiment, the functional variant of the CRE recombinase may comprise, or consist of,

the sequence SEQ ID NO: 2 (residues 32 to 340 of SEQ ID NO: 1), or

a sequence having at least 80% sequence identity with SEQ ID NO: 2, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity with SEQ ID NO: 2.

The functional variant of the CRE recombinase may also comprise, or consist of, a CRE recombinase of SEQ ID NO: 1 in which at most 31 successive amino acids have been deleted at the N-terminal and / or at most 3 successive amino acids have been deleted. deleted in C-terminal. In particular, the functional variant of the CRE recombinase may comprise, or consist of, a CRE recombinase of SEQ ID NO: 1 in which 18, 19, 26 or 31 successive amino acids have been deleted at the N-terminal. In certain embodiments, the functional variant of the CRE recombinase may have a substitution at the residue corresponding to the E340 residue of SEQ ID NO: 1 and / or a substitution at the residue corresponding to residue D341 of SEQ ID NO. The residues of the variant corresponding to residues E340 and D341 of SEQ ID NO: 1 are readily identified by those skilled in the art by means of a sequence alignment algorithm.

The substitution residue or residues are independently selected from the group consisting of A, G, V, L, I, P, M, W, C, N, Q, S, T and Y. Preferably, the residue or residues are substituted with an alanine residue.

According to a particular embodiment, the recombinase domain is a functional variant of the CRE recombinase, said variant comprising, or consisting of, a CRE recombinase of SEQ ID NO: 1 in which 18, 19, 26 or 31 amino acids have been deleted. N-terminal, and optionally having a substitution at the residue corresponding to residue E340 of SEQ ID NO: 1 and / or a substitution at the residue corresponding to residue D341 of SEQ ID NO: 1.

Preferably, the recombinase domain is a functional variant of the CRE recombinase, said variant comprising, or consisting of, a CRE recombinase of SEQ ID NO: 1 in which 19 or 31 amino acids have been deleted at the N-terminal, and exhibiting optionally a substitution at the residue corresponding to residue E340 of SEQ ID NO: 1 and / or a substitution at the residue corresponding to residue D341 of SEQ ID NO: 1

According to a preferred embodiment, the recombinase domain is

a functional variant of the CRE recombinase of SEQ ID NO: 1 obtained by deletion of 19 amino acids at the N-terminal and substitution of residues 340 and 341 of SEQ ID NO: 1 by an alanine (SEQ ID NO: 3) , or

a functional variant of the CRE recombinase of SEQ ID NO: 1 obtained by deletion of 31 amino acids at the N-terminal (SEQ ID NO: 4).

The photoreceptor domain of the fusion protein according to the invention is a domain obtained from a photoreceptor protein and which has the property of changing conformation when exposed to light. Preferably, the domain The photoreceptor is sensitive to blue light, a light having a wavelength between 400 and 500 nm.

Photoreceptor proteins are light-sensitive proteins involved in the perception and response to light of a wide variety of organisms. These light sensors have a photoreceptor domain that reacts and triggers a signal transduction cascade. Photoreceptors generally require the presence of co-factors that capture photons, such as flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN).

The photoreceptor domain of the fusion protein according to the invention can be obtained from any photoreceptor protein known to those skilled in the art such as rhodopsin, melanopsin, photopsin, protein kinase C, cryptochrome photolyases 1 and 2 , phototropins, phytochrome, photoconvertible fluorescent proteins, proteins containing a PAS domain (Per-ARNT-Sim) as described in Taylor, IB Zhulin, Microbiol. Mol. Biol. Rev. 63, 479 (1999) or BLUF domain-containing proteins as described in Wu, Q., and Gardner, K.H. (2009) Biochemistry (Mosc.) 48, 2620-2629.

Preferably, the photoreceptor domain of the fusion protein is an LOV (Light Oxygen Voltage) domain. The LOV domains belong to the superfamily of PAS domains and are flavin-dependent photoreceptors.

According to one embodiment, the photoreceptor domain of the fusion protein according to the invention comprises, or consists of, a LOV domain of a phototropin, or a variant or derivative thereof.

Phototropins are plant photoreceptors that have two LOV domains and one Ser / Thr kinase domain. In particular, the photoreceptor domain may be chosen from the LOV domains of Avena Sativa phototropins (UNIPROT Gene ID: 049003), Arabidopsis thaliana (Gene ID: 823721 and 835926), Neurospora crassa (GenBank ID: AAK08514.1), Brucella mellitus or Chlamydomonas reinhardtii (Zoltowski, BD, Vaccaro, B., and Skull, BR (2009) Nat Chem Biol., 5, 827-834).

Preferably, the photoreceptor domain is an LOV domain of phototropin d vena sativa, in particular the LOV1 or LOV2 domain of phototropin 1 d vena sativa, and more particularly preferably the LOV2 domain of phototropin 1 of Avena sativa. Avenu sativa LOV2 domain (asLOV2) consists of 143 amino acids (SEQ ID NO: 14) (residues 404 to 546 of phototropin 1, Genbank ID: AAC05083) and binds to FMN (Flavin MonoNucleotide). Like the other members of the LOV family, the asLOV2 structure reveals a PAS domain consisting of 2 b-strands (A and B) and 3 a-helices (C, D and E), supplemented by a helical connector Fa and 3 sheets b (G, H, I). The 5 strands b form a twisted antiparallel sheet b which with the loop Fa and the propellers allow the insertion of FMN. The structure of this photoreceptor shows a peculiarity at its C-terminal end, a helix Ja of about twenty amino acids as well as an A'a helix at the N-terminus (Halavaty and Moffat, 2007, Biochemistry (Mosc. 46, 14001-14009, Harper, 2003, Science 301, 1541-1544, Zayner et al., 2012, Mol Mol Biol 419, 61-74). Under the effect of blue light, the asLOV2 photoreceptor undergoes a succession of reactions resulting in a conformational change of the protein and a release and a relaxation of the helix Ja. When the domain is integrated in phototropin (its natural context), this modification triggers the kinase activity of phototropin.

The photoreceptor domain may also be a functional variant of an LOV phototropin domain, particularly an asLOV2 domain variant. The variant retains the capacity of the LOV domain to change conformation in response to light stimulation and has at least 80% sequence identity with the parent LOV domain, more preferably at least 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the parent LOV domain. The ability of the variant to change conformation in response to light stimulation can be easily tested by any technique known to those skilled in the art such as, for example, the asLOV2-SsrA binding assay to SspB described in the Lungu et al. 2012, Chem. Biol. 19, 507-517.

According to a preferred embodiment, the photoreceptor domain is a derivative of a LOV domain, preferably a derivative of the asLOV2 domain. Compared with parent LOV domains, these derivatives generally exhibit increased light reactivity, increased expression by the host cell, or different stability.

Numerous LOV domain derivatives, and in particular the asLOV2 domain, have been described in the literature. Such derivatives include, but are not limited to, the LOV-SsrA domain in which the bacterial SsrA peptide has been incorporated into the C-terminal helix of asLOV2 (Lungu et al., 2012, Chem Biol., 19, 507-517). ), the iLID domain (Improved Light Induced Dimer) (Guntas et al., 2015, Proc Natl Acad Sci 112, 112-117), mutants G528A and N358E as described in Strickland et al. Nature Methods (2010), 7 (8) 623-626, and the mutants described in the article by Kawano et al. PLoS One 2013 8 (12): e82693 such as V416T or V416L.

In a particular embodiment, the photoreceptor domain of the fusion protein is an iLID domain comprising, or consisting of, the following sequence: : 13), wherein X represents one or more amino acids.

X can be any amino acid sequence adopting an alpha helical conformation. Preferably, X represents one to 10 amino acids, more preferably one to 9, 8, 7, 6, 5 or 4 amino acids, and more preferably one to three amino acids.

According to certain particular embodiments, X is chosen from the group consisting of

an amino acid chosen from A, D, K and L,

a sequence of two amino acids chosen from IL, VV and AI, and

- the QPG sequence.

According to some preferred embodiments, X is selected from the group consisting of IL and AI.

The photoreceptor domain of the fusion protein may be fused to the N-terminal or C-terminal domain of the recombinase domain.

Preferably, the photoreceptor domain of the fusion protein is fused to the N-terminal of the recombinase domain.

The two domains can be immediately adjacent or separated by one or more link sequences. Preferably, the domains are separated by up to 10 amino acids, preferably at most 6, 5, 4, 3, 2 or 1 amino acids.

According to preferred embodiments, the binding sequence (s) between the two domains do not comprise a cleavage sequence recognized by a protease, in particular of a sequence recognized by a TEV protease. More particularly preferably, the two domains are directly adjacent, that is to say without a link sequence between them.

According to a particular embodiment, the fusion protein according to the invention comprises, or consists of, a sequence chosen from the sequences SEQ ID NO: 5 to 12.

The fusion protein according to the invention has a photoactivatable recombinase activity. This means that activation of the photoreceptor domain induces activation or enhancement of recombinase domain activity.

Activation of the photoreceptor domain is achieved by exposure to a light whose wavelength may vary depending on the nature of the photoreceptor. As mentioned above, the photoreceptor domain is preferably sensitive to blue light, ie a light having a wavelength of between 400 nm and 500 nm, preferably a light having a wavelength of between 440 nm and 480 nm.

The duration of exposure to light and the light intensity can be easily determined by those skilled in the art.

The fusion protein can be exposed to a light intensity of between 0.05 mW / cm ² and 50 mW / cm ² , preferably between 1.5 mW / cm ² and 40 mW / cm ² , and more particularly preferably between 15 mW / cm ² and 40 mW / cm ² .

According to one embodiment, the fusion protein is exposed to a luminous intensity of between 2 mW / cm ² and 40 mW / cm ² , preferably between 2 mW / cm ² and 10 mW / cm ² .

Preferably, the duration of exposure to light is between 2 minutes and three hours, preferably between 10 minutes and three hours, more preferably between 30 minutes and two hours. Most preferably, the exposure time is between 30 minutes and 90 minutes.

According to a particular embodiment, the recombinase activity of the fusion protein is induced by an exposure of 10 minutes to three hours, preferably from 30 minutes to two hours, to a blue light with an intensity of between 2 mW / cm. ² and 40 mW / cm ² , preferably between 2 mW / cm ² and 10 mW / cm ² .

According to another particular embodiment, the recombinase activity of the fusion protein is induced by an exposure of 2 minutes to two hours, preferably from 30 minutes to 90 minutes, to a blue light of an intensity between 1.5 mW / cm ² and 40 mW / cm ² , preferably between 15 mW / cm ² and 40 mW / cm ² .

As used herein, the term "photoactivatable" preferably means that exposing the fusion protein according to the invention to light achieves a minimum 15% increase in recombinase activity, preferably an increase in at least 25% of the recombinase activity, and more preferably at least 30%, 40% or 50% increase in recombinase activity, relative to the level without light exposure. More preferably, the exposure of the fusion protein according to the invention to a blue light of an intensity of about 2 mW / cm ² for 90 min allows to obtain an increase of at least 50% of recombinase activity, relative to the level without light exposure. Alternatively, the exposure of the fusion protein according to the invention to a blue light of an intensity of between 30 mW / cm ² and 40 mW / cm ² for 30 min makes it possible to obtain an increase of at least 50% of recombinase activity, relative to the level without light exposure.

The present invention also relates to a solid support on which is immobilized a fusion protein according to the invention. It also relates to a process for preparing such a solid support comprising providing a fusion protein according to the invention and a suitable solid support, and immobilizing said protein on said support.

The immobilization means are well known to those skilled in the art (see, for example, "Enzyme Technology" by Martin Chaplin and Christopher Bucke, Cambridge University Press, 1990). The fusion protein according to the invention may be immobilized on the solid support by any suitable means such as a covalent or non-covalent bond. A wide variety of materials can be used as an immobilizer. These materials are generally inert polymeric or inorganic matrices. The support may for example be membrane, particulate (e.g., beads, nanobeads, microbeads) or fibrous. Examples of carriers include, but are not limited to, polyurethane matrices, activated sepharose, alginate, Amberlite, Sephadex or Duolite resins, acrylic resins, polystyrene resins or macroreticulated resins.

The present invention also relates to a nucleic acid encoding a fusion protein according to the invention. The nucleic acid can be DNA (cDNA or gDNA), RNA or a mixture of both. It may be in single-stranded or duplex form or a mixture of both. It may comprise modified nucleotides, comprising, for example, a modified linkage, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to those skilled in the art, such as chemical synthesis, recombination and mutagenesis. The nucleic acid according to the invention can be deduced from the sequence of the fusion protein according to the invention and the use of the codons can be adapted according to the host cell in which the nucleic acid must be transcribed and / or translated. These steps can be performed according to methods well known to those skilled in the art and some of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Third Edition).

The present invention further relates to an expression cassette comprising a nucleic acid according to the invention,

The term "expression cassette" herein refers to a nucleic acid construct comprising a coding region comprising a nucleic acid according to the invention and one or more regulatory sequences, the coding region and the regulatory sequence (s) being operably linked.

The term "operably linked" indicates that the elements are combined so that expression of the coding region is under the control of the regulatory sequence (s).

The term "regulatory sequence" refers to a nucleic acid sequence necessary to express or control the expression of the coding region. Regulatory sequences may be endogenous or heterologous to the host cell. Such sequences are well known to a person skilled in the art who can easily select the regulatory sequences adapted to each application and in particular to the host cell in which the expression is desired. Without being limited thereto, these sequences may comprise in particular a promoter, a polyadenylation sequence, a transcription terminator, a 3'UTR region, a 5'UTR region and a signal peptide.

Preferably, the expression cassette according to the invention comprises, or consists of, a promoter making it possible to initiate transcription, a nucleic acid according to the invention, and a transcription terminator. Typically, the promoter sequence is placed upstream of the coding region and at a distance compatible with the control of the expression of said region.

The promoter contains transcriptional control sequences that initiate expression of the nucleic acid encoding a fusion protein of the invention. It may be a promoter recognized by a host cell or an in vitro expression system. The promoter may be any polynucleotide exhibiting transcriptional activity under the envisaged conditions of use, ie in vitro or in vivo, and may in particular be a promoter obtained from genes coding for endogenous or heterologous polypeptides at the host cell, or a mutant, truncated or hybrid promoter. The promoter may be a strong, weak, constitutive or inducible promoter. On the basis of his knowledge, a person skilled in the art can easily choose the adapted promoter.

The transcription terminator is recognized by a host cell to complete the transcription. The terminator is functionally linked to the 3 'end of the nucleic acid according to the invention. Any terminator that is functional in the host cell can be used in the present invention.

The present invention also relates to an expression vector comprising a nucleic acid or an expression cassette according to the invention. This expression vector can be used to transform a host cell and allow the expression of the nucleic acid according to the invention in said cell.

The vector may be a DNA or an RNA, circular or not, single- or double-stranded. It is advantageously chosen from a plasmid, a phage, a phagemid, a virus, a cosmid and an artificial chromosome.

In general, the vector is chosen to be compatible with the host cell into which it is to be introduced. The vector may be an autonomously replicating vector, i.e., an existing vector as an extra chromosomal entity whose replication is independent of chromosomal replication, or a vector which, when introduced into the host cell is integrated into the genome and replicated with the chromosome in which it has been integrated.

Advantageously, the expression vector comprises regulatory elements allowing the expression of the nucleic acid according to the invention. These elements may comprise, for example, transcription promoters, transcription activators, terminator sequences, initiation and termination codons. Methods for selecting these elements according to the host cell in which the expression is desired, are well known to those skilled in the art.

The vector may further comprise elements allowing its selection in the host cell, such as, for example, an antibiotic resistance gene or a selection gene ensuring the complementation of the respective gene deleted in the genome of the host cell. Such elements are well known to those skilled in the art and widely described in the literature.

In some embodiments, the vector comprises one or more elements for integrating the vector into the genome of the host cell. This integration may be based on homologous or non-homologous recombination. In the case of targeted integration, the vector may contain sequences homologous to a target region of the genome and which make it possible to target integration by homologous recombination at a specific location in the genome of the host cell.

When autonomous replication of the vector is desired, the vector may comprise a replication origin functional in the host cell.

The methods for selecting these different elements depending on the vector and the host cell in which the expression is desired are well known to those skilled in the art. The vectors according to the invention can be constructed by standard molecular biology techniques.

The present invention relates to the use of a nucleic acid, an expression cassette or an expression vector according to the invention to transform or transfect a cell. The host cell may be transiently / stably transfected / transfected and the nucleic acid, cassette or vector may be contained in the cell as an episome or chromosomal form.

The present invention also relates to a host cell comprising a nucleic acid, a cassette or an expression vector according to the invention.

The host cell may be a prokaryotic cell or a eukaryotic cell.

According to one embodiment, the host cell is a prokaryotic cell, preferably a bacterium.

According to another embodiment, the host cell is a eukaryotic cell, preferably a yeast, a mushroom cell, an algal cell, a cell of plant or animal cell, and more preferably an animal cell or yeast.

According to a particular embodiment, the host cell is an animal cell, preferably an insect or mammalian cell, and more preferably a mouse cell, a hamster cell (for example a CHO cell) or a human cell.

In a particular embodiment, the cell is non-human and / or is not an embryonic stem cell.

According to another particular embodiment, the host cell is a non-human mammalian cell.

According to another particular embodiment, the host cell is an animal cell, preferably human, and is not a human embryonic stem cell.

According to another particular embodiment, the host cell is a microorganism, preferably a bacterium or a yeast.

The term "host cell" also includes any offspring of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication.

The nucleic acid, expression cassette or expression vector according to the invention may be introduced into the host cell by any method known to those skilled in the art, such as infection, electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistics or transformation methods involving PEG, lipid agents, lithium acetate or liposomes.

According to the embodiments, one or more copies of a nucleic acid, a cassette or a vector according to the present invention may be inserted into a host cell.

The host cell may be an isolated cell (transformation / transfection ex vivo) or a cell present in an organism (transformation / transfection in vivo).

Preferably, the host cell is an isolated cell. As used herein, the term "isolated cell" refers to a separate cell and / or recovered from its natural environment. In particular, the cell can be isolated / recovered from a multicellular organism to be manipulated ex vivo. In another aspect, the present invention relates to a method of producing a fusion protein according to the invention comprising expressing a nucleic acid encoding said fusion protein and recovering it.

The present invention relates in particular to a process for the in vitro production of a fusion protein according to the invention comprising (a) bringing a nucleic acid, a cassette or a vector according to the invention into contact with an in vitro expression system, and (b) the recovery of the fusion protein. In vitro expression systems are well known to those skilled in the art and many systems are commercially available.

The present invention preferably relates to a method for producing a fusion protein according to the invention comprising

(a) culturing a host cell according to the invention in a suitable culture medium and under conditions suitable for producing said fusion protein; and

(b) recovering the fusion protein from the cell culture.

The host cells are cultured in a nutrient medium suitable for the production of the fusion protein using methods well known to those skilled in the art.

For example, the host cell may be cultured in a vial or fermentor in a suitable medium and under conditions allowing the fusion protein to be expressed and / or isolated. The culture is carried out in a suitable nutrient medium comprising in particular carbon, nitrogen and inorganic salt sources, using procedures well known to those skilled in the art. Such media are available from commercial suppliers or can be prepared according to published compositions (for example, in the catalogs of the American Type Culture Collection).

If the fusion protein is secreted into the nutrient medium, it can be recovered directly from the culture supernatant. If it is not secreted, it can be recovered from cell lysates or after permeabilization.

The fusion protein can be detected and recovered using any method known to those skilled in the art such as chromatography methods and / or electrophoretic methods. In another aspect, the present invention also relates to a method, preferably in vitro or ex vivo, for stimulating recombinase activity in a cell comprising

providing a cell expressing a fusion protein according to the invention or inserting a nucleic acid, an expression cassette or an expression vector according to the invention into a cell, and

The cell may be a host cell as defined above.

The insertion of the nucleic acid, the cassette or the vector according to the invention into the cell can be done by any technique known to those skilled in the art.

The cell may possibly have been previously genetically modified to insert one or more recognition sites recognized by the recombinase domain of the fusion protein, and optionally one or more nucleic acids whose expression, number and / or location in the genome will be modified by the action of the recombinase at said recognition sites.

In particular, the cell may be a microorganism, for example a bacterium or a yeast, in which one or more genes of interest have been introduced (for example genes coding for enzymes giving rise to the production of a compound of interest) in combination with recognition sites recognized by the recombinase domain of the fusion protein. The exposure of this cell to a light capable of activating the photoreceptor domain of the fusion protein generates a chromosomal rearrangement which, according to the configuration of the recognition sites of the recombinase, makes it possible, for example, to activate or inhibit the expression of the gene (s) of interest, and / or deleting, inserting or translocating one or more genes of interest of the host cell.

This type of modulation is well known to those skilled in the art. In contrast, the major advantage of the method according to the invention is to use light as an induction agent for recombinase activity and not a chemical inducer.

The present invention also relates to a method, preferably in vitro or ex vivo, for modifying one or more nucleic acids, comprising contacting a fusion protein according to the invention with one or more nucleic acids of interest comprising one or more recognition sites recognized by the recombinase domain of said fusion protein, and

exposing the fusion protein and the nucleic acid (s) to a light capable of activating the photoreceptor domain of said fusion protein and therefore the recombinase activity.

The modifications made to the nucleic acids can be translocations, inversions, insertions and / or deletions. These may in particular lead to inducing or blocking the expression of a gene, or to introducing a mutation in a coding sequence (wild-type sequence to mutated sequence or vice versa). This type of recombinase modification is well known to those skilled in the art.

The contacting of the fusion protein with the nucleic acid (s) of interest can take place in a cell, in vivo or ex vivo, or in vitro.

In the case of contacting in a cell, the fusion protein is preferably expressed by said cell which also comprises the nucleic acid (s) to be modified.

In the context of an in vivo use, the fusion protein according to the invention can be brought into contact with the nucleic acid (s) to be modified by administering either a vector or an expression cassette coding for said protein, or directly protein in the body, tissue or target cell. The activation of the recombinase activity can be carried out by phototherapy, photochemotherapy or via light sources offering spatial precision such as optical fibers. This type of application is of particular interest in gene therapy when it is necessary to modify the activity or viability of a cell or a group of cells involved in a pathology or in its evolution (see for example Greco and Gene Therapy Volume 13, pages 206-215 (2006)).

In the case of in vitro contacting, the fusion protein is preferably contacted with a reaction mixture comprising the nucleic acid (s) of interest in the form of a purified protein. The fusion protein can be used in solution or in immobilized form. Preferably, the reaction mixture also comprises an in vitro transcription and translation system.

According to one embodiment, this method is used to generate genetic diversity within a cell or a population of cells, preferably a population of bacteria or yeasts, or in a nucleic acid or mixture of nucleic acids. Optionally, the genome of the cell or the nucleic acid may be previously synthesized or modified to comprise a plurality of recognition sites inserted in a targeted or random manner and thus generating, under the action of the recombinase, a controlled genetic diversity or random.

The methods according to the invention can find application in a very large number of fields, in particular

to modulate the expression of a molecule of interest, for example in the context of a biomanufacturing process. The expression of the molecule of interest can in particular be induced, stopped, increased or decreased. The compound of interest may be in particular any fermentation product, a compound that changes the physiology or viability of the host cell or cells in direct or indirect contact therewith, a compound controlling a molecular or cellular property of said host cell or cells in direct or indirect contact with it. The compound of interest may also be a protein or nucleic acid having a mutation with respect to the version produced by the host cell before the action of the recombinase;

to induce genetic diversity in a host cell or a population of host cells, for example for directed evolution or screening applications. The cells thus obtained can then be screened to identify or select cells with new characteristics of interest, such as, for example, synthesis of a new compound, increased resistance to particular culture conditions or productivity. increased; or

to modify a biological activity in the context of a gene therapy, for example as described above.

According to another aspect, the present invention also relates to a kit comprising a fusion protein, a nucleic acid, a cassette or an expression vector according to the invention and / or a host cell according to the invention, and optionally a light source. capable of activating the photoreceptor domain of the fusion protein.

It also relates to the use of this kit for implementing a method according to the invention, in particular for inducing a recombinase activity in a cell and / or for modifying in vivo, ex vivo or in vitro one or more nucleic acids. All references cited in this specification are incorporated by reference in this application. Other features and advantages of the invention will appear better on reading the following examples given for illustrative and non-limiting.

EXAMPLES

Material and methods

Reporter system for quantifying CRE recombinase activity in vivo.

The reporter system is illustrated in Figure 1. This system is integrated with the HO locus of the yeast cell genome Saccharomyces cerevisiae. It consists of the following consecutive sequence elements:

PromTEF: promoter conferring constitutive expression (comes from the TEF1 gene of Saccharomyces cerevisiae).

- LoxP: recognition site of CRE recombinase.

-K1LEU2: coding sequence of the K1LEU2 gene of the yeast Kluyveromyces lactis, encoding a beta-isopropylmalate dehydrogenase.

-TermADH1: transcription terminator sequence from the ADH1 gene (Alcohol DeHydrogenase 1) of Saccharomyces cerevisiae. This sequence includes one or more STOP codons which also provide termination of the translation.

-LoxP: recognition site of CRE recombinase.

- GFP: coding sequence of the Green Fluorescent Protein

- TermCYCl: transcription terminator sequence from the CYC1 gene of Saccharomyces cerevisiae.

In the absence of CRE recombinase activity, cells carrying this construct are not fluorescent. In the presence of CRE recombinase activity, the recombination between the two loxP sites generates the excision of the KlLEU2-TermADH1 portion, and thus places the GFP portion under the transcriptional activity conferred by the PromTEF portion. Cells that have undergone this excision thus express the GFP protein and are fluorescent. Experimental conditions of CRE recombinase activity test.

Yeast cells corresponding to strain GY1761 of genotype MATalpha his3A200 leu2A1 trplA63 ura3A0 hoA :: loxKlLEU2loxGFP and carrying the reporter system described above are transformed with a plasmid carrying an expression cassette of a candidate protein likely to have an CRE recombinase activity. The yeast strain thus transformed is cultured overnight on a selective medium under conditions where the candidate protein is expressed. This medium is called preculture medium below. The next day, 100 ml of this culture are transferred to a flat-bottomed transparent plastic 96-well plate and, where appropriate, illuminated at room temperature under the desired conditions of time, wavelength and light energy. Part of the cells of this sample are then cultured for 4 hours in a medium corresponding to conditions where the candidate protein is not expressed. This medium is called the recovery medium below. A sample of these cells is then transferred to a medium called analysis medium below and analyzed at a rate of 400 events per second on a LACSCalibur flow cytometer (BDBiosciences) in which the GLP was excited with a 488 laser. nm and the fluorescence emitted through a 530/30 filter was quantified to produce data such as those shown in Figure 2A. Depending on the fluorescence of the cells, they are classified into two groups: OLE (low fluorescence) and ON (high fluorescence), which correspond to cells that do not have (OLE) or that have (ON) activated the reporter system. . The proportion of ON cells is used as an indicator of CRE recombinase activity, as shown in Figure 2B.

Media and buffers used

The TBS buffer is a tenth dilution in water of the TBS10X buffer, which corresponds to an aqueous solution (Tris 30 g / L, NaCl 80 g / L, KCI 2 g / L) adjusted to pH 7.4 with water. fuming hydrochloric acid.

The yeast cell culture media are prepared from a powdery mixture of amino acids and nucleotides called MixW whose composition is as follows: Adenine (Ad) 1 g, Uracil (U) 2 g, Alanine (A) 2 g, Arginine (R) 2 g, Aspartate (D) 2 g, Asparagine (N) 2 g, Cysteine (C) 2 g, Glutamate (E) 2 g, Glutamine (Q) 2 g, Glycine (G) 2 g, Histidine (H) 2 g, Isoleucine (I) 2 g, Leucine (L) 4 g, Lysine (K) 2 g, Methionine (M) 2 g, Phenylalanine (F) 2 g, Proline (P) 2 g, Serine (S) 2 g, Threonine (T) 2 g, Tyrosine (Y) 2 g, Valine (V) 2 g. Other mixtures, called MixW X where X denotes one of the compounds of the mixture, are prepared in the same manner but omitting the compound X. Thus, for example, the mixture MixW M does not contain methionine. Similarly, other mixtures called MixW XY are prepared by omitting two compounds designated X and Y.

IP Protocol:

The procedure described in the section 'Experimental conditions of the CRE recombinase activity test' above is applied, with the following media:

- Preculture medium: 6.7 g Yeast Nitrogen Base without Amino-Acids (Difco, BDBiosciences), 20 g of galactose, 20 g of raffinose, and 2 g of MixW described above diluted in 1L of distilled water, 1 mL of 1M NaOH is added and the solution is autoclaved at 0.5 bar.

Recovery medium: 6.7 g of Yeast Nitrogen Base without Amino Acids (Difco, BD Biosciences), 20 g of glucose, and 2 g of MixW described above diluted in 1 L of distilled water, 1 ml of 1 M NaOH is added and the solution is autoclaved at 0.5 bar.

- Analytical medium: TBS buffer to which is added 1 mM sodium azide.

P2 protocol:

- Preculture medium: 6.7 g of Yeast Nitrogen Base without Amino Acids (Difco, BDBiosciences), 20 g of glucose, and 2 g of MixW M described above diluted in 1L of distilled water, 1 ml of 1M NaOH is added and the solution is autoclaved at 0.5 bar.

Recovery medium: 6.7 g of Yeast Nitrogen Base without Amino Acids (Difco, BD Biosciences), 20 g of glucose, and 2 g of MixW H described above, diluted in 1 L of distilled water, 1 ml of 1 M NaOH. added and the solution is autoclaved at 0.5 bar.

- Analytical medium: TBS buffer. Protocol P3:

- Preculture medium: 6.7 g Yeast Nitrogen Base without Amino-Acids (Difco, BDBiosciences), 20 g glucose, and 2 g MixW M U described above diluted in

1L of distilled water, 1mL of 1M NaOH is added and the solution is autoclaved at 0.5 bar.

Recovery medium: 6.7 g of Yeast Nitrogen Base without Amino Acids (Difco, BD Biosciences), 20 g of glucose, and 2 g of MixW HU described above, diluted in 1 ml of distilled water, 1 ml of 1 M NaOH. added and the solution is autoclaved at 0.5 bar. - Analytical medium: TBS buffer.

Results

Effects of mutations of the C-terminal end of CRE recombinase

The Cre_Stop341 mutant was obtained by site-directed mutagenesis of a plasmid where codon D341 was replaced by a stop codon. The Cre_A340A341 mutant was obtained by site-directed mutagenesis of a plasmid where codons E340 and D341 were replaced by A340 and A341. In the plasmids thus obtained, the expression of the wild-type or mutant recombinase protein is placed under the control of the promoter of the GAL1 gene of Saccharomyces cerevisiae. The test conditions are those of the protocol Pl without illumination.

The results showing the CRE recombinase activity of these mutants are shown in FIG. 3. The Cre_Stop341 mutant mutant exhibits complete activity. This demonstrates that CRE recombinase deleted from 3 C-terminal amino acids is active. On the other hand, the results show that the Cre_A340A341 mutant has a slightly reduced activity compared to the wild-type CRE recombinase. Effects of mutations of the N-terminus of CRE recombinase

The mutants Cre_A2-21 and Cre_A2-28 were obtained by

i) PCR where the DNA template was the plasmid pGY 115 and where the primers were 1L80 whose sequence is 5'-tggtatctttatagtcctgtcgg-3 '(SEQ ID NO: 15) and, for Cre_A2-1L71 whose sequence is 5 '-gaacatgtccatcaggttcttgcgaacctccatttaacactcagataa-3' (SEQ ID NO: 16), and for Cre_A2-28 1L72 whose sequence is 5'-agaaaacgcctggcgatccctgaacatgtccatttaacactcagataa-3 '(SEQ ID NO: 17).

ii) digestion of the plasmid pGY155 with the Agel enzyme and purification on agarose gel 1% of the 6.3 Kb fragment

iii) co-transformation of the products of steps i) and ii) into a trplA yeast strain and selection on medium without tryptophan.

The denomination Dc-y here indicates the deletion of the amino acids located from the included position x to the included position y.

In the plasmids thus obtained, the expression of the wild-type or mutant recombinase protein is placed under the control of the promoter of the GAL1 gene of Saccharomyces cerevisiae. The test conditions are those of the protocol Pl without illumination. The results showing the activity of these mutants are shown in FIG. 4. The CRE activity of the Cre_A2-21 mutant is complete while that of the Cre_A2-28 mutant is reduced by less than 10%.

LOV2-CRE merger construction

The iLID photoreceptor derived from asLOV2 and described in the article by Guntas et al. 2015 P.N.A.S. 112 (1): 112-117 was fused to the N-terminal of several variants of the CRE recombinase protein. The method used is shown schematically in FIG. 5. It was transferred, by spontaneous homologous recombination into yeast cells, a DNA fragment amplified by PCR and containing the CRE variant sequence as well as, optionally, certain sequence modifications. N-terminal of said variant, in a plasmid containing an expression system and the coding sequence of iLID.

In particular, the iLID-Crel9 construct (fusion of the iLID domain to the residue at position 19 of SEQ ID NO: 1) was obtained by i) PCR where the DNA template was the plasmid pGY155 and where the primers were 1G42 with the sequence is 5'-AGAAAAGGAGAGGGCC AAGA-3 '(SEQ ID NO: 18) and 1M43 whose sequence is 5'-CTTATTAAGAAAACCGCATTTCAAATCgccacgagtgatgaggttcgcaag-3' (SEQ ID NO: 19), ii) digesting the plasmid pGY408 with the enzymes Mfel and BsiWI and agarose gel purification 1% of the fragment of 4.4Kb, iii) co-transformation of the products of steps i) and ii) in yeast strain GY 1761 and selection on medium without tryptophan. Similarly, the construct iLID-Cre27 (fusion of the iLID domain to the residue at position 27 of SEQ ID NO: 1) was obtained by applying steps i) to iii) above with the following modification of step i ): use of the 1M51 primer whose sequence is 5'-CTTATTAAGAAAACCGCATTT CAAATCgccctgatggacatgttcagggat-3 '(SEQ ID NO: 20) in place of the 1M43 primer.

Similarly, the construct iLID-Cre32 (fusion of the iLID domain to the residue at position 32 of SEQ ID NO: 1) contained in the plasmid pGY415 was obtained by applying steps i) to iii) above with the following modification. of step i): use of the 1M53 primer whose sequence is 5'-CTTATTAAGAAAACCGCA TTTCAAATCgccagggatcgccaggcgttttctg-3 '(SEQ ID NO: 21) in place of the 1M43 primer.

Similarly, construct iLID-CreAA20 (fusion of the iLID domain to the residue at position 20 of SEQ ID NO: 1 with the double mutation A340A341) contained in plasmid pGY416 was obtained by applying steps i) to v) above. above with the following modifications of step i): use of the 1M44 primer whose sequence is 5'-CTTATTAAG AAAACCGCATTTCAAATCgccagtgatgaggttcgcaagaac-3 '(SEQ ID NO: 22) in place of the 1M43 primer and use of the template pGY372 instead of the pGY15 matrix.

In the constructs thus obtained, the expression of the fusion protein is placed under the control of the promoter of the GAL1 gene of Saccharomyces cerevisiae.

Demonstration of the photoactivation of these fusion proteins.

The activity of the fusion proteins was evaluated by applying or not an illumination of the cells according to the P1 protocol. The results obtained are presented in FIG. 6. The iLID-Crel9 fusion exhibits an activity of 25% without illumination and 32% after illumination, the iLID-Cre27 fusion exhibits 50% activity without illumination and 60% after illumination, the iLID-Cre32 fusion exhibits 15% activity without illumination and 45% after illumination, and the iLID-CreAA20 fusion exhibits zero activity without illumination and 18% after illumination. Obtaining and building activities iLID (OJ) -x _n -Cre32

A series of mutants denoted iLID (QI) -xi-Cre32 was generated to introduce a random amino acid at the junction between iLID and Cre32. This series was obtained by PCR amplification of the sequence comprising a portion of CRE recombinase using the 1F14 antisense primer of the 5'-gtgatgacggtgaaaacctc-3 'sequence (SEQ ID NO: 23) and the sequence 1N24 sense primer. "-CTT ATT AAG AAA ACC GCA TT CAA ATC-VNN-agg gat cgc cag gcgttt tct g-3 '(SEQ ID NO: 24) where V is at random A, C or G, and N is at random A, T, C or G. The amplicon obtained was inserted by homologous recombination into the 4.4 kb vector resulting from the NcoI-BsiWI digestion of the plasmid pGY417.

Another series of mutants denoted iLID (QI) -X ₂ -Cre32 was similarly generated using the 1N25 sequence primer 5'-CTT ATT AAG AAA ACC GCA TTT CAA ATC-VNN VNN-agg gat cgc cag gcg ttt tct g-3 '(SEQ ID NO: 25) instead of primer 1N24.

Another series of mutants denoted iLID (QI) -X ₃ -Cre32 was generated in a similar manner using the 1N26 primer of 5'-CTT sequence ATT AAG AAA ACC GCA TTT CAA ATC-VNN VNN VNN-agg gat cgc cag gcg ttt tct g-3 '(SEQ ID NO: 26) instead of primer 1N24. In the constructs thus obtained, the expression of the fusion protein is placed under the control of the promoter of the GAL1 gene of Saccharomyces cerevisiae.

The activity of the fusion proteins was evaluated by applying or not an illumination according to the protocol Pl. FIG. 7 presents the activities without or with illumination for several constructions iLID (QI) -x _n -Cre32 thus generated. These constructions show a gain in activity after illumination.

Activity under different conditions of illumination

Illumination conditions of different times and intensities were applied to yeast cells containing the construct iLID_Cre32 or the construct iLID_CreAA20, placed in two types of expression cassette. The first cassette comprises the promoter of the GAL1 gene. The results obtained with this cassette and according to the protocol P1, by applying or not an illumination of the cells with varying intensities and durations, are presented in FIG. comprises the promoter of the MET17 gene. The results obtained with this cassette and according to the P2 protocol, by applying or not an illumination of the cells according to varying intensities and durations, are presented in FIGS. 8 and 9. These results show that the recombinase activity of the constructs is activatable in very different conditions of time and intensity.

Addition of an addressing peptide to the nucleus.

An NLS_iLID_CreAA20 construct was studied. This construction corresponds to the fusion at the N-terminus of iLID_CreAA20 of a peptide whose sequence VPKKKRKV (SEQ ID NO: 27) (Nuclear Localization Signal, NLS) is known to address proteins to the nucleus of the cells, as indicated in the article by Cheng et al. Endocrinology 157: 127-140, 2016, followed by the sequence GGSGG (SEQ ID NO: 28). This construct was obtained by performing PCR amplification with 1080 primers (5'-atgtgagttacctcactcat-3 '(SEQ ID NO: 29)) and 1084 (5'-GCTAAAGAACCGTGATGGTGGTGATGATGTGAACCTCTCATACCTCCGGAac caccgacttttctcttct-3' (SEQ ID NO: 30)) on the plasmid pGY491 and by co-transforming the product of said amplification with the SacI-EcoRI fragment of the plasmid pGY466 in a yeast strain. In the constructs thus obtained, the expression of the fusion protein is placed under the control of the promoter of the Saccharomyces cerevisiae MET 17 gene. The activity of this construction was evaluated by applying the P2 protocol. The activity of this construction is presented in the results of Figure 10.

Comparative test with the nMag / pMag-CRE system

An activatable recombinase system based on the photoinducible dimerization of peptides called nMag and pMag has been described in the article by Kawano et al. Nat. Chem. Biol. 2016 12 (12): 1059-1064. This system has two distinct fusion proteins that must be expressed in the same cell. In order to compare the performances of this system with those of the present invention, two plasmid constructs were made: pMet17-Crel8to59-nMag-NLS-URA (SEQ ID NO: 31) and pMet17-ATG-NLS-pMag-Cre60to343-TRP (SEQ ID NO: 32). In these constructs, the expression of each of the fusion proteins is placed under the control of the promoter of the MET gene. of Saccharomyces cerevisiae. The combination of these two plasmids is called nMag / pMag-CRE hereinafter.

The activity of this system was tested in comparison with the present invention according to the P3 protocol and the results obtained are shown in FIG. 11. These results confirm that the nMag / pMag-CRE system is photoactivatable (10% activity without illumination). and 35% after illumination). They also show that this system is less efficient than the system of the present invention, in particular that the mergers iLID_Cre32 and iLID_CreAA20: these have an activity differential related to the illumination which is higher.

Claims

A fusion protein comprising a recombinase domain fused to a photoreceptor domain and wherein the recombinase activity is photoactivatable.

The fusion protein of claim 1, wherein the recombinase domain is fused to the photoreceptor domain at the N-terminus.

A fusion protein according to any one of the preceding claims, wherein the photoreceptor domain comprises, or consists of, a LOV domain, preferably LOV2, of phototropin, or a variant or derivative thereof.

A fusion protein according to any one of the preceding claims, wherein the photoreceptor domain is a derivative of a LOV2 domain, preferably an iLID domain (improved light inductive dimer), and more preferably a derived iLID domain. of the LOV2 domain of phototropin 1 from Aven a sativa.

A fusion protein according to any one of the preceding claims, wherein the photoreceptor domain of the fusion protein is an iLID domain comprising, or consisting of, the sequence SEQ ID NO: 13 wherein X is one to three amino acids preferably X is A, D, K, L, IL, VV, AI or QPG.

A fusion protein according to any one of the preceding claims, wherein the photoreceptor domain of the fusion protein is an iLID domain comprising, or consisting of, the sequence SEQ ID NO: 13 wherein X is IL or AI.

A fusion protein according to any one of the preceding claims, wherein the recombinase domain is selected from the group consisting of CRE recombinase (SEQ ID NO: 1), and functional variants thereof, preferably variants functional groups having at least 80% sequence identity with SEQ ID NO: 1.

A fusion protein according to any one of the preceding claims, wherein the recombinase domain is a functional variant of the CRE recombinase comprising, or consisting of, the sequence SEQ ID NO: 2, or a sequence having at least 80% d sequence identity with SEQ ID NO: 2.

A fusion protein according to any one of the preceding claims, wherein the recombinase domain is a functional variant of the CRE recombinase, said variant comprising, or consisting of, a CRE recombinase of SEQ ID NO: 1 in which at most 31 amino acids were deleted at the N-terminus and / or at most 3 amino acids were deleted at the C-terminal.

A fusion protein according to any one of the preceding claims, wherein the recombinase domain is a functional variant of the CRE recombinase, said variant comprising, or consisting of, a CRE recombinase of SEQ ID NO: 1 in which 18, 19 26 or 31 amino acids, preferably 18, 26 or 31 amino acids, have been deleted at the N-terminus.

A fusion protein according to any one of the preceding claims, wherein the recombinase domain is a functional variant of the CRE recombinase, said variant comprising, or consisting of, a CRE recombinase of SEQ ID NO: 1 in which 19 amino acids have been deleted in N-terminal.

A fusion protein according to any one of claims 1 to 10, wherein the recombinase domain is a functional variant of the CRE recombinase, said variant comprising, or consisting of, a CRE recombinase of SEQ ID NO: 1 in which amino acids were deleted in N-terminal.

A fusion protein according to any one of the preceding claims, wherein the recombinase domain is a functional variant of the CRE recombinase which has a substitution at the residue corresponding to residue E340 of SEQ ID NO: 1 and / or a substitution at the level of the residue corresponding to residue D341 of SEQ ID NO: 1, preferably a substitution at the level of the residue corresponding to E340 residue of SEQ ID NO: 1 and a residue-level substitution corresponding to residue D341 of SEQ ID NO: 1.

The fusion protein of claim 13, wherein the one or more substitute residues are independently selected from the group consisting of A, G, V, L, I, P, M, W, C, N, Q, S, T and Y, preferably the residue or residues are substituted by an alanine residue.

A fusion protein according to any one of the preceding claims, wherein the recombinase domain is SEQ ID NO: 3 or SEQ ID NO:

4.

16. A fusion protein according to any one of the preceding claims, wherein the two domains are immediately adjacent.

17. The fusion protein according to any one of claims 1 to 15, wherein the two domains are separated by at most 6, 5, 4, 3, 2 or 1 amino acids.

18. A fusion protein according to claim 1 comprising or consisting of a sequence selected from SEQ ID NO: 5 to 12.

Nucleic acid encoding a fusion protein according to any one of the preceding claims. An expression cassette comprising a nucleic acid according to claim

19.

An expression vector comprising a nucleic acid according to claim 19 or an expression cassette according to claim 20.

22. A host cell comprising a nucleic acid according to claim 19, an expression cassette according to claim 20 or an expression vector according to claim 21.

23. A method for inducing recombinase activity in a cell comprising

supplying a cell expressing a fusion protein according to any one of claims 1 to 18 or inserting a nucleic acid according to claim 19, an expression cassette according to claim 20 or a vector of expression according to claim 21 in a cell and

exposing said cell to a light capable of activating the photoreceptor domain of said fusion protein and thus the recombinase activity. 24. Process for modifying a nucleic acid, comprising

contacting a fusion protein according to any one of claims 1 to 18 with said nucleic acid of interest comprising one or more recognition sites recognized by the recombinase domain of said fusion protein, and

exposing the fusion protein and said nucleic acid to a light capable of activating the photoreceptor domain of said fusion protein and thus the recombinase activity.

25. The method of claim 24, which is used

to modulate the expression of a molecule of interest in the host cell,

to induce genetic diversity in the host cell, or

to modify a biological activity in the context of a gene therapy.

A kit comprising a fusion protein according to any one of claims 1 to 18, a nucleic acid according to claim 19, an expression cassette according to claim 20, an expression vector according to claim 21 and / or a The host cell of claim 22, and optionally a light source capable of activating the photoreceptor domain of the fusion protein. Use of a kit according to claim 26 for inducing recombinase activity in a cell according to the method of claim 23 and / or for modifying one or more nucleic acids according to the method of claim 24 or 25.