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WO2024218137A1 - Nouvelles enzymes recombinase pour recombinaison d'adn spécifique à un site - Google Patents

Nouvelles enzymes recombinase pour recombinaison d'adn spécifique à un site Download PDF

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Publication number
WO2024218137A1
WO2024218137A1 PCT/EP2024/060392 EP2024060392W WO2024218137A1 WO 2024218137 A1 WO2024218137 A1 WO 2024218137A1 EP 2024060392 W EP2024060392 W EP 2024060392W WO 2024218137 A1 WO2024218137 A1 WO 2024218137A1
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seq
acid sequence
nucleic acid
protein
identity
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PCT/EP2024/060392
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Milica JELICIC
Lucas Theo SCHMITT
Frank Buchholz
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Technische Universität Dresden
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to the use of a protein with recombinase activity to catalyze a site-specific DNA recombination, as well as to a method for producing a site-specific DNA recombination.
  • the present invention is applicable alone or in combination with other recombinase systems for genetic manipulation, for example in medicine or medical research.
  • SSRs Site-specific recombinases
  • nuclease-based approaches are being intensively developed to expand their utility (z.e., base editors and prime editors ( Komor et al., 2016; Gaudelli et al., 2017; Anzalone et al., 2022; Anzalone et al., 2019), these systems still typically introduce DNA-nicks and rely on cell intrinsic repair mechanisms (Meinke et al., 2016).
  • SSRs can operate autonomously and the outcome of recombination is very predictable.
  • most SSRs are relatively small in size, rendering them more convenient for different delivery vectors (Meinke et al., 2016).
  • Tyrosine recombinases such as Cre and Flp are used extensively for genome engineering due to their simplicity and their ability to conduct efficient genome modifications in heterologous hosts (Sauer et al., 1988). Hence, these SSRs were extensively used to model and understand how these types of enzymes work.
  • the Cre/loxP system is derived from bacteriophage Pl and consists of the recombinase Cre and the 34-bp target sites loxP (Sternberg et al., 1981). loxP sites are palindromic sequences with two 13-bp inverted repeats separated by an 8-bp spacer.
  • Each half-site is bound by one recombinase protomer forming a tetrameric complex on two loxP sites, which together catalyze strand exchange within the spacer region (Duyne and Hamilton, 1981).
  • Cre is able to perform a variety of reactions such as excision, integration, inversion and translocations (Meinke et al., 2016).
  • Cre alone, or in combination with other SSRs can perform recombinase-mediated cassette exchange (RMCE) for precise replacement of a DNA fragment (Meinke et al., 2016; Anderson et al., 2012; Minorikawa and Nakayama, 2011).
  • DNA recombinase systems are the Nigri/nox system disclosed in EP 2 877 585 Bl, the Vika/vox system disclosed in EP 2 690 177 Bl, and the Panto/pox system disclosed in EP 3 263 708 Bl.
  • recombinase systems that are known in the art show different activities in cells of different origin. For many applications, such as the production of transgenic animals with conditional gene knockouts, two or more recombinase systems are used in combination with each other.
  • emerging complex genetics studies and applications require simultaneous use of multiple recombinases.
  • site-specific recombinases are equally applicable in all model organisms due to, e.g., genomic specificity (off-target activity on cryptic recognition target sites). For that reason, it is important that an optimal recombinase can be chosen depending on the target organism or experimental setup.
  • the method for producing a site-specific DNA-recombination comprises the steps of a) contacting a nucleic acid comprising at least a first and a second recognition site which are essentially identical or essentially reverse complementary to each other, with a protein having recombinase activity, and b) allowing the protein having recombinase activity to produce the site-specific DNA-recombination.
  • a recognition site comprises a first half-site, a spacer and a second half-site, and essentially identical or essentially reverse complementary to each other means that the nucleotide sequence of the first and the second half-site in the first recognition site may deviate in up to two nucleotides from the nucleotide sequence of the first and the second half-site in the second recognition site.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 7, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 16 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 16.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 12 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 12.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 10 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 10.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 11 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 11.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 13 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 13.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 5, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 14 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 14.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 6, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 15 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 15.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 8, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 17 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 17.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 9, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 15 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 15.
  • the protein having recombinase activity comprises at least two protein monomers.
  • the nucleic acid sequence that is recombined is present in a cell.
  • the method further comprises the step of introducing into the cell a nucleic acid encoding the protein having recombinase activity.
  • the cell comprises a nucleic acid encoding the protein having recombinase activity.
  • the nucleic acid encoding the protein having recombinase activity comprises a regulatory nucleic acid sequence, and wherein the expression of the nucleic acid encoding the protein having recombinase activity is regulated by the regulatory nucleic acid sequence.
  • the cell is a eukaryotic or a bacterial cell.
  • the present invention provides the use of a protein having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9 for producing a site-specific DNA- recombination.
  • the present invention provides the use of a protein having recombinase activity for catalyzing a site-specific DNA-recombination at recognition sites that essentially are identical or essentially reverse complementary to each other, wherein a recognition site comprises a first half-site, a spacer and a second half-site, and wherein essentially identical or essentially reverse complementary to each other means that the nucleotide sequence of the first and the second half-site in a first recognition site may deviate in up to two nucleotides from the nucleotide sequence of the first and the second half-site in a second recognition site.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 7, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 16 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 16.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 12 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 12.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 10 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 10.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 11 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 11.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 13 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 13.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 5, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 14 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 14.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 8, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 17 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 17.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 9, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 15 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 15.
  • the present invention provides a nucleic acid having a length of not more than 40 base pairs and comprising:
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 16 or a nucleic acid sequence reverse complementary thereto;
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 12 or a nucleic acid sequence reverse complementary thereto;
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or a nucleic acid sequence reverse complementary thereto;
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 11 or a nucleic acid sequence reverse complementary thereto;
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14 or a nucleic acid sequence reverse complementary thereto;
  • the present invention provides a vector comprising at least one and preferably at least two essentially identical or essentially reverse complementary nucleic acids of the present invention, wherein a DNA segment is preferably flanked by the two essentially identical or essentially reverse complementary nucleic acids.
  • the vector further comprises a nucleic acid encoding a protein having recombinase activity, wherein the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 9.
  • the present invention provides a vector comprising a nucleic acid encoding a protein having recombinase activity, wherein the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 9.
  • the present invention provides a protein having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9, or a vector according to the invention, for use in medicine.
  • the protein or the vector is for use in treating a genetic disease or disorder in a subject. More preferably, the genetic disease or disorder is characterized by modification of the subject’s genome.
  • the present invention provides an isolated host cell, comprising the following recombinant DNA fragments: at least one and preferably at least two nucleic acids according to the invention; and/or a vector according to the invention.
  • the isolated host cell further comprises (i) a nucleic acid encoding for a protein having recombinase activity, wherein the protein comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 9; or (ii) a vector according to the invention.
  • the present invention provides a non-human host organism, comprising: (i) at least one and preferably at least two nucleic acids according to the invention; or (ii) a vector according to the invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a protein having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9, the nucleic acid according to the invention, a vector according to the invention, the isolated host cell according to the invention, or the non-human host organism according to the invention, and optionally a pharmaceutically acceptable excipient
  • Fig. 1A shows an overview of the plasmid recombination assay. Important features, such as restriction sites, recombinase coding sequence and target sites (triangles) are shown. A schematic representation of expected recombination products on an agarose gel is also shown. Marker and expected sizes of recombined and unrecombined plasmids are indicated separately or together as usually seen on the gels.
  • Fig. IB shows the plasmid map of pEVO recombination reporter. Protein coding genes, the origin of replication (oriP15A) and the pBAD promoter are depicted as arrows.
  • the protein coding genes include the chloramphenicol resistance gene (cmR), the arabinose regulatory protein (araC) and the genes encoding for the recombinases of interest. Expression of the recombinase is driven by pBAD promoter upon addition of arabinose. Recombination between two lox sites leads to excision of about 700 bp stuffer sequence. BsrGI and Sbfl restriction enzymes are used for cloning of the recombinases as well as for linearization of the plasmids for test digest
  • Fig. 2 shows the recombination activity of seventeen tested putative Y-SSR/target site pairs. Each sample was tested with or without L-arabinose (100 pg/ml) added to the growth medium for recombinase expression, indicated with or "+”. Recombination is indicated by the band aligned with the single triangle and non-recombined plasmids are indicated by two triangles. Active recombinases are highlighted with a box.
  • M GeneRulerTM DNA Ladder Mix (Thermo Fisher).
  • Fig. 3 shows quantification and reproducibility of recombination activity of new SSRs in E. coli.
  • Recombinase expression was induced with rising concentrations (pg/ml) of L-Arabinose indicated along the x-axis.
  • Vika and Cre were included as positive controls.
  • Recombination was calculated from measuring the ratio of band intensities of unrecombined and recombined bands detected on agarose gels.
  • Fig. 4A shows the analysis of deep sequencing results.
  • Cross recombination events are displayed by a heatmap of recombination percentage for each possible combination.
  • Target sites are displayed horizontally and ordered based on their similarity.
  • Recombinases tested are aligned based on their homology on the vertical axis.
  • On-target recombination events are boxed with red squares.
  • Fig. 4B shows the validation of cross recombination events by plasmid-based recombination assay. Recombination activity of respective recombinases is shown on their on-target sites, and on off- targets. Recombination was assessed by agarose gel electrophoresis.
  • Fig. 5 shows the activity of new recombinases in mammalian cells.
  • Fig. 5A is a graphical representation of the mammalian recombination reporter and expression constructs. Important features are marked in the reporter and expression vectors. Upon recombination of the reporter vector the mCherry cassette will be excised allowing for the expression of GFP (green) from the pCAG promotor (arrow). Black triangles represent different lox sites for each corresponding recombinase. NLS, nuclear localization signal.
  • Fig. 5B shows fluorescence microscopy analysis in Hek293T cells.
  • Fig. 5C shows FACS analysis of the samples shown in Fig. 5B. Darker grey histograms depict control samples where non- recombined reporter plasmids were co-transfected with ‘empty’ expression plasmids, while the lighter gray histograms show samples transfected with corresponding recombinases. Fig.
  • 5D shows the plasmid maps of expression lentiviral vector (left) and reporter vector (right).
  • CMV, PGK and CAG promoters for mammalian expression of viral RNA, Recombinase-P2A-BFP cassette and mCherry cassette, respectively, are shown as white arrows.
  • Features for viral production on pLentiX vector are also depicted.
  • the lentiviral vector was either transfected into HEK293T cells for recombination assay, or used for virus production and infection as to test the effect of continuous expression of the recombinases.
  • BsrGI and Xbal restriction sites used for cloning of the recombinases are marked on the expression vector while Nhel and Hindlll sites labeled on pCAG-lox-mCherry-lox-GFP reporter plasmid were used for cloning of all the target sites.
  • Fig. 6 shows the effect on cell growth upon overexpression of the recombinases of the present invention in mammalian cells.
  • Fig. 6A is an overview of the experimental setup. Important steps are indicated by arrows.
  • Cells were transduced at a rate of ca. 50% with a bicistronic lentivirus expression construct, where the expression of respective recombinases was linked via P2A to BFP expression.
  • Cells were analyzed every 72 h by flow cytometry and the percentage of BFP-positive cells was recorded over the course of 2 weeks. Declining percentages of BFP-positive cells are indicative of a proliferation disadvantage of infected cells.
  • Fig. 6B shows the analysis of growth rates.
  • Fig. 7 shows the results of directed evolution of YR9 recombinase Fig. 7A.
  • Fig. 7C Activity of the most active clone YR9.10 in mammalian cells. FACS analysis of wt YR9 and YR9.10 recombinase are shown. Darker grey histograms depict control samples where nonrecombined reporter plasmids were co-transfected with ‘empty’ expression plasmids, while the lighter grey histograms show samples transfected with corresponding recombinases.
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  • the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same, i.e. that comprise the same sequence of nucleotides or amino acids. Sequences are “identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same.
  • At least 60% identical includes at least at least 61%, at least at least 62%, at least at least 63%, at least at least 64%, at least at least 65%, at least at least 66%, at least at least 67%, at least at least 68%, at least at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity over the specified sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison
  • the term "at least XY% sequence identity" is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide.
  • a protein having recombinase activity and comprising an amino acid sequence having at least 80% identity to a given SEQ ID NO preferably means that said protein has an amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the given SEQ ID NO.
  • a nucleic acid sequence having at least 60% sequence identity to a given SEQ ID NO or a nucleic acid sequence reverse complementary thereto preferably means that said nucleic acid has a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the given SEQ ID NO or a nucleic acid sequence reverse complementary to said SEQ ID NO.
  • sequence comparison is used herein to refer to the process wherein one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm When using a sequence comparison algorithm, test and reference sequences are entered into a computer, if necessary, subsequence coordinates are designated, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by one of the SEQ ID NOs of the present invention, if not specifically indicated otherwise.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • B the BLOSUM62 scoring matrix
  • B the BLOSUM62 scoring matrix
  • nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.
  • the invention also comprises the corresponding RNA sequences (in which thymine is replaced by uracil), complementary sequences and sequences with modified nucleic acid backbone or 3 'or 5 '-terminus. Nucleic acids in the form of DNA are however preferred.
  • protein having recombinase activity comprises any enzyme that is capable of manipulating the structure of a genome. More specifically, the term refers to respective enzymes that can catalyse a recombination selected among an excision, integration, inversion and translocation reaction. Proteins having recombinase activity are well known in the art (cf. discussion of the background above) and specifically include DNA recombinases such as site-specific recombinases and more specifically tyrosine recombinases. A protein having recombinase activity can be present as a monomer, a dimer or a tetramer.
  • Dimers and tetramers can comprise two or four monomers of the same protein having recombinase activity (homodimer or homotetramer), respectively, or alternatively two or more different monomers having recombinase activity (heterodimer or heterotetramer).
  • recognition site refers to a specific nucleotide sequence which a recombinase enzyme recognizes, and at which DNA breakage and strand exchange occur. These sequences typically range between 30 and 200 base pairs in length and are comprised of two inversely repeated recombinase binding regions flanking a central spacer sequence (Meinke et al., 2016). An example of such a recognition site can be seen in the SSR Cre/loxP binding complex, where the Cre recombinase is bound to the 34 base pair loxP target sequence.
  • the loxP recognition site comprises two 13 base pair inverted repeat Cre binding elements flanking an 8 base pair spacer region.
  • the recombinase complex recognizes a first recognition site and a second recognition site on a DNA double strand.
  • the recognition sites are also referred to as upstream and downstream recognition sites, depending on their location on the DNA double strand.
  • the first half-site (e.g. the left half-site) and the second half-site (e.g. the right half-site) are identical and palindromic (reverse complement).
  • the first half-site (e.g. the left half-site) and the second half-site (e.g. the right halfsite) are not identical and not palindromic, i.e. they differ from each other in at least one nucleotide.
  • the term "functional mutant” as used herein means that one or more nucleic acids can be added to, inserted, deleted or substituted from the nucleic acid sequence to which the term refers.
  • the term “functional mutant” means that one or more amino acids can be added to, inserted, deleted or substituted from the amino acid sequence to which the term refers.
  • Preferred mutations are point mutations or an exchange of a particular nucleic acid or amino acid. Specifically in cases of functional mutants of a recognition site, it is known that the exchange of the spacer region of a recognition site does not influence its activity as a target for the specific recombinase.
  • functional mutants of recognition sites explicitly include mutations in the spacer region up to a full substitution of the spacer region.
  • Functional mutants of recognition sites also include mutations in the half sites of a recognition site.
  • a functional mutant of a recognition site as disclosed herein may include one, two, three, four, five, six, seven or eight mutations in its nucleotide sequence compared to its reference nucleotide sequence, from which the functional mutant is derived.
  • a functional mutant of a recognition site includes one or two mutations in the nucleotide sequence of the first half site, in the nucleotide sequence of the second half site, or in the nucleotide sequences of both half sites, compared to its reference nucleotide sequence, from which the functional mutant is derived.
  • reporter vector includes a plasmid, virus or other nucleic acid carriers, that comprise a nucleic acid sequence according to the invention by genetic recombination (recombinantly), e.g. by insertion or incorporation of said nucleic acid sequence.
  • Prokaryotic vectors as well as eukaryotic vectors, for example artificial chromosomes, such as YAC (yeast artificial chromosomes), are applicable for the invention.
  • terapéuticaally effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
  • composition refers to a substance and/or a combination of substances being used for the identification, prevention or treatment of a disease or tissue status.
  • the pharmaceutical composition is formulated to be suitable for administration to a patient in order to prevent and/or treat a disease.
  • a pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for therapeutic use.
  • a carrier is also referred to as being pharmaceutically acceptable.
  • Pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalative, rectal, sublingual, transdermal, subcutaneous or vaginal application routes according to their chemical and physical properties.
  • Pharmaceutical compositions comprise solid, semisolid, liquid, transdermal therapeutic systems (TTS).
  • Solid compositions are selected from the group consisting of tablets, coated tablets, powder, granulate, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also comprised are liquid compositions, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention.
  • Semisolid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.
  • pharmaceutically acceptable embraces both human and veterinary use:
  • pharmaceutically acceptable embraces a veterinarily acceptable compound or a compound acceptable in human medicine and health care.
  • subject refers to an animal, preferably a mammal, most preferably a human.
  • the method for producing a site-specific DNA- recombination comprises the steps of a) contacting a nucleic acid comprising at least a first and a second recognition site which are essentially identical or essentially reverse complementary to each other, with a protein having recombinase activity, and b) allowing the protein having recombinase activity to produce the site-specific DNA-recombination.
  • a recognition site is a nucleotide sequence comprising a first half-site, a second half-site, and a spacer separating the first and the second half-sites.
  • the term "essentially identical” or “essentially reverse complementary to each other” means that the nucleotide sequence of the first- and the second half-site in the first recognition site may deviate in up to two nucleotides from the nucleotide sequence of the first and the second half-site in the second recognition site.
  • it is referred to the recognition sites as identified herein and as shown in Fig. 4B.
  • both recognition sites have the sequence of SEQ ID NO: 10.
  • one of the two recognition sites has the sequence of SEQ ID NO: 10, and the other has the sequence of TTAATTTCTGAGA ACTGTCAT TCTCGGAAATTGA (SEQ ID NO: 19).
  • one of the recognition sites has SEQ ID NO: 10 but with up to two diverging nucleotides in the sequences of the first and the second half-site.
  • the first half-site of a recognition site comprises a nucleotide sequence that differs from the nucleotide sequence of the first half-site of the other recognition site by two nucleotides, and the second half site and the spacer are identical in both recognition sites
  • the first halfsite of a recognition site comprises a nucleotide sequence that differs from the nucleotide sequence of the first half-site of the other recognition site by one nucleotide
  • the second half site of the same recognition site comprises a nucleotide sequence that differs from the nucleotide sequence of the second half-site of the other recognition site by one nucleotide
  • the spacer sequences identical in both recognition sites or
  • the second half-site of a recognition site comprises a nucleotide sequence that differs from the nucleotide sequence of the second half-site of the other recognition site by two nucleotides, and the first half site and the spacer are identical in both recognition sites.
  • a first recognition site has the sequence of SEQ ID NO: 10, and the other recognition site is the reverse complement thereto, i.e. SEQ ID NO: 19.
  • the first recognition site may have the sequence of SEQ ID NO: 10
  • the second recognition site may have the sequence of SEQ ID NO: 19, with the proviso that SEQ ID NO: 19 comprises up to two diverging nucleotides in the sequences for the first and the second half-sites, but not in the sequence of the spacer.
  • the two diverging nucleotides may either be in the first half-site, the second half-site, or there is one different nucleotide in each the first and the second half-site.
  • the first and second recognition sites are identical or reverse complementary to each other in their half site sequences, meaning that the first and second recognition sites do not have any sequence deviations between their first half sites and between their second half sites.
  • the spacer sequences of the first and the second recognition sites do not have to be identical or reverse complementary to each other.
  • the first and second recognition sites are identical or reverse complementary to each other over their entire sequence including the first and second half sites and the spacer region, meaning that the first and second recognition sites do not have any sequence deviations between each other.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 7, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 16 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 16.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 12 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 12.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 10 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 10.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 11 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 11.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 13 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 13.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 5, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 14 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 14.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 6, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 15 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 15.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 8, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 17 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 17.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 9, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 15 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 15.
  • a nucleic acid encoding the recognition sites according to the invention comprises a maximum of 40, preferably 34, base pairs.
  • This nucleic acid according to the invention includes the recognition sites of the recombinase proteins of the present invention. Accordingly, the present invention provides a nucleic acid having a length of not more than 40 base pairs and comprising:
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or a nucleic acid sequence reverse complementary thereto;
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 11 or a nucleic acid sequence reverse complementary thereto;
  • nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14 or a nucleic acid sequence reverse complementary thereto;
  • a protein having recombinase activity and comprising an amino acid sequence having at least 80% identity to a given SEQ ID NO preferably means that said protein has an amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the given SEQ ID NO.
  • a nucleic acid sequence having at least 60% sequence identity to a given SEQ ID NO or a nucleic acid sequence reverse complementary thereto preferably means that said nucleic acid has a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the given SEQ ID NO or a nucleic acid sequence reverse complementary to said SEQ ID NO.
  • the recombinase protein is contacted with at least two of its recognition sites preferably inside a cell. Upon binding of the recombinase protein to the recognition sites, site-specific DNA-recombination occurs.
  • the recombinase YR6 having SEQ ID NO: 1 is contacted with at least two lox6 sites having SEQ ID NO: 10 inside a cell. Upon binding of the recombinase protein to said lox6 sites, site-specific DNA-recombination occurs.
  • the method according to the invention can be carried out in vitro or in vivo. In case the invention is carried out in an animal (including humans), it can be carried out for a non-therapeutic use. According to one embodiment, the method is not for the therapeutic treatment of a human being or an animal.
  • the method is applicable in all areas where site specific recombinases are conventionally used (including inducible knock-out or knock-in mice and other transgenic animal models).
  • the site-specific recombination results in integration, deletion, inversion, translocation or exchange of DNA.
  • the method is used for creating animal models, which are useful for biomedical research, e.g. as models for human diseases.
  • the protein having recombinase activity is present as a monomer.
  • the protein having recombinase activity comprises at least two protein monomers, i.e. is in a dimeric form (dimer).
  • a dimer can comprise two monomers of the same type (i.e. two identical protein monomers, homodimer) or two monomers of a different type (i.e. two different protein monomers, heterodimer).
  • the protein having recombinase activity comprises at least four protein monomers, i.e. is in a tetrameric form (tetramer).
  • Such a tetramer can comprise four monomers of the same type (i.e. four identical protein monomers, homotetramer), or monomers of a different type such as two, three or four different monomers (heterotetramer).
  • the protein having recombinase activity comprises a heterodimer or heterotetramer comprising different protein monomers, i.e. is a heterodimer or a heterotetramer.
  • the protein having recombinase activity is a recombinase, more preferably a DNA recombinase.
  • the nucleic acid sequence that is to be recombined is already present in a cell.
  • the nucleic acid sequence to be recombined can be introduced into the cell by conventional means known to the skilled person, such as by recombinant techniques.
  • the nucleic acid sequence encoding the recombinase protein of the present invention is either already present in the cell or is introduced into the cell by conventional means known to the skilled person, such as by recombinant techniques.
  • Such a method thus further includes the step of introducing into the cell a nucleic acid encoding for the recombinase protein of the invention.
  • the cell already comprises a nucleic acid encoding the protein of the invention having recombinase activity.
  • the nucleic acid encoding for the recombinase protein further comprises a regulatory nucleic acid sequence, preferably a promoter region.
  • a regulatory nucleic acid sequence preferably a promoter region.
  • the regulatory nucleic acid sequence (preferably the promoter region) is either introduced into the cell, preferably together with the sequence encoding for the recombinase protein, or the regulatory nucleic acid sequence is already present in the cell in the beginning of the method according to the invention.
  • the nucleic acid encoding for the recombinase protein is introduced into the cell (and placed under the control of the regulatory nucleic acid sequence).
  • regulatory nucleic acid sequence refers to gene regulatory regions of DNA. In addition to promoter regions, this term encompasses operator regions more distant from the gene as well as nucleic acid sequences that influence the expression of a gene, such as ciselements, enhancers or silencers.
  • promoter region refers to a nucleotide sequence on the DNA allowing a regulated expression of a gene. The promoter region allows regulated expression of the nucleic acid encoding for the respective protein. The promoter region is located at the 5'-end of the gene and thus before the RNA coding region. Both, bacterial and eukaryotic promoters are applicable for the invention.
  • the recognitions sites are either included in the cell or introduced into the cell, preferably by recombinant techniques.
  • the methods of the present invention may include the steps of introducing into a cell the following nucleic acids: a) a first nucleic acid (first recognition site) comprising a nucleic acid sequence according to or reverse complementary to one of SEQ ID NOs: 10 to 17; or a nucleic acid sequence that is a functional mutant thereof; b) a second nucleic acid (second recognition site) comprising a nucleic acid sequence essentially identical or essentially reverse complementary to the nucleic acid sequence of the first nucleic acid (first recognition site).
  • a nucleic acid encoding for the recombinase protein of the invention and at least two recognition sites are introduced into the cell.
  • This method includes the following steps: a) introducing into a cell the following nucleic acids:
  • nucleic acid encoding for the recombinase protein, wherein the nucleic acid is introduced into the DNA such that a regulatory nucleic acid sequence (preferably a promoter region) controls the expression of the nucleic acid encoding for the recombinase protein,
  • a second nucleic acid comprising a nucleic acid sequence according to or reverse complementary to one of SEQ ID NOs: 10 to 17; or a nucleic acid sequence that is a functional mutant thereof;
  • a third nucleic acid comprising a nucleic acid sequence essentially identical or essentially reverse complementary to the nucleic acid sequence defined in (ii), and b) activating the regulatory nucleic acid sequence (preferably the promoter region) to induce expression of the first nucleic acid for the synthesis of the protein with recombinase activity.
  • the nucleic acid sequence encoding for the recombinase protein is introduced into a cell and at least two recognition sites are introduced into the genomic or episomal DNA of the cell.
  • the steps (i) to (iii) can be performed in any order.
  • the introduction of the nucleic acids into the cells is performed using techniques of genetic manipulation known by a person skilled in the art. Among suitable methods are cell transformation, transfection or viral infection, whereby a nucleic acid sequence encoding the protein is introduced into the cell as a component of a vector or part of virus-encoding DNA or RNA.
  • the cell culturing is carried out by methods known to a person skilled in the art for the culture of the respective cells. Therefore, cells are preferably transferred into a conventional culture medium, and cultured at temperatures and in a gas atmosphere that is conducive to the survival of the cells.
  • the present invention also includes nucleic acid sequences or polynucleotides in which the coding sequence for the recombinase protein is fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a protein from a host cell.
  • a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell may be fused to the sequence encoding the recombinase protein.
  • the polypeptide or protein having such a leader sequence is termed a pre-protein or a pre-proprotein and may have the leader sequence cleaved by the host cell to form the mature form of the protein.
  • polynucleotides may have a 5' extended region so that it encodes a proprotein, which is the mature protein plus additional amino acid residues at the N-terminus.
  • the expression product having such a pro-sequence is termed a pro-protein, which is an inactive form of the mature protein; however, once the pro-sequence is cleaved, an active mature protein remains.
  • the additional sequence may also be attached to the protein and be part of the mature protein.
  • the polynucleotides of the present invention may encode polypeptides, or proteins having a pro-sequence, or proteins having both, a pro-sequence and a pre-sequence (such as a leader sequence).
  • the nucleic acids of the present invention may also have the coding sequence fused in frame to a marker sequence, which allows for purification of the proteins of the present invention.
  • the marker sequence may be an affinity tag or an epitope tag such as a polyhistidine tag, a streptavidin tag, a Xpress tag, a FLAG tag, a cellulose or chitin binding tag, a glutathione-S transferase tag (GST), a hemagglutinin (HA) tag, a c-myc tag or a V5 tag.
  • the HA tag would correspond to an epitope obtained from the influenza hemagglutinin protein (Wilson et al., 1984), and the c-myc tag may be an epitope from human Myc protein (Evans et al., 1985).
  • the nucleic acid of the invention is a mRNA, in particular for use as a medicament, the delivery of mRNA therapeutics can be facilitated by the significant progress that has been achieved in maximizing the translation and stability of mRNA, preventing its immune-stimulatory activity and the development of in vivo delivery technologies.
  • the 5' cap and 3' poly(A) tail are the main contributors to efficient translation and prolonged half-life of mature eukaryotic mRNAs. Incorporation of cap analogs such as ARCA (anti-reverse cap analogs) and poly(A) tail of 120-150 bp into in vitro transcribed (IVT) mRNAs has markedly improved expression of the encoded proteins and mRNA stability.
  • New types of cap analogs such as 1,2-dithiodiphosphate-modified caps, with resistance against RNA decapping complex, can further improve the efficiency of RNA translation.
  • Replacing rare codons within mRNA protein-coding sequences with synonymous frequently occurring codons, so-called codon optimization, also facilitates better efficacy of protein synthesis and limits mRNA destabilization by rare codons, thus preventing accelerated degradation of the transcript.
  • engineering 3' and 5' untranslated regions which contain sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs, can enhance the level of protein product.
  • UTRs can be deliberately modified to encode regulatory elements (e.g., K-tum motifs and miRNA binding sites), providing a means to control RNA expression in a cell-specific manner.
  • regulatory elements e.g., K-tum motifs and miRNA binding sites
  • Some RNA base modifications such as Nl-methyl-pseudouridine have not only been instrumental in masking mRNA immune-stimulatory activity but have also been shown to increase mRNA translation by enhancing translation initiation.
  • base modifications and codon optimization affect the secondary structure of mRNA, which in turn influences its translation.
  • Respective modifications of the nucleic acid molecules of the invention are also contemplated by the invention.
  • RNA or plurality of RNAs preferably encode the recombinase enzyme of the present invention or any of its subunits.
  • Specific methods for delivering and expressing nucleic acids and specifically RNAs are disclosed e.g. in EP2590676 and EP3115064, which are herein incorporated by reference.
  • the RNA may be present in a particle and is preferably self-replicating. After in vivo administration of the particles, RNA is released from the particles and is translated inside a cell to provide the DNA recombining enzyme or any of its monomeric subunits.
  • a self-replicating RNA molecule can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself).
  • These daughter RNAs, as well as collinear sub- genomic transcripts may be translated by themselves to provide in situ expression of an encoded polypeptide, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the polypeptide.
  • the overall results of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded polypeptide becomes a major polypeptide product of the cells.
  • a preferred self-replicating RNA molecule encodes (i) an RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule, and (ii) a recombinase protein of the present invention.
  • the polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsPl, nsP2, nsP3 and nsP4. It is preferred that the self-replicating RNA molecules of the invention do not encode alphavirus structural proteins.
  • a preferred self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions.
  • a self-replicating RNA molecule useful in the context of the present invention may have two open reading frames.
  • the first (5 1 ) open reading frame encodes a replicase
  • the second (3 1 ) open reading frame encodes a polypeptide of the present invention.
  • the RNA may have additional (e.g. downstream) open reading frames e.g. for further encoding accessory polypeptides.
  • RNA is particularly suitable for the general use in gene therapy, and specifically for use in the treatment of genetic disorder or disease.
  • the method according to the invention can be performed using eukaryotic and prokaryotic cells.
  • Preferred prokaryotic cells are bacterial cells. Particularly preferred prokaryotic cells are cells of Escherichia coli.
  • Preferred eukaryotic cells are yeast cells (preferably Saccharomyces cerevisiae). insect cells, non-insect invertebrate cells, amphibian cells, or mammalian cells (preferably somatic or pluripotent stem cells, including embryonic stem cells and other pluripotent stem cells, like induced pluripotent stem cells, and other native cells or established cell lines, including NIH3T3, CHO, HeLa, HEK293, hiPS).
  • cells are preferably obtained without destroying human embryos, e.g. by outgrowth of single blastomeres derived from blastocysts as described by Chung et al., 2008, by parthenogenesis, e.g. from a one-pronuclear oocyte as described by Lin et al. 2007, or by parthenogenetic activation of human oocytes as described by Mai et al. 2007.
  • cells of a non-human host organism preferably non-human germ cells, somatic or pluripotent stem cells, including embryonic stem cells, or blastocytes.
  • the cell is a eukaryotic or a bacterial cell.
  • the cell is not a human germ cell.
  • the present invention provides the use of a protein having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9 for producing a site-specific DNA- recombination.
  • a protein having at least 80% identity to a given SEQ ID NO preferably means that said protein has an amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the given SEQ ID NO.
  • the present invention provides the use of a protein having recombinase activity for catalyzing a site-specific DNA-recombination at recognition sites that essentially are identical or essentially reverse complementary to each other, wherein a recognition site comprises a first half-site, a spacer and a second half-site, and wherein essentially identical or essentially reverse complementary to each other means that the nucleotide sequence of the first and the second half-site in a first recognition site may deviate in up to two nucleotides from the nucleotide sequence of the first and the second half-site in a second recognition site.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 7, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 16 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 16.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 12 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 12.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 10 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 10.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 11 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 11.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 13 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 13.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 5, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 14 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 14.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 6, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 15 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 15.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 8, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 17 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 17.
  • the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 9, and the at least one two recognition sites comprise a nucleic acid sequence according to or reverse complementary to SEQ ID NO: 15 or to a functional mutant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO: 15.
  • a protein having recombinase activity and comprising an amino acid sequence having at least 80% identity to a given SEQ ID NO preferably means that said protein has an amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the given SEQ ID NO.
  • a nucleic acid sequence having at least 60% sequence identity to a given SEQ ID NO or a nucleic acid sequence reverse complementary thereto means that said nucleic acid has a sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the given SEQ ID NO or a nucleic acid sequence reverse complementary to said SEQ ID NO.
  • the present invention provides a vector comprising at least one and preferably at least two essentially identical or essentially reverse complementary nucleic acids of the present invention, wherein a DNA segment is preferably flanked by the two essentially identical or essentially reverse complementary nucleic acids.
  • the vector further comprises a nucleic acid encoding a protein having recombinase activity, wherein the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 9.
  • the present invention provides a vector comprising a nucleic acid encoding a protein having recombinase activity, wherein the protein having recombinase activity comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 9.
  • the invention provides vectors comprising the nucleic acid sequences of the present invention.
  • such vector comprises at least one, preferably at least two essentially identical or essentially reverse complementary nucleic acid sequences as described herein for the recognition sites.
  • a vector according to the present invention comprises a nucleic acid encoding for a protein having recombinase activity, preferably wherein the protein comprises an amino acid sequence exhibiting at least 80%, preferably at least 85%, at least 90%, at least 95%, and most preferably at least 99% sequence identity to one of SEQ ID NOs: 1 to 9.
  • the present invention further provides a vector (also referred to herein as "reporter vector”) comprising at least one nucleic acid comprising a nucleic acid sequence according to or reverse complementary to one of SEQ ID NOs: 10 to 17, or a nucleic acid sequence that is a functional mutant thereof.
  • a vector also referred to herein as "reporter vector” comprising at least one nucleic acid comprising a nucleic acid sequence according to or reverse complementary to one of SEQ ID NOs: 10 to 17, or a nucleic acid sequence that is a functional mutant thereof.
  • the at least two recognition sites are preferably not located consecutively in the vector. Rather, the at least two recognition sites are positioned such that they are flanking a DNA segment of interest, which upon recognition of the recognition sites by the respective recombinase protein is recombined, preferably excised or inverted.
  • the DNA segment of interest can preferably contain a gene or a promoter region.
  • the DNA segment of interest is excised when it is flanked by two recognition sites having the same orientation (same nucleic acid sequence).
  • An inversion of the DNA segment of interest is catalyzed by a recombinase protein when the DNA segment is flanked by two recognition sites arranged in opposite orientations, i.e. the recognition sites comprise nucleic acid sequences that are reverse complementary to one another.
  • the present invention provides an isolated host cell, comprising the following recombinant DNA fragments: at least one and preferably at least two nucleic acids according to the invention; and/or a vector according to the invention.
  • the isolated host cell further comprises a nucleic acid encoding for a protein having recombinase activity, wherein the protein comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 9.
  • the isolated host cell further comprises a vector according to the invention.
  • isolated host cells that comprise the nucleic acids or the vector as mentioned herein naturally.
  • the invention concerns only those isolated host cells that comprise the above mentioned nucleic acids or vectors recombinantly and not naturally, i.e. by genetic modification of the host cell.
  • the present invention provides a non-human host organism, comprising at least one and preferably at least two nucleic acids according to the invention. According to a further aspect, the present invention provides a non-human host organism, comprising the vector according to the invention.
  • the invention includes an isolated host cell or an isolated host organism comprising (i) at least one, preferably at least two, nucleic acids according to the invention comprising a recognition site as defined above (preferably two nucleic acids according to the invention that include a recognition site, respectively, and which flank a further DNA segment) and/or a nucleic acid according to the invention encoding for a protein with recombinase activity, as defined above, or (ii) a vector according to the invention comprising at least two nucleic acids comprising a recognition site as defined above (preferably two nucleic acids according to the invention that include a recognition site, respectively, and which flank a further DNA segment) and/or a vector according to the invention comprising a nucleic acid encoding for a protein with recombinase activity as defined above.
  • the present invention also includes an isolated host cell comprising the following recombinant DNA fragments: (i) at least one, preferably at least two, nucleic acids according to the invention comprising a recognition site and/or a nucleic acid according to the invention encoding for a recombinase protein or (ii) a vector according to the invention comprising at least two nucleic acids comprising a recognition site (preferably two nucleic acids according to the invention that include a recognition site and flank a further DNA segment of interest) and/or a vector according to the invention comprising a nucleic acid encoding for a recombinase protein.
  • the invention concerns only those isolated host cells that comprise the above mentioned nucleic acids or vectors recombinantly and not naturally, i.e. by genetic modification of the host cell.
  • isolated host cells that contain both, a nucleic acid encoding for the recombinase protein of the present invention and at least two of its recognition sites (which are either oriented in the same or in opposite direction).
  • a host cell within the meaning of the invention is a naturally occurring cell or a cell line (optionally transformed or genetically modified) that comprises at least one vector according to the invention or a nucleic acid according to the invention recombinantly, as described above.
  • the invention includes transient transfectants (e.g. by mRNA injection) or host cells that include at least one expression vector according to the invention as a plasmid or artificial chromosome, as well as host cells in which an expression vector according to the invention is stably integrated into the genome of said host cell.
  • Suitable host cells in the context of the present invention are in particular eukaryotic cells, including stem cells like hematopoietic stem cells, neuronal stem cells, adipose tissue derived stem cells, fetal stem cells, umbilical cord stem cells, induced pluripotent stem cells and embryonic stem cells.
  • stem cells like hematopoietic stem cells, neuronal stem cells, adipose tissue derived stem cells, fetal stem cells, umbilical cord stem cells, induced pluripotent stem cells and embryonic stem cells.
  • stem cells like hematopoietic stem cells, neuronal stem cells, adipose tissue derived stem cells, fetal stem cells, umbilical cord stem cells, induced pluripotent stem cells and embryonic stem cells.
  • fetal stem cells fetal stem cells
  • umbilical cord stem cells fetal stem cells
  • induced pluripotent stem cells and embryonic stem cells.
  • embryonic stem cells there are preferably not derived
  • the present invention also includes a non-human host organism comprising the following recombinant DNA fragments: at least one, preferably at least two, nucleic acids according to the invention comprising a recognition site (preferably two nucleic acids according to the invention that include a recognition site, respectively, which flank a further DNA segment of interest) and/or a nucleic acid according to the invention encoding for a recombinase protein.
  • a recognition site preferably two nucleic acids according to the invention that include a recognition site, respectively, which flank a further DNA segment of interest
  • a nucleic acid according to the invention encoding for a recombinase protein.
  • Explicitly included are non-human host organisms that only comprise a recombinant nucleic acid encoding for a recombinase protein of the present invention (and which do not comprise a nucleic acid including a recognition site respectively).
  • the invention includes non-human host organisms that only comprise at least one, preferably at least two recognition sites (and which do not comprise a nucleic acid encoding for a recombinase protein of the invention).
  • the offspring includes host organisms expressing the recombinase protein and further including the recognition sites, so that a site-specific DNA-recombination, like a tissue-specific conditional knock-out, is possible.
  • non-human host organisms may comprise a vector according to the invention or a nucleic acid according to the invention as described above that is, respectively, stably integrated into the genome of the host organism or individual cells of the host organism.
  • Preferred host organisms according to the present invention are plants, invertebrates and vertebrates, particularly Bovidae. Drosophila melanogaster, Caenorhahditis elegans. Xenopus laevis. medaka, zebrafish, or Mus musculus, or embryos of these organisms.
  • the present invention also provides a novel recombinase system suitable for producing a sitespecific recombination in cells of various cell types.
  • a novel recombinase system suitable for producing a sitespecific recombination in cells of various cell types.
  • Such a system includes the respective recombinase protein (one of SEQ ID NOs: 1 to 9) and at least two respective recognition sites as identified herein.
  • a diverse range of genetic manipulations can be realized, particularly rearrangements of the DNA fragments flanked by the recognition sites of the present invention in same orientation (excision), opposite orientation (inversion), or when one specific recognition site is present on each of two DNA molecules with one - if in circular form - in any orientation (integration).
  • Exemplary manipulations are the excision of a DNA segment that is flanked by two recognition sites oriented in the same direction mediated by the respective recombinase protein as identified herein.
  • the recombinase systems according to the invention provide the possibility to excise a target DNA, such as a stopper DNA fragment, flanked by two recognition sites, which target DNA is located 5’ of the gene and 3’ of the corresponding to the gene promoter. Without recombination, the stopper sequence prevents gene expression, whereas upon recombinase-mediated excision of the stopper via the two flanking recognition sites, the gene is located to the proximity of the promoter and will therefore be expressed.
  • promoter regions that regulate the expression of the recombinase allow, inter aha, conditional DNA recombination, when for example a tissue or organism-specific or inducible promoter region is used to express the recombinase protein.
  • the recombinase systems according to the invention are applicable for use in combination with other recombinase systems and become a particular valuable tool for genetic experiments where multiple recombinases are required simultaneously or sequentially.
  • the present invention further provides the use of a protein with recombinase activity, wherein the protein comprises an amino acid sequence exhibiting at least 85%, at least 90%, at least 95%, preferably at least 95%, even more preferably at least 99% amino acid sequence identity to one of the amino acid sequences according to SEQ ID NOs: 1 to 9 to catalyze a site-specific DNA recombination.
  • the aforementioned site-specific recombinase is thus used for site-specific DNA recombination at least one and preferably at least two recognition sites of the present invention that are essentially identical or essentially reverse complementary to each other.
  • the at least one recognition site comprises a nucleic acid sequence according to or reverse complementary to the nucleic acid sequence according to SEQ ID NOs: 10 to 17, respectively, or a nucleic acid sequence that is a functional mutant thereof.
  • the present invention provides a protein having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9, or a vector according to the invention, for use in medicine.
  • the protein or the vector is for use in treating a genetic disease or disorder in a subject.
  • the genetic disease or disorder is characterized by modification of the subject’s genome.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the recombinase protein of the present invention, the vector or the host cell of the present invention, or one or more nucleic acids according to the present invention, and optionally a pharmaceutically acceptable carrier.
  • compositions that contain a therapeutically active agent according to the invention may be in any form that is suitable for the selected mode of administration.
  • a pharmaceutical composition of the present invention is administered parenterally.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrastemal injection and infusion.
  • the therapeutically active agents as referred to herein include but are not limited to the recombinase proteins of the present invention and the recognition sites of the present invention.
  • the therapeutically active agents of the invention can be administered, as sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • the pharmaceutical compositions contain carriers (also termed vehicles) which are pharmaceutically acceptable for a formulation capable of being injected.
  • carriers also termed vehicles
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising the therapeutically active agents as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the therapeutically active agents can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be as solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by fdtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fdtered solution thereof.
  • solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. Multiple doses can also be administered.
  • the therapeutically active agents described herein may be formulated in any suitable vehicle for delivery. For instance, they may be placed into a pharmaceutically acceptable suspension, solution or emulsion. Suitable mediums include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • colloidal dispersion system may also be used for targeted gene delivery.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • each unit dosage of the genetically engineered DNA recombining enzyme expressing vector may comprise a composition including a viral expression vector in a pharmaceutically acceptable fluid at a concentration ranging from 10 11 to 10 16 viral genomes per ml, for example.
  • the effective dosages and the dosage regimens for administering a genetically engineered DNA recombining enzyme of the invention or of its subunits in the form of a recombinant polypeptide depend on the disease or condition to be treated and may be determined by the persons skilled in the art.
  • a physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the therapeutically active agents of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a composition of the present invention will be that amount of the delivery system which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above.
  • Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target.
  • the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a delivery system of the present invention to be administered alone, it is preferable to administer the delivery system as a pharmaceutical composition as described above.
  • kits comprising a therapeutically active agent as described herein.
  • the kit provides the therapeutically active agents prepared in one or more unitary dosage forms ready for administration to a subject, for example in a preloaded syringe or in an ampoule.
  • the therapeutically active agents are provided in a lyophilized form.
  • the present invention provides a method for treating or preventing a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a protein having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9, of the nucleic acid according to the invention, of the vector according to the invention, of the isolated host cell according to the invention, of the non-human host organism according to the invention, or of the pharmaceutical composition according to the invention.
  • the present inventors screened over 500 putative recombinase candidates of which a selection of 17 candidates were experimentally tested and eight novel recombinase systems were molecularly characterized in detail. To optimally use different recombinase systems, it is essential to characterize their applied properties. The determination of the in vivo recombinase activity may be crucial for planning an experiment.
  • the eight newly characterized recombinases showed varying activity on their predicted target sites in bacteria, from over 90% in the case of YR1, YR4, YR6 to as low as around 15% in the case of YR8 and YR9.
  • Possible explanations for this low activity may include the requirement for additional cofactors, or optimal temperature for more efficient recombination of the target site.
  • the specificity of the recombinases was profiled, providing a valuable overview of possible cross recombination events on all target sites.
  • the new recombinases were tested for their activity and compatibility in mammalian cells. All the recombinases showed high activity in a plasmid-based assay in HEK293T cells. Interestingly, YR8 showed high recombination rates in mammalian cells, whereas this recombinase had only weak activity in bacteria, suggesting that activity profiles of recombinases can vary in heterologous hosts.
  • Y-SSRs were identified by using tblastn from BLAST+ 2.10.1 (https://www.ncbi.nlm.nih.gov/books/NBK131777/) with the protein sequences of Cre, Vika (Karimova et al., 2012), Nigri, and Panto (Karimova et al. 2016) as references to search the NCBI nucleotide collection database (v5). The results were filtered for below 90% identity and a sequence length of 300 to 400 amino acids with GNU awk. Protein sequences were acquired with efetch (https://dataguide.nlm.nih.gov/edirect/efetch.html).
  • the phylogeny tree of known and putative recombinases was generated by performing an all- against-all pairwise sequence alignment of the protein sequences using EMBOSS needle (Needleman and Wunsch, 1970), followed by complete hierarchical clustering of the sequence dissimilarities. Visualization of the tree was done with R packages tidygraph and ggraph. For brevity, the tree was cut at the 98% sequence similarity, to represent almost identical proteins as a single node. Human and mouse genomic sequences with high similarity to potential target sites were identified using PatMaN (Priifer et al., 2008). The search was performed on half-sites only, allowing for up to 2 mismatches.
  • Genomic coordinates manipulation and sequence extraction steps were performed with the BEDTools suite (Quinlan and Hall, 2010).
  • recombinase expression was induced with increasing concentrations of L-arabinose (0, 1, 10 or 100 pg/ml medium) overnight in 6 ml culture volume.
  • the test digest was prepared for each induction level and recombination efficiency was estimated by agarose gel electrophoresis.
  • HEK293T cells were plated at a density of 2 x 10 5 cells per well in 24-well dishes and cultured in glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco®), supplemented with 10% fetal bovine serum (Invitrogen), 1% Penicillin- Streptomycin (10,000 U/ml, Thermo Fisher).
  • DMEM Modified Eagle’s Medium
  • Penicillin- Streptomycin 10,000 U/ml, Thermo Fisher.
  • HEK293T were washed once with PBS and then detached using Trypsin (Gibco). The cells were then resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco®) and analyzed with the MACSQuant® VYB Flow Cytometer (Miltenyi). Analysis of the data was performed using Flow JoTM 10 (BD).
  • pPGK-Recombinase-P2A-BFP vectors were used to produce lentiviral particles as described previously (Suriin et al., 2020).
  • NIH/3T3 mouse fibroblasts were seeded at a density of 4 x 10 4 cells per well in 24-well plates and grown at 37°C and 5% CO2. The next day, fibroblasts were transduced with the different lentiviruses with a MOI of 0,5 in order to achieve about 50% of infection rate.
  • the percentage of BFP expressing cells from at least 2 x 10 4 cells was tracked over the course of 15 days using MACSQuant® VYB Flow Cytometer (Miltenyi).
  • the recombinases and Cre, Vika, Panto, Dre (Anastassiadis et al., 2009), and VCre (Suzuki and Nakayama, 2011) were amplified from pEVO vectors, cleaned using the Isolate II PCR and Gel Cleanup Kit (Bioline) and mixed together in a 1: 1 ratio. All respective target sites were cloned into the pEVO vectors with Cold Fusion Cloning kit as previously described and the resulting vectors were also mixed with equal molar ratio.
  • Both, the mix of recombinases and pEVO backbones were digested with BsrGI and Sbfl and ligated in a single reaction, thus creating a library of different recombinase/ target site pairs. Plasmids were transformed in XL 1 -Blue electrocompetent E. coli cells and grown overnight with 100 pg/ml L-arabinose to induce recombinase expression. On the next day, plasmids were linearized with BsrGI and Seal and fragments carrying the recombinase sequence and target sites were isolated by agarose gel excision using the Isolate II PCR and Gel Cleanup Kit (Bioline).
  • the target sites were identified using exonerate v2.2.0 using the affmedocal model (https://www.ebi.ac.uk/about/vertebrate- genomics/software/exonerate).
  • the read ID and the matching references were then extracted from both alignments and combined in R with the dplyr package. Visualization of the data was performed with the R package ggplot2.
  • Example 7 New recombinases recombine their predicted target sites in bacteria
  • candidate recombinase was tested on the predicted target sequence.
  • candidates were selected and their coding sequence individually cloned into the L-arabinose inducible pEVO recombination reporter vector (Buchholz and Stewart, 2001) harboring two copies of the respective predicted target sites (Fig. IB).
  • the plasmids were then transformed into E. coli and cultured overnight in medium containing L-arabinose to induce recombinase expression.
  • successful recombination leads to excision of a -700 bp DNA fragment from the plasmid. This size difference was visualized by agarose electrophoresis of linearized plasmids (Fig. 1A).
  • YRl 2 recombinase showed a mostly constant recombination rate raging from -50% at 0 pg/ml L-arabinose and peaking at 70% when induced with 100 pg/ml of L-arabinose.
  • YR8 and YR9 showed the weakest activity and only recombined their target sites to 25% and 17%, respectively, at the highest L-arabinose concentration.
  • Recombinase YR9 was further subjected to substrate-linked directed evolution (Buchholz and Stewart, 2001), thereby managing to increase the activity of YR9 recombinase significantly.
  • the best performing clone named YR9.2 (SEQ ID NO: 9) showed about seven-fold improvement in activity in bacteria (Fig. 7A), and eight-fold improvement in mammalian cells (Fig. 7C).
  • Example 8 Profiling target-site selectivity of Cre-like recombinases
  • plasmid DNA was retrieved and fragments carrying the recombinase sequence on the 3’-end and target site(s) on the 5’-end were cut out.
  • a total of 417,769 reads was obtained containing both, the specified recombinases and target sites. All possible 169 combinations of Y-SSRs and target sites were identified with a minimum coverage of 224 reads.
  • the recombination rates for the individual recombinases on all the target sites was calculated, providing a specificity profile for each recombinase (Fig. 4B).
  • Example 9 Activity of recombinases in human cells
  • HEK293T cells were co-transfected with recombinase expression plasmids (as described in Example 3 for the mammalian recombination assay) alongside recombination reporter plasmids harboring the corresponding target sites (Fig. 5A).
  • the reporter plasmids the mCherry cassette, driven by a CAG promoter, is flanked by the target (lox) sites.
  • the mCherry cassette is deleted from the plasmid and the CAG promoter then drives the expression of a GFP cassette (Fig. 5A).
  • the activity of the recombinases on their predicted target sites can be visualized by fluorescent microscopy and quantified by flow cytometry.
  • all of the recombinases tested displayed GFP positive cells (Fig. 5B), demonstrating that these recombinases are active in HEK293T cells, whereas no GFP-positive cells were observed when co-transfections were done with an “empty” expression vector, lacking recombinase coding sequences (Fig. 5B).
  • Example 10 Influence of recombinase expression on cell proliferation
  • lentiviral vectors were constructed, which allow co-expression of the recombinases and tagBFP (Fig. 6A).
  • viral particles for overexpression of either Cre, an inactive Cre variant (CreY324F), Vika and tagBFP alone were used.
  • NIH3T3 cells were infected and the change in percentage of BFP -positive cells was monitored for 15 days. A reduction in BFP-positive cells over time indicates a negative effect on cell proliferation due to recombinase overexpression (Schmidt et al., 2000; Pugach et al., 2015).
  • Dre recombinase is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis Model Meeh, 2, 508-515.
  • PatMaN rapid alignment of short sequences to large databases. Bioinformatics, 24, 1530-1531.

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Abstract

La présente invention concerne le domaine des enzymes de recombinaison et propose un procédé de production d'une recombinaison d'ADN spécifique à un site, comprenant les étapes consistant à a) mettre en contact un acide nucléique comprenant au moins un premier et un second site de reconnaissance qui sont essentiellement identiques ou sensiblement inverses l'un par rapport à l'autre avec une protéine présentant une activité recombinase, et b) permettre à la protéine présentant une activité recombinase de produire la recombinaison d'ADN spécifique à un site, un site de reconnaissance comprenant un premier demi-site, un espaceur et un second demi-site, et sensiblement identique ou sensiblement inverse l'un à l'autre signifie que la séquence nucléotidique du premier et du second demi-site dans le premier site de reconnaissance peut dévier de deux nucléotides maximum de la séquence nucléotidique du premier et du second demi-site dans le second site de reconnaissance, la protéine présentant une activité recombinase comprenant une séquence d'acides aminés présentant au moins 80 % d'identité avec n'importe lequel des SEQ ID No : 1 à 9, et les au moins deux sites de reconnaissance comprenant une séquence d'acide nucléique correspondant ou étant complémentairement inverse à n'importe lequel des SEQ ID No : 10 à 17 ou à un mutant fonctionnel correspondant, le mutant fonctionnel comprenant une séquence d'acide nucléique présentant au moins 60 % d'identité de séquence avec des SEQ ID No : 10 à 17.
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