TW202507002A - Novel recombinase enzymes for site-specific dna-recombination - Google Patents

Abstract

The present invention is in the field of recombining enzymes and provides a method for producing a site-specific DNA-recombination, comprising 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, 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 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, 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, and wherein the at least two recognition sites comprise a nucleic acid sequence according to or reverse complementary to any one of SEQ ID NOs: 10 to 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: 10 to 17.

Description

Novel recombinase enzymes for site-specific DNA recombination

The present invention relates to the use of proteins having recombinase activity for catalyzing site-specific DNA recombination, and methods for producing site-specific DNA recombination. The present invention can be applied alone or in combination with other recombinase systems for gene manipulation, for example in medicine or medical research.

Site-specific recombinases (SSRs) are reliable tools for targeted modification of the genome with a variety of applications in research, medicine, and biotechnology. SSRs are evolutionarily and mechanistically divided into two distinct enzyme families: tyrosine and serine recombinases (Meinke et al., 2016). Nevertheless, unlike systems that modify DNA (such as CRISPR-Cas and other nuclease-based technologies), SSRs can catalyze the cleavage and immediate resealing of DNA strands without the help of additional proteins (Meinke et al., 2016). Although nuclease-based approaches are being vigorously developed to expand their utility (i.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 generally introduce DNA nicks and rely on the cell's intrinsic repair machinery (Meinke et al., 2016). In contrast, SSRs can operate autonomously, and the outcome of recombination is very predictable. In addition, the relatively small size of most SSRs makes them more convenient for different delivery vectors (Meinke et al., 2016).

Tyrosine recombinases (Y-SSRs) such as Cre and Flp are widely used for genome engineering due to their simplicity and ability to perform efficient genome modification in heterologous hosts (Sauer et al., 1988). Therefore, these SSRs are widely used to model and understand how these types of enzymes work. The Cre/loxP system is derived from bacteriophage P1 and consists of the recombinase Cre and a 34-bp target site loxP (Sternberg et al., 1981). The loxP site is a palindromic sequence with two 13-bp inverted repeats separated by an 8 bp spacer. Each half-site is bound by a recombinase unit (protomer), forming a tetrameric complex at the two loxP sites, which together catalyze strand exchange within the spacer region (Duyne and Hamilton, 1981). Depending on the relative orientation of the spacer, Cre can perform various reactions, such as excision, incorporation, inversion, and translocation (Meinke et al., 2016). In addition, when two heterospecific target sites are present in the genome, Cre alone or in combination with other SSRs can perform recombinase-mediated cassette exchange (RMCE) to precisely replace DNA fragments (Meinke et al., 2016; Anderson et al., 2012; Minorikawa and Nakayama, 2011).

In addition to the most widely used tyrosine site-specific recombinase systems, Cre/loxP and Flp/FRT systems, other recombinase systems are also known in the art. US 7,422,889 and US 7,915,037 disclose the so-called Dre/rox system, which comprises a Dre recombinase isolated from the enterobacterial phage D6, which recognizes a site called the rox site. More known recombinase systems are the VCre/VloxP system isolated from the Vibrio plasmid p0908 and the sCre/SloxP system (WO 2010/143606 A1; Suzuki and Nakayama, 2011).

Other site-specific DNA recombinase systems are the Nigri/nox system disclosed in EP 2 877 585 B1, the Vika/vox system disclosed in EP 2 690 177 B1, and the Panto/pox system disclosed in EP 3 263 708 B1.

The recombinase systems known in the art show different activities in cells of different origin. For many applications (e.g. the production of transgenic animals with conditional gene knockouts), two or more recombinase systems are used in combination with each other. However, emerging complex genetic studies and applications require the simultaneous use of multiple recombinases. At the same time, not all well-described site-specific recombinases are equally applicable to all model organisms due to, for example, genome specificity (hidden off-target activities at the identified target site). For this reason, it is important to select the best recombinase depending on the target organism or the experimental setup. Therefore, there is a need in the art to provide additional specific recombinase systems that can be used to catalyze site-specific DNA recombination on short targets in a variety of cell types with high activity and low toxicity.

Therefore, one object of the present invention is to provide a novel recombinase system for site-specific gene recombination that can be used in various cell types. Another object of the present invention is to provide a novel, highly specific recombinase system for site-specific gene recombination with little or substantially no inhibitory effect on cell proliferation.

The basic object of the present invention can be solved by providing a method for generating site-specific DNA recombination. According to the present invention, the method for generating site-specific DNA recombination comprises the following steps: a) contacting a nucleic acid comprising at least a first recognition site and a second recognition site that are substantially identical or substantially inversely complementary to each other with a protein having recombinase activity, and b) allowing the protein having recombinase activity to generate site-specific DNA recombination. According to the method, the recognition site comprises a first half site, a spacer, and a second half site, and substantially identical or substantially inversely complementary to each other means that the nucleotide sequence of the first half site and the second half site in the first recognition site may differ from the nucleotide sequence of the first half site and the second half site in the second recognition site by at most two nucleotides.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 16 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 16 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 16.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 12 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 12 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 12.

According to one aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 10 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 10.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 11 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 11 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 11.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 13 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 13 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 13.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 5, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 14 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 14 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 14.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 6, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 8, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 17 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 17 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 17.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

According to one embodiment, the protein having recombinase activity comprises at least two protein monomers.

According to one embodiment, the recombinant nucleic acid sequence is present in a cell.

According to a preferred embodiment, the method further comprises the step of introducing a nucleic acid encoding a protein having recombinase activity into the cell.

According to another embodiment, the cell comprises a nucleic acid encoding a protein having recombinase activity.

According to a preferred embodiment, 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.

According to yet another embodiment, the cell is a eukaryotic cell or a bacterial cell.

According to another aspect, 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 generating site-specific DNA recombination.

According to yet another aspect, the present invention provides a use of a protein having recombinase activity for catalyzing site-specific DNA recombination at recognition sites that are substantially identical or substantially reverse complementary to each other, wherein the recognition sites comprise a first half site, a spacer, and a second half site, and wherein substantially identical or substantially reverse complementary to each other means that the nucleotide sequences of the first half site and the second half site in the first recognition site may differ from the nucleotide sequences of the first half site and the second half site in the second recognition site by at most two nucleotides.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 16 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 16 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 16.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 12 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 12 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 12.

According to one aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 10 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 10.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 11 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 11 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 11.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 13 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 13 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 13.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 5, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 14 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 14 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 14.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 6, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 8, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 17 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 17 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 17.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

According to another aspect, the present invention provides a nucleic acid having a length of no more than 40 base pairs and comprising: (i) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 16 or a nucleic acid sequence that is reverse complementary to it; or (ii) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 12 or a nucleic acid sequence that is reverse complementary to it; or (iii) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 10 or a nucleic acid sequence that is reverse complementary to it; or (iv) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 11 or a nucleic acid sequence that is reverse complementary to it; or (v) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 13 or a nucleic acid sequence that is reverse complementary to it; or (vi) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 14 or a nucleic acid sequence that is reverse complementary to it; or (vii) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 15 or a nucleic acid sequence that is reverse complementary to it; NO: 15 has at least 80% sequence identity or a nucleic acid sequence that is reverse complementary to it; or (viii) a nucleic acid sequence that has at least 80% sequence identity with SEQ ID NO: 17 or a nucleic acid sequence that is reverse complementary to it.

According to another aspect, the present invention provides a vector comprising at least one, and preferably at least two, substantially identical or substantially reverse complementary nucleic acids of the present invention, wherein the DNA segment is preferably flanked by two substantially identical or substantially reverse complementary nucleic acids.

According to one embodiment, 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: 1-9.

According to another aspect, 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 with any one of SEQ ID NOs: 1 to 9.

According to a preferred embodiment of the present invention, use of the carrier of the present invention in the method of the present invention is provided.

According to yet another aspect, the present invention provides a protein or vector of the present invention 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 use in medicine. Preferably, the protein or vector is used to treat a genetic disease or condition in an individual. More preferably, the genetic disease or condition is characterized by modification of the genome of the individual.

According to another aspect, the present invention provides an isolated host cell comprising the following recombinant DNA fragments: at least one, and preferably at least two nucleic acids of the present invention; and/or the vector of the present invention.

According to one embodiment, the isolated host cell further comprises (i) a nucleic acid encoding 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 of the present invention.

According to another aspect, the present invention provides a non-human host organism comprising: (i) at least one, and preferably at least two nucleic acids of the present invention; or (ii) a vector of the present invention.

According to one aspect, the present invention provides a pharmaceutical composition comprising a protein having at least 80% identity with 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; a nucleic acid of the present invention; a vector of the present invention; an isolated host cell of the present invention, or a non-human host organism of the present invention, and optionally a pharmaceutically acceptable excipient.

Further aspects and embodiments of the present invention will become apparent from the appended claims and the following detailed description.

Sequence Listing SEQ ID NO: 1 (YR6): MIENQLSLLGDFSGVRPDDVKAAVQAAQKKGINVAENEQFKAVFDHLLGEFKKREERYSPNTLRRLESAWTCFVDWCLAHHRHSLPATPDTVEAFFIERSETLHRNTLSVYRWAISRVHRVAGCPDPCLDIYVEDRLKAISRKKVREGETVKQASPFNEQHLLKLTSLWYLSDKLLLRRNLALLAVAYESM LRAAELANIRVSDLELSGDGTAVLTIPITKTNHSGEPDTCILSQDVVSSLLMDYTEAGRLDMRADGYLFVGISKHNTCINPKRDADTGECLHKPITTKTVEGVFYSAWQALELERQGVKPFTAHSARVGAAQDLLKKGYNTLQIQQSGRWSSGTMVARYGRAILARDGAMAHSRVKTRNVSIDWGSGGSKNTI SEQ ID NO: 2 (YR1): MIENQLSLLGDFTDVRPSDVKTAIEKAQKKGVVVAEDHVFQAAINHLLNEFKKREDRYSPNTLRRLESAWGCFVEWCLDNKRHSLPASPDTTEKFLIYKAESVHRNTLSIYKWAISRVHRVAGCPNPCNDVFVEDRYKALVRVKVQSGEAIKQASPFNELHLNALVEKWKQHERVLERRNLALLGVAYE SMLRAAELANIKLSDIELAGDGTAILTIPITKTNHSGDPDTCILSHDVVGLIMDYIEAGELHLKQDGYLFTGVSKHNKCTKPKVDKETGEVTYKPITTKTVEGIFKAAWSELELGRQGVKPFTGHSARVGATQDLLRKGYNTLQIQQSGRWSSEVMVARYGRAILARESAMAQSRVKTKNIDLSWGSKR SEQ ID NO: 3 (YR2): MNNEIIHSTSNTSLSQYPAEHIQKALANGDIPTDSHLFQSAADHLINEYRSREGLAENTFLALDTGWSLFVDWCVEHNRVSLPASSKTVEDYVKSISKVLRRNTIRVRKWAITKIHKICGLPNPFDSEFVTQTISGIYKKKLHEDEITEQASPFNETHLEALELLYADSTLKKRRDLLM MTIAYESLLRSSELCNIKLKHLRLIGKEIHITIPVTKTNHSGNPDVVALSEHATNQVLEYLNDHSMKLSGDGYLFRRLRRNGLAYPSTKQAMSNQSVIDVFNSVHNDLGGSDVLHCEPFTSHSCRVGGAQDLLAAGYSILQVQQAGRWSDPSMVYRYGRGIFAAKSAMAHFRRNRQKPRN SEQ ID NO: 4 (YR4): MSELLPLTPLTVDRNSDITERLRQFVQDKEAFSPNTWRQLLSVMRICNRWSEDNQRSFLPMSADDLRDYLSFLAESGRASSTVTSHAALISMLHRNAGLPVPNVSPLVFRTMKKINRVAVINGERAGQAVPFRLSDLLALDEEWSGSDNLQALRDLAFLHVAYATLLRISEL SRLRVRDVMRAGDGRIILDVAWTKTIVQTGGLIKALSARSTQRLEEWIEASGLSSQPDAWLFTAVHRSGRPLIAEKPMSTRALEQIFSRAWRTAGKEGAVKANKNRYTGWSGHSARVGAAQDMADKGYPIARIMQEGTWKKPETLMRYIRHVDAHKGAMVEFMEQYGDPDYPG SEQ ID NO: 5 (YR8): MGKLSPTNQTLPAIQAEEDVLARLKEFVQDKEAFSPNTWRQLMSVMRICHRWSIENSRSFLPMLPADLRDYLNWLQESGRASSTIATHGSLISMLHRNAGLIPPNTSPLVFRAVKKINRVAVVTGERTGQAVPFRLEDLLELDALWSDSISLRHKRDLAFLHVAYSTLLRISEL ARLRVRDISRATDGRIILNVSYTKTIVQTGGLIKSLSSQSSRRLTEWMSVSGINAEPDAFLFCPVHRSGSATLSVTRPLSTPAIESIFAQAWLTIGAGEPIIPNKGRYTAWTGHSARVGAAQDMAGRGYAVAQIMQEGTWKKPETLMRYIRNLQAHEGAMTDIMEKSTLDHNNTK SEQ ID NO: 6 (YR9): MLAVLHEDLERAAAYKKAARAAATHRAYNSDWIIYTDWCRTRGLEAMPAHPEQIAAFVANQAASGLKPSTIERRVAAIGHHHRTSNYPAPAAHPEAGGLREALAGIRNEKRAKKTRKEPADATALRDMLAQIKGDGLRARRDRAALAIGMAAALRRSELVAL TLENVGILEHGIELYLGATKTDQAGEGTTIAIPEGTRLRPKALLLDWISAVRVLEAGVVRTPAQEAAVPLFRRLTRSDQLTGEPMSDKAVARLVKRYAGAAGYDAAKFSGHSLRAGFLTEAANQGATIFKMQEVSRHKTVQVLSDYVRSADRFRDHAGERFL SEQ ID NO: 7 (YR11): MVGGMSFVRRDVVVIPDNPDLNDEVIRNLNAFMKDREAFAENTWKQLMMAVRLWCHWCIAKGRPYLPVDADYLRDYLLELHDNGLAPATISNYAAMLNLLHRQAGLIPAGESQKVKRVLKKISRTSIIKGETVGQAIPFRIADLNQVDEAWEASDRLKTIRNLAFLFVAYNT LLRISNIAHLKVKDLAFDHDGSVMLNIGYTKTLVDGKGITKALSPRASARVLKWLHVSGLLDHPDAYLFCKVYRTNKASVTTDKPLTLHPLESIFSEAWAVIHGEKVGIKNKGRYATWTGHSARVGAAQDMTESGYSLAQIMHEGTWKAPKTVLGYTRNLEAKKSVMIDLVG SEQ ID NO: 8 (YR12): MTEMIVANPLLAQFSASDDISAKLASFVRDREAFSSNTWRQLLSVMRICWRWSEENHRSFLPMAPEDLRDYLLHLQCIGRASSTISTHAALISMLHRNAGLVPPNVSPDVFRVVKKINRAAVIAGERTGQAVPFCRQDLKKLDTAWQGSPRLQQLRDLAFMHVAYSTLLRL SELSRLRVRDISRAADGRMILDVAWTKTIVQSGGIVKALSTQSSQRLTDWIVAAGLTGEPDAMIFCPVHRSNRMTKKIFSPMSTPCLEDIFLRAREAAGVAALSRTNKGRYAGWSGHSARVGAAQDMARKGFSVAQIMQEGTWTRTETVMRYIRMVEAHKGAMIGLMEEDE SEQ ID NO: 9 (YR9. 2): MLAILHEDLERAAAYKKAARAAATHRAYNSDWIIYTNWCRTRGLEAMPAHPEQIAAFVANQAASGLKPSTIERRVAAIGHYHRTSNYPAPAAHPEAGGLREVLAGIRNEKRAKKTRKEPADATALRDMLAQIKGDGLRARRDRAVLAIGMAAALRRSELVAL TLENVGILEHGIELYLGATKTDQAGEGTTIAIPKGTRLRPKALLLDWISAVRVLEAGVVRTPAQEAAVPLFRRLTRSDQLTGEPMSDKAVARLVKRYAGAAGYDAAKFSGHSLRAGFLTEAANQGATIFKMQEVSRHKTVQVLSDYVRSADRFRDHAGERFL SEQ ID NO Identification sites and spacer sequences are bold and underlined The site is called 10 TCAATTTCCGAGAATGACAGTTCTCAGAAATTAA lox6 11 TCAATTTCTGAGA AGTGTAAT TCTCAGAAATTGA lox1 12 ATAACTTGAGATAACGCGAATTATCACAAGTTAA lox2 13 TGACTTCGTATA AGAACAAT TATACGAAGTTA lox4 14 CTAACTTTATATA AGTCCCAT TATATAATGTTAG lox8 15 CGTCTGTCCGATA ATCTTTCT TATCGGACATACT lox9 16 GTCAACTTCACATA AGTGTTATTATGTGGAGTTGAC lox11 17 ATAACCTAATATA ATTGTATT TATATTAGGTCAG lox12 18 TTAATTTCTGAGAACTGTCATTCTCGGAAATTGA lox6 rev. compl.

Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the scope of the attached patent application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kölbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated number or step or group of numbers or steps but not the exclusion of any other number or step or group of numbers or steps. In the following paragraphs, various aspects of the invention are further defined. Each aspect so defined may be combined with any other aspect(s) unless expressly indicated to the contrary. Any feature indicated as optionally present, preferably or advantageous may be combined with any other feature indicated as optionally present, preferably or advantageous.

Several documents are cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, specifications, etc.), whether supra or infra, is incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. Some of the documents cited herein are characterized as "incorporated by reference." In the event of a conflict between the definitions or teachings of these incorporated references and the definitions or teachings cited in this specification, the text of this specification controls.

The elements of the present invention are described below. These elements are listed in specific embodiments; however, it should be understood that they may be combined in any manner and in any amount to create additional embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to the embodiments expressly described. This description should be understood to support and encompass embodiments that combine the expressly described embodiments with any number of disclosed and/or preferred elements. In addition, unless the context indicates otherwise, any arrangement and combination of elements described throughout this application should be considered to be disclosed by the description of this application. Definitions

Some definitions of terms frequently used in this specification are provided below. These terms will have their respective defined meanings and preferred meanings in the remainder of the specification, in each case where they are used.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

"Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison range, wherein the portion of the sequence in the comparison range may include additions or deletions compared to the reference sequence (which does not include additions or deletions) to achieve optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison range, and multiplying the result by 100 to obtain the percentage of sequence identity.

The term "identical" is used herein in the context of two or more nucleic acid or polypeptide sequences to refer to two or more sequences or subsequences that are identical, i.e., contain the same sequence of nucleotides or amino acids. Sequences are "identical" to one another if they have a certain percentage of identical nucleotide or amino acid residues. According to the present invention, at least 60% identity includes at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, 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 to a specified sequence when compared and aligned to obtain maximum correspondence over a comparison range or a specified region. These definitions also relate to the complementary sequences of the test sequences. Thus, the term "at least XY% sequence identity" is used throughout the specification with respect to polypeptide and polynucleotide sequence comparisons. This term preferably refers to 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% sequence identity to a respective reference polypeptide or to a respective reference polynucleotide.

In the context of the present invention, 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 the 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.

Likewise, in the context of the present invention, a nucleic acid sequence having at least 60% sequence identity with a given SEQ ID NO, or a nucleic acid sequence that is the reverse complement thereof, preferably means a sequence of the nucleic acid 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 with a given SEQ ID NO, or a nucleic acid sequence that is the reverse complement of the SEQ ID NO.

The term "sequence comparison" as used herein refers to a process in which one sequence serves as a reference sequence and a test sequence is compared to it. When a sequence comparison algorithm is used, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters are usually used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters. If two sequences are compared and a comparison reference sequence to which the percentage of sequence identity is calculated is not specified during comparison, if not otherwise specifically stated, the sequence identity will be calculated with reference to the longer of the two sequences to be compared. If a reference sequence is specified, then if not otherwise specifically stated, the sequence identity is determined based on the full length of the reference sequence indicated by one of the SEQ ID NOs of the present invention.

Methods for comparing sequences are well known in the art. Optimal alignment of sequences for comparison can be achieved, 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 by similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The algorithm first identifies high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold score T when aligned with a word of the same length in a database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word results act as seeds for initiating searches to find longer HSPs containing them. The word results are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of word results in each direction ceases if: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score falls to zero or below due to the accumulation of one or more negative scoring residual 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. The BLASTN program (for nucleotide sequences) uses the defaults wordlength (W) of 11, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses a default word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) with a comparison (B) of 50, an expectation value (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). A similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability of a match occurring accidentally between two nucleotide or amino acid sequences. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when the test nucleic acid is compared 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 terms "nucleic acid" and "nucleic acid molecule" are used synonymously herein and are understood to be widely accepted in the art, i.e., single-stranded or double-stranded oligomers or polymers of deoxyribonucleotides or ribonucleotide bases or both. The term "nucleic acid" as used herein includes not only deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), but also all other linear polymers in which the bases are adenine (A), cytosine (C), guanine (G), and thymine or uracil (U) arranged in a corresponding sequence (nucleic acid sequence). The present invention also encompasses corresponding RNA sequences in which thymine is replaced by uracil, complementary sequences, and sequences with modified nucleic acid backbones or 3' or 5'-ends. However, nucleic acids in the form of DNA are preferred.

The term "protein with recombinase activity" includes any enzyme that can manipulate the structure of the genome. More specifically, the term refers to each enzyme that can catalyze the reorganization selected from excision, incorporation, inversion and translocation reactions. Proteins with recombinase activity are well known in the art (see the discussion of the background above) and specifically include DNA recombinases, such as site-specific recombinases and more specifically tyrosine recombinases. Proteins with recombinase activity can exist in the form of monomers, dimers or tetramers. Dimers and tetramers can respectively include two or four monomers (homodimers or homotetramers) of the same protein with recombinase activity, or two or more different monomers (heterodimers or heterotetramers) with recombinase activity.

As used herein, the term "recognition site" (sometimes also referred to as "target site") refers to a specific nucleotide sequence that is recognized by the recombinase enzyme and at which DNA cleavage and strand exchange occurs. These sequences are typically between 30 and 200 base pairs in length and consist of two inverted repeat recombinase binding regions flanked by 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 binds to a 34 base pair loxP target sequence. The loxP recognition site contains two 13 base pair inverted repeat Cre binding elements flanked by 8 base pair spacer regions. The left half site is a 13 base pair binding element on the left side of the spacer, while the right half site is a 13 base pair binding element on the right side of the spacer. Depending on the number and relative orientation of the recognition sites and their spacers, DNA recombinases perform excision, incorporation, inversion or substitution of genetic content (reviewed by Meinke et al., 2016). Therefore, a "recognition site" according to the present invention is a nucleotide sequence comprising a first half site, a second half site and a spacer separating the first half site and the second half site.

For recombination events to occur, the recombinase complex recognizes the first recognition site and the second recognition site on the DNA double strand. The recognition sites are also called upstream recognition sites and downstream recognition sites, depending on their location on the DNA double strand.

In a symmetric recognition site, 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 sequence). In an asymmetric recognition site, the first half site (e.g., the left half site) and the second half site (e.g., the right half site) are not identical and are 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, inserted, deleted or replaced from the nucleic acid sequence referred to by the term. In the case of polypeptides and proteins, the term "functional mutant" means that one or more amino acids can be added, inserted, deleted or replaced to the amino acid sequence referred to by the term. Preferred mutations are point mutations or exchanges of specific nucleic acids or amino acids. Specifically, in the case of functional mutants of recognition sites, it is known that the exchange of spacer regions of the recognition sites will not affect its activity as a specific recombinase target. Therefore, the functional mutants of the recognition sites explicitly include mutations in the spacer regions until the complete replacement of the spacer regions. The functional mutants of the recognition sites also include mutations in the half sites of the recognition sites. For example, the functional mutant of the identification site disclosed herein may include one, two, three, four, five, six, seven or eight mutations in its nucleotide sequence compared to the reference nucleotide sequence from which the functional mutant is derived. According to a preferred embodiment of the present invention, the functional mutant of the identification site includes one or two mutations in the nucleotide sequence of the first half site, the nucleotide sequence of the second half site, or the nucleotide sequence of both half sites from which the functional mutant is derived.

The term "reporter vector" as used herein includes plasmids, viruses or other nucleic acid vectors, which contain the nucleic acid sequence according to the present invention by genetic recombination (recombinantly), for example by insertion or incorporation of the nucleic acid sequence. Prokaryotic vectors as well as eukaryotic vectors (e.g. artificial chromosomes, such as YAC (yeast artificial chromosomes)) are suitable for use in the present invention.

As used herein, the term "therapeutically effective amount" means that amount of an active compound or agent that will elicit the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, physician or other clinician, including a reduction in symptoms of the disease or condition being treated.

The term "pharmaceutical composition" as used herein refers to a substance and/or combination of substances used to identify, prevent or treat a disease or tissue state. A pharmaceutical composition is formulated to be suitable for administration to a patient to prevent and/or treat a disease. In addition, a pharmaceutical composition refers to a combination of an active agent and an inert or active carrier so that the composition is suitable for therapeutic use. Such a carrier is also referred to as pharmaceutically acceptable. The pharmaceutical composition can be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous or vaginal administration depending on its chemical and physical properties. Pharmaceutical compositions include solid, semisolid, liquid, and transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powders, granules, pills, capsules, blister tablets or transdermal therapeutic systems. Also included are liquid compositions selected from the group consisting of solutions, syrups, infusion solutions, extracts, solutions for intravenous administration, solutions for infusion or solutions of the carrier system of the present invention. Semisolid compositions that can be used in the context of the present invention include emulsions, suspensions, creams, lotions, gels, globules, buccal tablets and suppositories.

As used herein, the term "pharmaceutically acceptable" encompasses both human and veterinary uses: for example, the term "pharmaceutically acceptable" encompasses a compound that is acceptable in veterinary medicine or a compound that is acceptable in human medicine and healthcare.

As used herein, the term "individual" refers to an animal, preferably a mammal, and most preferably a human. Description of the Embodiments

According to the present invention, a method for generating site-specific DNA recombination comprises the steps of: a) contacting a nucleic acid comprising at least a first recognition site and a second recognition site that are substantially identical or substantially reverse complementary to each other with a protein having a recombinase, and b) allowing the protein having recombinase activity to generate site-specific DNA recombination.

According to the method and as defined above, the identification site is a nucleotide sequence comprising a first half site, a second half site, and a spacer separating the first half site and the second half site.

As used in the context of the present invention, the term "substantially identical" or "substantially complementary to each other" means that the nucleotide sequence of the first half site and the second half site in the first recognition site may differ from the nucleotide sequence of the first half site and the second half site in the second recognition site by at most two nucleotides. As an example, it refers to the recognition sites identified herein and as shown in Figure 4B. Taking the recombinase YR6 (SEQ ID NO: 1) as an example, its recognition site has the sequence of TCAATTTCCGAGA ATGACAGT TCTCAGAAATTAA (SEQ ID NO: 10), wherein the spacer sequence is shown in bold and underlined. In the case where the first recognition site and the second recognition site are identical, both recognition sites have the sequence of SEQ ID NO: 10. In the case where the sequences are reverse complements of each other, 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). In the case of substantially identical sequences, one of the recognition sites has the sequence of SEQ ID NO: 10 and the other has SEQ ID NO: 10 with at most two different nucleotides in the sequence of the first and second half sites. This means that the first half of the recognition site comprises a nucleotide sequence that differs by two nucleotides from the nucleotide sequence of the first half of the other recognition site, while the second half and the spacer are identical in both, the first half of the recognition site comprises a nucleotide sequence that differs by one nucleotide from the nucleotide sequence of the first half of the other recognition site, while the second half of the same recognition site comprises a nucleotide sequence that differs by one nucleotide from the nucleotide sequence of the second half of the other recognition site, and the spacer sequence is identical in both, or the second half of the recognition site comprises a nucleotide sequence that differs by two nucleotides from the nucleotide sequence of the second half of the other recognition site, and the first half and the spacer of the two recognition sites are identical. The same principle applies to the case where the two recognition sites comprise sequences that are essentially reverse complements of each other. In this case, the reverse complement sequence must be compared to the non-reverse complement sequence. Taking the recognition sites of YR6 as an example, the first recognition site has the sequence of SEQ ID NO: 10, and the other recognition site is its reverse complement sequence, i.e., SEQ ID NO: 19. In the case where the two recognition sequences are substantially reverse complements of each other, the first recognition site may have the sequence of SEQ ID NO: 10, and the second recognition site may have the sequence of SEQ ID NO: 19, provided that SEQ ID NO: 19 contains two different nucleotides with respect to the sequence of the first half site and the second half site, but is not in the sequence of the spacer. Similar to the example above for substantially identical recognition sites, the two different nucleotides may be in the first half site, in the second half site, or one different nucleotide in each of the first half site and the second half site.

According to one embodiment of the present invention, the first recognition site and the second recognition site are identical or reverse complementary to each other in their half site sequences, meaning that the first recognition site and the second recognition site do not have any sequence deviation between their first half sites and between their second half sites. In such an embodiment, the spacer sequences of the first recognition site and the second recognition site are not necessarily identical or reverse complementary to each other. According to another embodiment of the present invention, the first recognition site and the second recognition site are identical or reverse complementary to each other in their entire sequence including the first half site and the second half site and the spacer, meaning that the first recognition site and the second recognition site do not have any sequence deviation between each other.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 16 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 16 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 16.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 12 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 12 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 12.

According to one aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 10 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 10.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 11 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 11 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 11.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 13 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 13 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 13.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 5, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 14 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 14 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 14.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 6, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 8, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 17 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 17 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 17.

According to another aspect of the method of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

The nucleic acid encoding the recognition site according to the invention comprises a maximum of 40, preferably 34 base pairs. This nucleic acid of the invention comprises the recognition site of the recombinase protein of the invention. Therefore, the present invention provides a nucleic acid having a length of no more than 40 base pairs and comprising the following: (i) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 16 or a nucleic acid sequence that is reverse complementary to it; or (ii) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 12 or a nucleic acid sequence that is reverse complementary to it; or (iii) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 10 or a nucleic acid sequence that is reverse complementary to it; or (iv) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 11 or a nucleic acid sequence that is reverse complementary to it; or (v) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 13 or a nucleic acid sequence that is reverse complementary to it; or (vi) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 14 or a nucleic acid sequence that is reverse complementary to it; or (vii) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 15 or a nucleic acid sequence that is reverse complementary to it; NO: 15 has at least 80% sequence identity or a nucleic acid sequence that is reverse complementary to it; or (viii) a nucleic acid sequence that has at least 80% sequence identity with SEQ ID NO: 17 or a nucleic acid sequence that is reverse complementary to it.

In the methods of the present invention, a protein having recombinase activity and comprising an amino acid sequence having at least 80% identity with a given SEQ ID NO preferably means that the 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 with the given SEQ ID NO.

Similarly, in the methods of the present invention, a nucleic acid sequence having at least 60% sequence identity with a given SEQ ID NO, or a nucleic acid sequence that is the reverse complement thereof, preferably means that the 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 with a given SEQ ID NO, or a nucleic acid sequence that is the reverse complement of the SEQ ID NO.

In the method of the present invention, the recombinase protein is brought into contact with at least two of its recognition sites, preferably inside the cell. When the recombinase protein binds to the recognition sites, site-specific DNA recombination occurs. For example, the recombinase YR6 having SEQ ID NO: 1 is brought into contact with at least two lox6 sites having SEQ ID NO: 10 inside the cell. When the recombinase protein binds to the lox6 sites, site-specific DNA recombination occurs.

The methods of the invention may be performed in vitro or in vivo. Where the invention is practiced in animals (including humans), it may be used for non-therapeutic purposes. According to one embodiment, the method is not intended for therapeutic treatment of humans or animals.

The method is applicable to all fields where site-specific recombinases are routinely used (including induced knockout or insertion mice and other transgenic animal models). In the preferred method of the present invention, site-specific recombination results in DNA incorporation, deletion, inversion, translocation or exchange. According to one embodiment of the present invention, the method is used to create an animal model, which can be used in biomedical research, for example as a model of human disease.

According to one embodiment of the present invention, the protein with recombinase activity exists as a monomer. According to a preferred embodiment of the present invention, the protein with recombinase activity comprises at least two protein monomers, i.e., in the form of a dimer (dimer). Such a dimer can comprise two monomers of the same type (i.e., two identical protein monomers, homodimers), or two monomers of different types (i.e., two different protein monomers, heterodimers). According to another preferred embodiment of the present invention, the protein with recombinase activity comprises at least four protein monomers, i.e., in the form of a tetramer (tetramer). Such a tetramer can comprise four monomers of the same type (i.e., four identical protein monomers, homotetramers), or monomers of different types, such as two, three or four different monomers (heterotetramers). According to a particularly preferred embodiment, the protein having recombinase activity comprises a heterodimer or heterotetramer comprising different protein monomers, i.e., a heterodimer or a heterotetramer.

According to a preferred embodiment of the present invention, the protein having recombinase activity is a recombinase, more preferably a DNA recombinase.

According to a preferred embodiment, the nucleic acid sequence to be recombined already exists in the cell. Alternatively, the nucleic acid sequence to be recombined can be introduced into the cell by conventional methods known to those of ordinary skill (e.g., by recombinant technology).

According to one embodiment, the nucleic acid sequence encoding the recombinase protein of the present invention is already present in the cell or is introduced into the cell by conventional means known to those of ordinary skill (such as by recombinant technology). Therefore, such a method further comprises the step of introducing the nucleic acid encoding the recombinase protein of the present invention into the cell.

Alternatively, according to one embodiment of the present invention, the cell already contains a nucleic acid encoding a protein of the present invention having recombinase activity.

Regarding the expression of nucleic acid encoding a protein with recombinase activity, the nucleic acid encoding the recombinase protein further comprises a regulatory nucleic acid sequence, preferably a promoter region. Therefore, the expression of nucleic acid encoding a protein with recombinase activity is activated or regulated by activating the regulatory nucleic acid sequence. Thus, in order to induce DNA recombination, the regulatory nucleic acid sequence (preferably the promoter region) is activated to express the gene encoding the recombinase protein. Preferably, the regulatory nucleic acid sequence (preferably the promoter region) is preferably introduced into the cell together with the sequence encoding the recombinase protein, or the regulatory nucleic acid sequence is already present in the cell at the beginning of the method of the present invention. In the second case, only the nucleic acid encoding the recombinase protein is introduced into the cell (and is controlled by the regulatory nucleic acid sequence).

The term "regulatory nucleic acid sequence" as used herein refers to the gene regulatory region of DNA. In addition to the promoter region, this term also includes operator regions farther away from the gene and nucleic acid sequences that affect gene expression, such as cis elements, enhancers or introns. The term "promoter region" as used herein refers to a nucleotide sequence on DNA that allows a gene to be regulated and expressed. The promoter region allows the nucleic acid encoding the corresponding protein to be regulated and expressed. The promoter region is located at the 5' end of the gene, therefore before the RNA coding region. Both bacterial and eukaryotic promoters are applicable to the present invention.

According to one embodiment of the present invention, the recognition site is incorporated into a cell or introduced into a cell (preferably by recombinant technology). Therefore, the method of the present invention may include the step of introducing the following nucleic acids into a cell: a)     a first nucleic acid (first recognition site) comprising a nucleic acid sequence according to one of SEQ ID NOs: 10 to 17 or a reverse complement thereof; or a nucleic acid sequence that is a functional mutant thereof; b)     a second nucleic acid (second recognition site) comprising a nucleic acid sequence that is substantially identical to or substantially reverse complement to the nucleic acid sequence of the first nucleic acid (first recognition site).

According to a preferred embodiment of the present invention, a nucleic acid encoding the recombinase protein of the present invention and at least two recognition sites are introduced into a cell. This method comprises the following steps: a)     introducing the following nucleic acids into a cell: (i)    a first nucleic acid encoding a recombinase protein, wherein the nucleic acid is introduced into DNA so that a regulatory nucleic acid sequence (preferably a promoter region) controls the expression of the nucleic acid encoding the recombinase protein, (ii)   a second nucleic acid comprising a nucleic acid sequence according to one of SEQ ID NOs: 10 to 17 or a reverse complement thereof; or a nucleic acid sequence that is a functional mutant thereof; (iii)  a third nucleic acid comprising a nucleic acid sequence that is substantially identical to or substantially reverse complement to the nucleic acid sequence defined in (ii), and b)     activating the regulatory nucleic acid sequence (preferably a promoter region) to induce the expression of the first nucleic acid for use in synthesizing a protein having recombinase activity.

According to this method, a nucleic acid sequence encoding a recombinase protein is introduced into a cell and at least two identification sites are introduced into the genome or episomal DNA of the cell. Steps (i) to (iii) may be performed in any order.

Nucleic acids are introduced into cells using gene manipulation techniques known to those of ordinary skill in the art. Suitable methods include cell transformation, transfection or viral infection, whereby nucleic acid sequences encoding proteins are introduced into cells as components of vectors or as part of DNA or RNA encoding viruses. Cell culture is performed by culture methods for individual cells known to those of ordinary skill in the art. Therefore, it is preferred that the cells be transferred to a known culture medium and cultured at a temperature and atmosphere that is conducive to cell survival.

The present invention also includes nucleic acid sequences or polynucleotides in which the coding sequence of the recombinase protein is fused in the same reading frame to a polynucleotide sequence that facilitates the expression and secretion of the protein from the host cell. For example, a leader sequence that acts as a secretory sequence to control the transport of the polypeptide out of the cell may be fused to a sequence encoding the recombinase protein. A polypeptide or protein having such a leader sequence is called a preprotein or preproprotein and may have a leader sequence that is cut by the host cell to form a mature form of the protein. These polynucleotides may have a 5' extension region so that they encode a proprotein, which is a mature protein plus an additional amino acid residue at the N-terminus. The expression product having such a presequence is called a proprotein, which is an inactive form of the mature protein; however, once the presequence is cut off, the active mature protein will remain. Additional sequences may also be attached to the protein and become part of the mature protein. Thus, for example, a polynucleotide of the invention may encode a polypeptide, or a protein having a prosequence, or a protein having both a prosequence and a presequence (e.g., a leader sequence).

The nucleic acids of the invention may also have a coding sequence fused in frame to a marker sequence that allows purification of the protein of the invention. The marker sequence may be an affinity tag or an epitope tag, such as a polyhistidine tag, a streptavidin tag, an Xpress tag, a FLAG tag, a cellulose or chitin binding tag, a glutamine sulfoxide-S transferase tag (GST), a hemagglutinin (HA) tag, a c-myc tag, or a V5 tag.

The HA tag would correspond to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984), while the c-myc tag would be an epitope derived from the human Myc protein (Evans et al., 1985).

If the nucleic acid of the present invention is mRNA, especially for use as a pharmaceutical agent, the delivery of mRNA therapeutics can be facilitated by the great progress that has been made in maximizing the translation and stability of mRNA, avoiding its immunostimulatory activity, and developing in vivo delivery technologies. The 5' cap and 3' poly(A) tail are major contributors to the efficient translation and extended half-life of mature eukaryotic mRNA. Incorporation of cap analogs (such as ARCA (anti-reverse cap analogs)) and 120-150 bp poly(A) tails into in vitro transcribed (IVT) mRNAs has significantly enhanced the expression of the encoded protein and mRNA stability. Novel cap analogs, such as those modified with 1,2-dithiobisphosphate, are resistant to RNA decapping complexes and can further improve the efficiency of RNA translation. Replacing rare codons in protein-encoding mRNA sequences with frequently occurring synonymous codons, a process known as codon optimization, can also help improve the efficiency of protein synthesis and limit mRNA instability caused by rare codons, thereby preventing accelerated transcript degradation. Similarly, engineering the 3' and 5' untranslated regions (UTRs), which contain sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs, can increase the amount of protein production. Interestingly, UTRs can be deliberately modified to encode regulatory elements, such as K-turn motifs and miRNA binding sites, providing a way to control RNA expression in a cell-specific manner. Some RNA base modifications (such as N1-methyl-pseudouridine) not only help mask the mRNA immunostimulatory activity, but have also been shown to increase mRNA translation by improving translation initiation. In addition to the effects observed on protein translation, base modifications and codon optimization can also affect the secondary structure of mRNA and thus its translation. The present invention also contemplates individual modifications of the nucleic acid molecules of the present invention.

RNA or a plurality of RNAs preferably encodes any of the recombinase enzymes of the present invention or its subunits. Specific methods for delivering and expressing nucleic acids and in particular RNA are disclosed in, for example, EP2590676 and EP3115064, which are incorporated herein by reference. The RNA may be present in particles and is preferably self-replicating. After the particles are administered in vivo, the RNA is released from the particles and translated inside the cells to provide the DNA recombinase or any of its monomeric subunits.

When a self-replicating RNA molecule (replicon) is delivered to a vertebrate cell, multiple daughter RNAs may be generated by transcription from itself (via antisense copies generated from itself), even in the absence of any protein. These daughter RNAs, as well as the colinear subgenomic transcripts, may be transcribed by themselves to provide in situ expression of the encoded polypeptide, or may be transcribed to provide further transcripts of the same meaning as the delivered RNA, which are transcribed to provide in situ expression of the polypeptide. The overall result of this transcriptional sequence is a huge expansion in the amount of the introduced replicon RNA, so that the encoded polypeptide becomes the major polypeptide product of the cell.

Preferred self-replicating RNA molecules encode (i) an RNA-dependent RNA polymerase that 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, for example comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3 and nsP4. Preferred self-replicating RNA molecules of the present invention do not encode alphavirus structural proteins. Therefore, preferred self-replicating RNA can cause the production of its own genome RNA copies in cells, but will not produce viral particles containing RNA. Self-replicating RNA molecules that can be used in the context of the present invention may have two open reading frames. The first (5') open reading frame encodes the replicase, and the second (3') open reading frame encodes the polypeptide of the present invention. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, e.g., for further encoding additional polypeptides.

Such RNA is particularly suitable for general use in gene therapy, and particularly for the treatment of genetic disorders or diseases.

The method according to the present invention can be performed using eukaryotic cells and prokaryotic cells. Preferred prokaryotic cells are bacterial cells. Particularly preferred prokaryotic cells are Escherichia coli cells. Preferred eukaryotic cells are yeast cells (preferably brewing yeast), insect cells, non-insect invertebrate cells, amphibian cells or mammalian cells (preferably somatic cells or pluripotent stem cells, including embryonic stem cells and other pluripotent stem cells, such as induced pluripotent stem cells), as well as other natural cells or established cell lines (including NIH3T3, CHO, HeLa, HEK293, hiPS). In the case of human embryonic stem cells, the cells are preferably obtained without destroying the human embryo, for example by outgrowth of a single blastomere derived from a blastocyst (as described by Chung et al., 2008), by parthenogenesis (e.g. from a monopronuclear oocyte, as described by Lin et al. 2007), or by parthenogenetic activation of a human oocyte (as described by Mai et al. 2007). Also preferred are cells of a non-human host organism, preferably non-human germ cells, somatic cells or pluripotent stem cells, including embryonic stem cells or blastocysts.

According to one embodiment, the cell is a eukaryotic cell or a bacterial cell.

According to another embodiment, the cell is not a human germ cell.

According to another aspect, 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 generating site-specific DNA recombination.

In the context of the present invention, a protein having at least 80% identity to a given SEQ ID NO preferably means that the 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.

According to yet another aspect, the present invention provides a use of a protein having recombinase activity for catalyzing site-specific DNA recombination at recognition sites that are substantially identical or substantially reverse complementary to each other, wherein the recognition sites comprise a first half site, a spacer, and a second half site, and wherein substantially identical or substantially reverse complementary to each other means that the nucleotide sequence of the first half site and the second half site in the first recognition site may differ from the nucleotide sequence of the first half site and the second half site in the second recognition site by at most two nucleotides.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 16 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 16 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 16.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 12 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 12 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 12.

According to one aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 10 or a functional variant thereof, or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 10.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 11 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 11 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 11.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 13 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 13 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 13.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 5, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 14 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 14 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 14.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 6, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 8, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 17 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 17 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 17.

According to another aspect of the use of the present invention, the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional mutant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15.

In the use of the present invention, a protein having recombinase activity and comprising an amino acid sequence having at least 80% identity with a given SEQ ID NO preferably means that the 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 with the given SEQ ID NO.

Similarly, in the use of the present invention, a nucleic acid sequence having at least 60% sequence identity to a given SEQ ID NO, or a nucleic acid sequence that is the reverse complement thereof, means that the 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 a given SEQ ID NO, or a nucleic acid sequence that is the reverse complement of the SEQ ID NO.

According to yet another aspect, the present invention provides a vector comprising at least one, preferably at least two substantially identical or substantially reverse complementary nucleic acids of the present invention, wherein the DNA segment is preferably flanked by two substantially identical or substantially reverse complementary nucleic acids.

According to one embodiment, 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: 1-9.

According to another aspect, 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 with any one of SEQ ID NOs: 1 to 9.

According to a preferred embodiment of the present invention, use of the carrier of the present invention in the method of the present invention is provided.

In addition, the present invention provides vectors comprising the nucleic acid sequences of the present invention. According to one embodiment, such vectors comprise at least one, preferably at least two, substantially identical or substantially reverse complementary nucleic acid sequences as described herein for the identification sites. According to yet another embodiment, the vector according to the present invention comprises a nucleic acid encoding 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 with one of SEQ ID NOs: 1 to 9.

The present invention further provides a vector (also referred to herein as a "reporter vector") comprising at least one nucleic acid comprising a nucleic acid sequence according to one of SEQ ID NOs: 10 to 17 or a nucleic acid sequence that is reverse complementary to one of SEQ ID NOs: 10 to 17, or a nucleic acid sequence that is a functional mutant thereof. Typically, an expression vector comprises a replication origin, a promoter, and a specific gene sequence that allows phenotypic selection of a host cell comprising the reporter vector. In a preferred embodiment of the present invention, the vector comprises at least two recognition sites, i.e., at least two nucleic acid sequences that independently exhibit a nucleic acid sequence according to one of SEQ ID NOs: 10 to 17 or a functional mutant thereof, or a nucleic acid sequence that is reverse complementary to a nucleic acid sequence of one of SEQ ID NOs: 10 to 17 or a functional mutant thereof, or a nucleic acid sequence that is a functional mutant thereof. At least two recognition sites are preferably located discontinuously in the vector. In contrast, at least two recognition sites are positioned so that they flank the DNA fragment of interest, and when the recognition sites are recognized by the respective recombinase proteins, the DNA fragment is recombined, preferably excised or inverted. The DNA fragment of interest may preferably contain a gene or a promoter region. When the DNA fragment of interest is flanked by two recognition sites with the same orientation (same nucleic acid sequence), the DNA fragment is excised. When the DNA fragment of interest is flanked by two recognition sites arranged in opposite orientations (i.e., the recognition sites contain nucleic acid sequences that complement each other in reverse), the inversion of the DNA fragment is catalyzed by the recombinase proteins.

The use of any of the vectors of the present invention in a method for producing site-specific DNA recombination is also included in the present invention.

According to another aspect, the present invention provides an isolated host cell comprising the following recombinant DNA fragments: at least one, and preferably at least two nucleic acids of the present invention; and/or the vector of the present invention.

According to one embodiment, the isolated host cell further comprises a nucleic acid encoding 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: 1-9.

According to yet another embodiment, the isolated host cell further comprises a vector of the present invention.

Preferably, isolated host cells that naturally contain the nucleic acids or vectors mentioned herein are excluded. According to one embodiment, the present invention relates only to isolated host cells that recombinantly and non-naturally (i.e., through genetic modification of the host cell) contain the above-mentioned nucleic acids or vectors.

According to another aspect, the present invention provides a non-human host organism comprising at least one, and preferably at least two nucleic acids of the present invention. According to another aspect, the present invention provides a non-human host organism comprising a vector of the present invention.

Furthermore, the present invention comprises 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, each comprising a recognition site and flanked by a further DNA fragment), and/or a nucleic acid according to the invention encoding a protein having 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, each comprising a recognition site and flanked by a further DNA fragment), and/or a vector comprising a nucleic acid encoding a protein having recombinase activity as defined above.

The present invention also includes an isolated host cell comprising the following recombinant DNA segments: (i) at least one, preferably at least two, nucleic acids of the present invention comprising a recognition site and/or a nucleic acid of the present invention encoding a recombinase protein, or (ii) a vector of the present invention comprising at least two nucleic acids of the present invention comprising a recognition site (preferably two nucleic acids of the present invention comprising a recognition site and flanked by another DNA segment of interest) and/or a vector of the present invention comprising a nucleic acid encoding a recombinase protein.

The present invention relates only to those isolated host cells that contain the above-mentioned nucleic acids or vectors (in a recombinant and non-natural manner, i.e., by genetically modifying the host cell).

Particularly preferred are isolated host cells containing nucleic acid encoding a recombinase protein of the invention and at least two recognition sites thereof, oriented in the same or opposite directions.

Host cells within the meaning of the present invention are naturally occurring cells or cell strains (transformed or genetically modified as appropriate) that recombinantly comprise at least one vector of the present invention or the nucleic acid of the present invention as described above. Thus, the present invention includes transient transfectants (e.g., by mRNA injection) or host cells that comprise at least one expression vector of the present invention as a plasmid or artificial chromosome, as well as host cells in which the expression vector of the present invention is stably incorporated into the genome of the host cell.

Suitable host cells in the context of the present invention are in particular eukaryotic cells, including stem cells, such as hematopoietic stem cells, neural stem cells, stem cells derived from adipose tissue, fetal stem cells, umbilical cord stem cells, induced pluripotent stem cells and embryonic stem cells. In the case of human embryonic stem cells, preferably they are not derived from a destroyed embryo. In addition, it is preferred to exclude the modification of the human germ line and human gametes as host cells.

Using the present invention, tissue-specific or site-specific recombination can also be induced in host organisms (such as mammals). Thus, the present invention also includes non-human host organisms comprising the following recombinant DNA fragments: at least one, preferably at least two nucleic acids of the present invention comprising a recognition site (preferably two nucleic acids of the present invention, each comprising a recognition site flanked by another DNA fragment of interest) and/or a nucleic acid of the present invention encoding a recombinase protein.

Specifically included are non-human host organisms that comprise only a recombinant nucleic acid encoding a recombinase protein of the invention (and separately not a nucleic acid comprising a recognition site).

In addition, the present invention includes non-human host organisms that only contain at least one, preferably at least two, recognition sites (and do not contain nucleic acids encoding recombinase proteins of the present invention). When two non-human host organisms are crossed, wherein the first host organism contains a recombinant nucleic acid encoding a recombinase protein of the present invention, and the second host organism contains at least two recombination recognition sites (preferably flanked by another DNA segment of interest), the progeny include host organisms that express the recombinase protein and further include the recognition sites, so that site-specific DNA recombination (such as tissue-specific conditional knockouts) is possible.

Also provided are non-human host organisms which may comprise a vector of the invention or a nucleic acid of the invention as described above, which are stably incorporated into the genome of the host organism or into individual cells of the host organism, respectively.

Preferred host organisms of the present invention are plants, invertebrates and vertebrates, especially bovines, Drosophila melanogaster, Cryptobacilus elegans, Xenopus laevis, Gymnosoma turcica, zebrafish or mice, or embryos of these organisms.

The present invention also provides novel recombinase systems suitable for producing site-specific recombination in cells of various cell types. Such systems comprise a corresponding recombinase protein (one of SEQ ID NOs: 1 to 9) and at least two corresponding recognition sites as defined herein. Using such systems, a variety of genetic manipulations can be achieved, in particular the rearrangement of DNA segments flanked by the recognition sites of the present invention, with the recognition sites being in the same orientation (excision), in opposite orientations (inversion), or when one specific recognition site is present on each of two DNA molecules and one (if circular) is present in any orientation (incorporation). An exemplary manipulation is the excision of a DNA segment flanked by two recognition sites oriented in the same direction mediated by a respective recombinase protein as defined herein. Among other things, the recombinase system of the present invention provides the possibility of excising a target DNA, such as a terminator DNA fragment flanked by two recognition sites, which is located 5' of a gene and corresponds to 3' of the promoter of the gene. Without recombination, the terminator sequence would block gene expression, whereas upon recombinase-mediated excision of the terminator via the two flanking recognition sites, the gene is located near the promoter and will therefore be expressed. Different types of promoter regions that regulate recombinase expression allow in particular conditional DNA recombination when, for example, tissue- or organism-specific or inducible promoter regions are used to express the recombinase protein.

The recombinase system of the present invention is suitable for use in combination with other recombinase systems and becomes a particularly valuable tool for genetic experiments requiring multiple recombinases simultaneously or sequentially.

The present invention further provides the use of a protein having recombinase activity, wherein the protein comprises an amino acid sequence that exhibits at least 85%, at least 90%, at least 95%, preferably at least 95%, even more preferably at least 99%% amino acid sequence identity with one of the amino acid sequences of SEQ ID NOs: 1 to 9, to catalyze site-specific DNA recombination. Thus, the above-mentioned site-specific recombinase is used to perform site-specific DNA recombination on at least one, preferably at least two, substantially identical or substantially reverse complementary recognition sites of the present invention. At least one recognition site comprises a nucleic acid sequence according to SEQ ID NOs: 10 to 17, respectively, or a nucleic acid sequence that is reverse complementary to a nucleic acid sequence of SEQ ID NOs: 10 to 17, or a nucleic acid sequence that is a functional mutant thereof.

According to another aspect, the present invention provides a protein or a vector of the present invention 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 use in medicine. According to a preferred embodiment, the protein or vector is used to treat a genetic disease or condition in an individual. Preferably, the genetic disease or condition is characterized by modification of the individual's genome.

The present invention also provides a pharmaceutical composition comprising the recombinant enzyme protein of the present invention, the vector or host cell of the present invention, or one or more nucleic acids of the present invention, and optionally a pharmaceutically acceptable carrier.

Pharmaceutical compositions containing the therapeutically active agents of the invention may be in any form suitable for the chosen mode of administration.

In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.

As used herein, the phrases "parenteral administration" and "administered parenterally" refer to 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, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnical, intraspinal, intracranial, intrathoracic, intradural, and intrasternal injection and infusion.

As mentioned herein, therapeutic agents include but are not limited to the recombinase proteins of the present invention and the recognition sites of the present invention. The therapeutic agents of the present invention can be administered to animals and humans as a single active agent or in combination with other active agents in a unit administration form, such as a mixture with a conventional pharmaceutical support.

In a further embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier (also referred to as a vehicle) for a formulation that can be injected. These may be in particular isotonic, sterile aqueous saline solutions (sodium dihydrogen phosphate or sodium dihydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride or mixtures of these salts), or dry (in particular lyophilized) compositions, added depending on if sterile water or physiological saline is used, injectable solutions can be prepared.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or propylene glycol in water; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, 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 (e.g., bacteria and fungi).

Solutions containing the therapeutically active agent in the form of a free base or a pharmacologically acceptable salt 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 agent can be formulated as a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and those formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

The carrier can also serve as a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. 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 dispersions, and by the use of a surfactant. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars or sodium chloride. The absorption of the injectable composition can be prolonged by the use in the composition of an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active polypeptide in the required amount with several of the other ingredients listed above (as required) in an appropriate solvent and then filtering and sterilizing. Generally speaking, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof.

After formulation, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, such as the injectable solution type described above, but drug release capsules and the like can also be used. Multiple doses can also be administered. If appropriate, the therapeutically active agents described herein may be formulated in any medium suitable for delivery. For example, they can be placed in a pharmaceutically acceptable suspension, solution or emulsion. Suitable media include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, but are not limited to, water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include liquids and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), and the like.

Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like.

Colloidal dispersion systems may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, hybrid micelles, and liposomes.

The appropriate treatment regimen can be determined by the physician and will depend on the individual's age, sex, weight, and stage of the disease. For example, to deliver a nucleic acid sequence encoding a genetically engineered DNA recombinase of the present invention using a viral expression vector, each unit dose of the genetically engineered DNA recombinase expression vector may comprise a composition comprising the viral expression vector in a pharmaceutically acceptable fluid at a concentration ranging from, for example, 10 ¹¹ to 10 ¹⁶ viral genomes/ml.

The effective dosage and dosage regimen for administering the genetically engineered DNA recombinase of the present invention or its subunit in the form of a recombinant polypeptide depends on the disease or condition to be treated and can be determined by one of ordinary skill in the art.

A physician or veterinarian with ordinary knowledge in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian may start the dosage of the therapeutically active agent of the present invention employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of the composition of the present invention will be the amount of the delivery system that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Administration may, for example, be intravenous, intramuscular, intraperitoneal, or subcutaneous, and, for example, administered close to the target site. If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six or more divided doses at appropriate intervals throughout the day, optionally in unit dosage form. Although the delivery system of the present invention can be administered alone, the delivery system is preferably administered as a pharmaceutical composition as described above.

Further provided are kits comprising a therapeutic agent as described herein. In one embodiment, the kit provides the therapeutic agent prepared in one or more unit dosage forms, ready for administration to a subject, such as in a pre-filled syringe or in an ampoule. In another embodiment, the therapeutic agent is provided in a lyophilized form.

According to another aspect, 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, a nucleic acid of the present invention, a vector of the present invention, an isolated host cell of the present invention, a non-human host organism of the present invention, or a pharmaceutical composition of the present invention.

The inventors screened more than 500 putative recombinase candidates, of which 17 candidates were selected for experimental testing, and eight novel recombinase systems were molecularly characterized in detail. In order to optimally use different recombinase systems, their application properties must be characterized. Determining the recombinase activity in vivo may be crucial for planning experiments. The eight newly characterized recombinases showed varying activities at the predicted target sites in bacteria, ranging from over 90% in YR1, YR4, YR6 to as low as around 15% in YR8 and YR9. Possible explanations for this low activity may include the need for additional cofactors, or an optimal temperature for more efficient recombination of the target site. The specificity of the recombinases was dissected, providing a valuable overview of possible crossover recombination events at all target sites. For applications in higher organisms, the novel recombinases were tested for activity and compatibility in mammalian cells. All recombinases demonstrated high activity in plastid-based assays in HEK293T cells. Interestingly, YR8 showed high recombination rates in mammalian cells, whereas this recombinase was only weakly active in bacteria, suggesting that the activity profile of recombinases may differ in heterologous hosts.

With regard to applications in heterologous cells, it is also important to consider possible pseudosites present in the host genome. All known Cre-type recombinases in the human and mouse genomes were screened for lox-like sites. This information is useful in two ways: i) it provides an estimate of potential off-target sites that may compromise the experiment; and ii) it provides potential endogenous target sites that may be useful for genome engineering exercises (e.g. for targeted delivery of DNA cargo into safe harbor loci).

The disclosure and characterization of different naturally occurring recombinases is particularly useful for accelerating the development of novel enzymes via directed evolution. It has been demonstrated that shuffling of related genes can accelerate the evolutionary process (Crameri et al., 1998). Therefore, by family shuffling, novel recombinases with desired properties may be obtained in a shorter time. Examples Example 1 : Identification of putative recombinases and their target sites

Potential novel Y-SSRs were identified by searching the NCBI nucleotide collection database (v5) using tblastn in BLAST+ 2.10.1 (https://www.ncbi.nlm.nih.gov/books/NBK131777/) and the protein sequences of Cre, Vika (Karimova et al., 2012), Nigri and Panto (Karimova et al. 2016) as references. Results with less than 90% identity and sequence length of 300 to 400 amino acids were filtered using GNU awk. Protein sequences were obtained using efetch (https://dataguide.nlm.nih.gov/edirect/efetch.html). The complete genome sequences of potential SSRs were collected using bastdbcmd (part of BLAST+). Potential target sites were identified by searching the genome sequence 1000 bp upstream and downstream of potential Y-SSRs for palindromes. Palindrome searches were performed using EMBOSS Palindrome (v6.6.0.0) (Rice et al., 2000) with a minimum palindrome length of 13 to 15 base pairs, a gap limit of 8, and one mismatch allowed. The program output was then converted to a tabular format using GNU awk and combined with protein data in R using the dplyr package. Potential SSRs with a Levenshtein distance to the reference (stringdist R package, https://journal.r-project.org/archive/2014/RJ-2014-011/index.html) less than 10 were removed. Potential SSRs were grouped based on Levenshtein distance using complete hierarchical clustering (base R) and clusters were formed with a cutoff distance of 11. The same clustering approach was also used for potential half-sites of palindromic sequences, where the cutoff distance for clustering was 2. The top few test candidates considered to be clustered were selected, along with their organisms and their distance from the reference recombinase.

Phylogenetic trees of known and putative recombinases were generated using the EMBOSS needle (Needleman and Wunsch, 1970) by full pairwise alignment of protein sequences followed by complete hierarchical clustering of sequence differences. Visualization of the trees was done using the R packages tidygraph and ggraph. For simplicity, nearly identical proteins were represented as a single node at a cutoff of 98% sequence similarity.

Human and mouse genomic sequences with high similarity to potential target sites were identified using PatMaN (Prüfer et al., 2008). Only half sites were searched, allowing up to 2 mismatches. If two genomic sequences matching the same half site were found, located on opposite strands with a distance of 8 bp between them, they were called potential target sites for the respective recombinases. Genomic coordinate manipulation and sequence extraction steps were performed using the BEDTools suite (Quinlan and Hall., 2010). Example 2 : Plasmid construction

For expression in E. coli, codon-optimized DNA sequences of 17 predicted candidates were synthesized by Twist Biosciences and cloned into the pEVO vector via BsrGI and XbaI restriction sites (Figure 1B) (Buchholz and Stewart., 2001). The target site was introduced via primers designed to carry the desired target site and overlap with the pEVO vector. The PCR fragment generated when using the pEVO vector as a template was then cloned into the pEVO backbone (System Biosciences) digested with BglII by Cold Fusion.

To express the recombinase in mammalian cells, a lentiviral PGK-NLS-BFP plasmid was used (Figure 5D). The recombinase sequence was amplified from the pEVO vector and cloned via the BsrGI and XbaI restriction sites. The aforementioned lentiviral vectors carrying the recombinase can be transfected into HEK293T cells for transient expression or used for virus production and infection for continuous expression.

To construct the recombinant reporter, the pCAG-loxP-mCherry-loxP-GFP "red and green light" vector (Karpinski et al. 2016) was used. Oligonucleotides containing the respective target sites were used to amplify the mCherry cassette, and the fragment was subsequently ligated into the pCAG plasmid via the NheI and HindIII restriction sites. Example 3 : Analysis of recombinant reporters

To visualize the recombinase activity at its predicted target site, a plasmid-based assay was used as described previously (Karimova et al., 2012 and 2016) (Figure 1A). Briefly, the pBAD promoter was induced to express the recombinase with L-arabinose (Sigma-Aldrich Chemie GmbH). Single clones containing pEVO plasmids with the recombinase and the recombinase target site were cultured overnight at 37°C and 200 rpm in 6 ml LB medium with 25 μg/ml Cm and 0 or 100 μg/ml L-arabinose. After digestion with BsrGI and SbfI restriction enzymes, recombinase-mediated excision events were detected by agarose gel electrophoresis. The size of the recombinant plasmids was smaller compared to the non-recombinant plasmids. Therefore, after gel electrophoresis, a non-recombinant band (~5.0 kb) that migrates slowly and a band (~4.3 kb) that migrates faster can be seen from the recombinant plasmid (Figure 1A).

To compare the recombination efficiency of active recombinases at their native target sites, recombinase expression was induced overnight in 6 ml culture volumes with increasing concentrations of L-arabinose (0, 1, 10, or 100 μg/ml medium). Test digests were prepared for each level of induction, and recombination efficiency was estimated by agarose gel electrophoresis. To quantify recombinase activity, image processing was performed using Fiji-ImageJ to determine the ratio of band intensities. Quantified recombinases were plotted in R 4.0.3 using dplyr v1.0.7 and visualized using ggplot2 v3.3.5. All test digests were performed in triplicate (n = 3).

For mammalian recombinant reporter assays, HEK293T cells were plated at 2 x 10 ⁵ cells per well in 24-well culture dishes and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco ^® ) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin/streptomycin (10,000 U/ml, Thermo Fisher). At 70-80% confluency, cells were co-transfected with pPGK-NLS-Recombinase-P2A-BFP plasmids expressing recombinase and pCAG-lox-mCherry-lox-GFP red and green light reporters using Lipofectamine® 2000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions. 0.5 μg of DNA (0.25 μg per plasmid) and 2.5 μl of Lipofectamine® 2000 reagent diluted in 100 μl of Opti- ^MEM® Reduced Serum Media were used per well. The medium was changed the next day and the cells were further cultured at 37°C and 5% CO2. After recombination between targets, the mCherry cassette is cleaved and the CAG promoter begins to drive downstream green fluorescent protein (GFP) expression. Two days after transfection, cells were analyzed using fluorescence activated cell assay and then imaged using a fluorescence microscope (EVOS FL Imaging System; Thermo Fisher Scientific). Example 4 : Fluorescence activated cell assay

HEK293T cells were washed once with PBS and then detached using trypsin (Gibco). Cells were then resuspended in Dulbecco's modified Eagle's medium (DMEM, Gibco®) and analyzed using a MACSQuant® VYB flow cytometer (Miltenyi). Data analysis was performed using FlowJo™ 10 (BD). Example 5 : Overexpression studies in mammalian cells

Regarding viral delivery, lentiviral particles were produced using the pPGK-Recombinase-P2A-BFP vector as described previously (Sürün 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% CO _2. The following day, fibroblasts were transduced with different lentiviruses at an MOI of 0.5 to achieve an infection rate of approximately 50%. The percentage of BFP-expressing cells in at least 2 x 10 ⁴ cells was tracked over a period of 15 days using a MACSQuant ^® VYB flow cytometer (Miltenyi). The difference in the percentage of BFP cells between the last time point (day 15) and the first time point was calculated and visualized using GraphPad Prism, and statistical significance relative to the BFP control was calculated by performing a one-way ANOVA using 95% confidence intervals (CI). Example 6 : Cross-recombination analysis: Nanopore sequencing

Recombinases and Cre, Vika, Panto, Dre (Anastassiadis et al., 2009), and VCre (Suzuki and Nakayama, 2011) were amplified from the pEVO vector, cleaned up using the Isolate II PCR and Gel Cleanup Kit (Bioline) and mixed together in a 1:1 ratio. All individual target sites were cloned into the pEVO vector using the Cold Fusion Cloning Kit as described previously, and the resulting vectors were also mixed in an equimolar ratio. The mixture of recombinases and the pEVO backbone was digested with BsrGI and SbfI and ligated in a single reaction to create a library of different recombinase/target site pairs. Plasmids were transformed into XL1-Blue electrocompetent E. coli cells and grown overnight with 100 μg/ml L-arabinose to induce recombinase expression. On the next day, plasmids were linearized with BsrGI and ScaI, and fragments carrying the recombinase sequence and the target site were isolated by agarose gel excision using the Isolate II PCR and Gel Cleanup Kit (Bioline). These DNA fragments were then prepared for nanopore sequencing using the SQK-LSK110 Kit on a MinION R9.4.1 Flow Cell (Oxford Nanopore Technologies) according to the "Amplicons by Ligation" protocol. Base calling of sequence data was performed using guppy v5.0.7 with the high precision model (Oxford Nanopore Technologies). Sequence reads with a read length of at least 1800 bp and a minimum average phred score of ten were then filtered using filtlong (https://github.com/rrwick/Filtlong). To identify recombinases, reads were aligned to reference recombinase sequences using minimap2 v2.17 (Li, 2018). Target sites were identified using Exonerate v2.2.0 using the affine:local model (https://www.ebi.ac.uk/about/vertebrate-genomics/software/exonerate). Read IDs and matching references were then taken from both alignments and combined with R with the dplyr package. Data visualization was performed using the R package ggplot2 .

Candidate recombinases were tested for activity on the predicted target sequence. Briefly, candidates were selected and their coding sequences were individually cloned into the L-arabinose-inducible pEVO recombinase reporter vector (Buchholz and Stewart, 2001), which carries two copies of the respective predicted target site (Figure 1B). The plasmids were then transformed into E. coli and cultured overnight in medium containing L-arabinose to induce recombinase expression. Following expression, successful recombinase excised a ~700 bp DNA fragment from the plasmid. This size difference was visualized by agarose electrophoresis of linearized plasmids (Figure 1A). Eight of the seventeen candidates showed activity at their predicted target sites, as evidenced by the appearance of a ~4 kb recombinant band (Figure 2). Five of the candidates demonstrated efficient recombining in samples without L-arabinose added to the medium (YR1, YR2, YR4, YR6, and YR12), indicating that these enzymes are active even when expressed at very low levels. The other recombinases (YR8, YR9, and YR11) recombined plastids only when L-arabinose was present in the growth medium, indicating that they require higher induction to be active in this assay. These results confirm the successful identification of novel Y-SSRs and their respective target sites.

To characterize the active Y-SSRs in more detail, their recombination efficiency was quantified at different levels of expression in order to study dosing responses and obtain more side-by-side comparisons of their efficiency compared to the well-established recombinases Cre and Vika (Karimova et al., 2012). pEVO plasmids with the desired recombinase/target site pairs were transformed into E. coli and cultured overnight in the presence of different concentrations of L-arabinose to induce recombinase expression (Guzman et al., 1995). Extracted plasmid DNA was then analyzed for recombination on agarose gels. Quantification of band intensities indicated that the novel recombinases had different activity profiles at their respective target sites (Figure 3). Although most recombinases were highly active when grown at high L-arabinose concentrations, showing recombination rates between 87% and 100% (YR1, YR2, YR4, YR6, and YR11), their performance was quite different when expressed at low L-arabinose concentrations. While YR1, YR2, YR4, and YR11 showed lower recombination rates when induced with 1 or 10 µg/ml L-arabinose, YR6 was highly active even at such low induction levels, with an activity profile similar to that of Cre and Vika (Figure 3). Interestingly, the YR12 recombinase showed a generally constant recombination rate of ~50% at 0 µg/ml L-arabinose, peaking at 70% when induced with 100 µg/ml L-arabinose. On the other hand, YR8 and YR9 showed the weakest activity at the highest L-arabinose concentration, with only 25% and 17% of their target sites recombined, respectively.

The recombinase YR9 was further subjected to substrate-linked directed evolution (Buchholz and Stewart, 2001), which significantly improved the activity of the YR9 recombinase. The best performing strain, designated YR9.2 (SEQ ID NO: 9), showed approximately sevenfold improvement in activity in bacteria (Figure 7A) and eightfold improvement in mammalian cells (Figure 7C). Example 8 : Dissecting the target site selectivity of Cre -like recombinases

SSRs with different sequence specificities are often used in combination to perform complex genomic or synthetic biology experiments (Feil, 2007; Sheets et al., 2020; Merrick et al., 2018; Livet et al., 2007; Snippert et al., 2010). For such applications, it is important to understand the specificity of the enzymes and consider possible cross-reactivity (Fenno et al., 2014; Weinberg et al., 2017). In order to test all possible combinations, high-throughput sequencing methods have been developed, in which the activities of known (Panto, Dre, Cre, Vika, and VCre) and novel Y-SSRs (YR1, YR2, YR4, etc.) can be quantified for all target sites in a single experiment. A two-step cloning scheme was established to generate all combinations of 13 recombinases and their respective 13 target sites on 169 (13x13) individual vectors. The 13 target sequences were individually cloned into the pEVO vector. The resulting constructs were then pooled and linearized to be cloned into a pool of 13 recombinase encoding sequences in one ligation reaction (Figure 4A). After overnight culture and induction of recombinase expression, plasmid DNA was recovered and a fragment with the recombinase sequence was cut at the 3' end and a fragment with the target site was cut at the 5' end. Using the long read sequencing platform of Oxford Nanopore Technologies, a total of 417,769 reads were obtained, all of which contained the specified recombinase and target site. All possible 169 combinations of Y-SSR and target site were identified with a minimum coverage of 224 reads. Using this data, the recombination rates of individual recombinases at all target sites were calculated and a profile of the specificity of each recombinase was provided (Figure 4B). Example 9 : Activity of recombinases in human cells

The activity of the novel Y-SSR was tested in human cell lines. Briefly, HEK293T cells were co-transfected with a recombinase expression plasmid (as described in Example 3 of mammalian recombination analysis) together with a recombinase reporter plasmid carrying the corresponding target site (Figure 5A). In the reporter plasmid, the mCherry cassette driven by the CAG promoter was flanked by the target (lox) sites. After recombination, the mCherry cassette was deleted from the plasmid, and the CAG promoter then drove the expression of the GFP cassette (Figure 5A). Therefore, the activity of the recombinase on its predicted target site can be visualized by fluorescence microscopy and quantified by flow cytometry. When co-transfection experiments were analyzed, all recombinases tested exhibited GFP-positive cells (Figure 5B), confirming that these recombinases are active in HEK293T cells, whereas no GFP-positive cells were observed when co-transfections were performed using an "empty" expression vector (lacking the recombinase coding sequence) (Figure 5B). Flow cytometric analysis showed that in almost all samples, more than 90% of cells co-transfected with the recombinase expression plasmid and reporter (cells double positive for BFP and mCherry) were also GFP-positive, indicating that these recombinases are highly active in this setting (Figure 5C). Example 10 : Effects of recombinase expression on cell proliferation

The study of SSRs in heterologous hosts is important to define their application properties. Recombinases can recognize cryptic (pseudo) recombination sites in the genome that may recombine and cause off-target effects, possibly leading to growth arrest or apoptosis. Indeed, active pseudo-loxP sites have been described in human and mouse genomes (Thyagarajan et al., 2000). Therefore, when Cre is overexpressed in human or mouse cells, it may impair cell proliferation (Loonstra et al., 2001; Schmidt et al., 2000; Pugach et al., 2015).

To test the potential effects of the newly identified Y-SSR on cell proliferation when overexpressed, a lentiviral vector was constructed that allows co-expression of the recombinase and tagBFP (Figure 6A). Viral particles overexpressing Cre, an inactive Cre variant (CreY324F), Vika, and tagBFP alone were used as controls. NIH3T3 cells were infected and the percentage of BFP-positive cells was monitored for 15 days. The decrease in BFP-positive cells over time suggests that overexpression of the recombinase has a negative effect on cell proliferation (Schmidt et al., 2000; Pugach et al., 2015). When the Cre recombinases were tested in this assay, the number of BFP-positive cells progressively decreased, whereas the catalytically inactive form of Cre had no effect (Fig. 6B). In contrast to Cre, YR4 and R8 showed a less pronounced decrease in the percentage of BFP-positive cells (~15%; p<0.0001 and ~17%; p<0.0001, respectively), indicating that overexpression of these recombinases slightly inhibited cell proliferation (Fig. 6B). In contrast, the percentage of BFP-positive cells did not change significantly in cells expressing the other recombinases (Fig. 6B), indicating that overexpression of these recombinases is well tolerated in the cells. Cited non-patent literature Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F. and Stewart, AF (2009) Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis Model Mech, 2, 508–515. Anderson,RP, Voziyanova,E. and Voziyanov,Y. (2012) Flp and Cre expressed from Flp–2A–Cre and Flp–IRES–Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange. Nucleic Acids Res, 40, e62–e62. Anzalone,AV, Gao,XD, Podracky,CJ, Nelson,AT, Koblan,LW, Raguram,A., Levy,JM, Mercer,JAM and Liu,DR (2022) Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat Biotechnol, 40, 731–740. Anzalone,AV, Randolph,PB, Davis,JR, Sousa,AA, Koblan,LW, Levy,JM, Chen,PJ, Wilson, C., Newby, GA, Raguram, A., et al. (2019) Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576, 149–157. Buchholz, F., and Stewart, AF (2001). Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat Biotechnol 19, 1047–1052. Crameri,A., Raillard, S.-A., Bermudez, E. and Stemmer, WPC (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature, 391, 288–291. Duyne, GDV (2001) A Structural View of Cre- loxP Site-Specific Recombination. Annu Rev Bioph Biom, 30, 87–104. Feil,R. (2007) Conditional Mutagenesis: An Approach to Disease Models. Handb Exp Pharmacol, 10.1007/978-3-540-35109-2_1. Fenno,LE, Mattis,J., Ramakrishnan,C., Hyun,M., Lee,SY, He,M., Tucciarone,J., Selimbeyoglu,A., Berndt,A., Grosenick,L., et al. (2014) Targeting cells with single vectors using multiple-feature Boolean logic. Nat Methods, 11, 763–772. Gaudelli,NM, Komor,AC, Rees,HA, Packer,MS, Badran,AH, Bryson,DI and Liu,DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 551, 464–471. Guzman,LM, Belin,D., Carson,MJ and Beckwith,J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol, 177, 4121–4130. Karimova,M., Abi-Ghanem,J., Berger,N., Surendranath,V., Pisabarro,MT and Buchholz, F. (2012) Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system. Nucleic Acids Res, 41, e37–e37. Karimova, M., Splith, V., Karpinski, J., Pisabarro, MT and Buchholz, F. (2016) Discovery of Nigri/nox and Panto/pox site-specific Recombinase systems facilitates advanced genome engineering. Sci Rep-uk, 6, 30130. Karpinski,J., Hauber,I., Chemnitz,J., Schäfer,C., Paszkowski-Rogacz,M., Chakraborty,D., Beschorner,N., Hofmann-Sieber,H., Lange,UC, Grundhoff,A., et al. (2016) Directed evolution of a recombination that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotechnol, 34, 401–409. Komor,AC, Kim,YB, Packer,MS, Zuris,JA and Liu,DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533, 420–424. Li,H. (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics, 34, 3094–3100. Livet,J., Weissman,TA, Kang,H., Draft,RW, Lu,J., Bennis,RA, Sanes,JR and Lichtman,JW (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature, 450, 56–62. Loonstra,A., Vooijs,M., Beverloo,HB, Allak,BA, Drunen,E. van, Kanaar,R., Berns,A. and Jonkers,J. (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc National Acad Sci, 98, 9209–9214. Meinke,G., Bohm,A., Hauber,J., Pisabarro,MT and Buchholz,F. (2016) Cre Recombinase and Other Tyrosine Recombinases. Chem Rev, 116, 12785–12820. Merrick,CA, Zhao,J. and Rosser,SJ (2018) Serine Integrases: Advancing Synthetic Biology. Acs Synth Biol, 7, 299–310. Minorikawa,S. and Nakayama, M. (2011) Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems. Biotechniques, 50, 235–246. Needleman,SB and Wunsch,CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol, 48, 443–453. Prüfer,K., Stenzel,U., Dannemann,M., Green,RE, Lachmann,M. and Kelso,J. (2008) PatMaN: rapid alignment of short sequences to large databases. Bioinformatics, 24, 1530–1531. Pugach,EK, Richmond,PA, Azofeifa,JG, Dowell,RD and Leinwand,LA (2015) Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic. J Mol Cell Cardiol, 86, 54–61. Quinlan,AR and Hall,IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 26, 841–842. Sauer,B. and Henderson,N. (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc National Acad Sci, 85, 5166–5170. Schmidt, EE, Taylor, DS, Prigge, JR, Barnett, S. and Capecchi, MR (2000) Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc National Acad Sci, 97, 13702–13707. Sternberg, N. and Hamilton, D. (1981) Bacteriophage P1 site-specific recombination I. Recombination between loxP sites. J Mol Biol, 150, 467–486. Sürün, D., Schneider, A., Mircetic, J., Neumann, K., Lansing, F., Paszkowski-Rogacz, M., Hänchen, V., Lee-Kirsch, MA and Buchholz, F. (2020) Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. Genes-basel, 11, 511. Rice,P., Longden,I. and Bleasby,A. (2000) EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet, 16, 276–277. Sheets,MB, Wong,WW and Dunlop, MJ (2020) Light-Inducible Recombinases for Bacterial Optogenetics. Acs Synth Biol, 9, 227–235. Snippert, HJ, Flier, LG van der, Sato, T., Es, JH van, Born, M. van den, Kroon-Veenboer, C., Barker, N., Klein, AM, Rheenen, J. van, Simons, BD, et al. (2010) Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells. Cell, 143, 134–144. Suzuki,E. and Nakayama,M. (2011) VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering. Nucleic Acids Res, 39, e49–e49. Thyagarajan,B., Guimarães,MJ, Groth,AC and Calos,MP (2000) Mammalian genomes contain active recombinase recognition sites. Gene, 244, 47–54. Weinberg,BH, Pham,NTH, Caraballo,LD, Lozanoski,T., Engel,A., Bhatia,S. and Wong,WW (2017) Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat Biotechnol, 35, 453–462.

without

The present invention is further described by the following figures and examples, but is not limited thereto.

Figure 1A shows an overview of plastid recombination analysis. Important features such as restriction sites, recombinase coding sequences, and target sites (triangles) are shown. A schematic of the expected recombinant products is also shown on the agarose gel. Markers and the expected sizes of recombinant and non-recombinant plastids are shown individually or together as normally seen on the gel. Figure 1B shows a plastid map of the pEVO recombinant reporter. Protein coding genes, the origin of replication (oriP15A), and the pBAD promoter are indicated by arrows. Protein coding genes include the chloramphenicol resistance gene (cmR), arabinose regulatory protein (araC), and the gene encoding the recombinase of interest. Upon addition of arabinose, expression of the recombinase is driven by the pBAD promoter. Recombination between the two lox sites results in the excision of approximately 700 bp of stuffer sequence. BsrGI and SbfI restriction enzymes were used for selection of the recombinases and for linearization of test digested plasmids.

Figure 2 shows the recombinase activity of seventeen tested putative Y-SSR/target site pairs. Each sample was tested for recombinase expression in the presence or absence of L-arabinose (100 µg/ml) added to the growth medium, indicated by "-" or "+". Recombination is indicated by a band aligned with a single triangle, while non-recombinant plasmids are indicated by two triangles. Active recombinases are highlighted by boxes. M = GeneRuler ^TM DNA Ladder Mix (Thermo Fisher).

Figure 3 shows the quantification and reproducibility of novel SSR recombinant activity in E. coli. Recombinase expression was induced with increasing L-arabinose concentrations (μg/ml) indicated along the x-axis. Vika and Cre were included as positive controls. Recombination was calculated by measuring the ratio of the band intensities of the non-recombinant and recombinant bands detected on agarose gel. Bacterial analysis was performed in triplicate (n = 3).

Figure 4A shows the analysis of deep sequencing results. Crossover recombination events are displayed by heatmaps of the recombination percentage for each possible combination. Target sites are displayed horizontally and ranked according to their similarity. The tested recombinases are arranged according to their homology on the vertical axis. On-target recombination events are framed by red squares. Figure 4B shows the validation of crossover recombination events by plastid-based recombination analysis. The recombination activity of individual recombinases is shown at their on-target and off-target sites. Recombination was assessed by agarose gel electrophoresis. Each sample was tested with or without L-arabinose-induced recombinase expression, indicated by "-" or "+" (100 µg/ml L-arabinose). Recombination is represented by a band aligned with a single triangle, while non-recombination bands are represented by two triangles. M = GeneRulerTM DNA Ladder Mix (Thermo Fisher). Portions of the figure were created using BioRender.com.

Figure 5 shows the activity of the novel recombinases in mammalian cells. Figure 5A is a schematic representation of the mammalian recombinase reporter and expression constructs. Important features are marked in the reporter and expression vectors. After recombination of the reporter vector, the mCherry cassette will be excised, allowing the pCAG promoter (arrow) to express GFP (green). The black triangles represent the different lox sites for each corresponding recombinase. NLS, nuclear localization signal. Figure 5B shows fluorescence microscopy analysis in Hek293T cells. Cells transfected with an empty expression plasmid and a non-recombinant reporter plasmid with eight novel target sites (top panel), or co-transfected with the reporter and an expression plasmid with the respective recombinase (bottom panel) are shown. Vika/vox and Cre/loxP were included as positive controls. Ctrl, negative control; Rec, recombinase. Figure 5C shows FACS analysis of the samples shown in Figure 5B. The dark gray histogram depicts control samples in which non-recombinant reporter plasmids were co-transfected with "empty" expression plasmids, while the light gray histograms show samples transfected with the corresponding recombinase. Figure 5D shows plasmid maps of expression lentiviral vectors (left) and reporter vectors (right). The CMV, PGK and CAG promoters used for mammalian viral RNA expression, the recombinase-P2A-BFP cassette and the mCherry cassette are shown as white arrows, respectively. The characteristics of virus production on the pLentiX vector are also depicted. Lentiviral vectors were transfected into HEK293T cells for recombinant analysis or used for virus production and infection to test the effect of sustained expression of the recombinase. BsrGI and XbaI restriction sites for cloning recombinases are marked on the expression vector, while NheI and HindIII sites marked on the pCAG-lox-mCherry-lox-GFP reporter plasmid are used to clone all target sites. Ori - ColE1 origin of replication in bacteria; AmpR - ampicillin resistance gene; bGH poly(A) signal - bovine growth hormone polyadenylation signal; rbGlob - polyA - rabbit β-globin polyadenylation signal.

Figure 6 shows the effect of overexpressing the recombinases of the present invention on cell growth in mammalian cells. Figure 6A is an overview of the experimental configuration. Important steps are indicated by arrows. Cells were transduced at a rate of approximately 50% with a bicistronic lentiviral expression construct, in which expression of the respective recombinase was linked to BFP expression via P2A. Cells were analyzed by flow cytometry every 72 hours, and the percentage of BFP-positive cells was recorded within 2 weeks. A decrease in the percentage of BFP-positive cells indicates a proliferative disadvantage of the infected cells. Figure 6B shows an analysis of growth rate. The difference in the percentage of BFP-positive cells between day 3 and day 15 is plotted (biological replicates are shown as points, n = 3). Error bars represent standard deviations (SD) of the mean. Statistical significance relative to BFP control was calculated by one-way ANOVA test. (****): P ≦ 0.0001.

Figure 7 shows the results of directed evolution of the YR9 recombinase. Figure 7A , Activity analysis of the plastid-based wt YR9 recombinase and three clones randomly picked from the evolution library. The agarose gel images on the right show the best clones that recombined lox9 at four different expression levels (0 µg/ml, 1 µg/ml, 10 µg/ml, and 100 µg/ml) compared to wt YR9. The quantification and reproducibility of the recombination are shown on the right. The recombination efficiency was calculated by comparing the ratio of the intensity of the recombinant band to the intensity of the non-recombinant band in the gel on the left. The experiment was performed in triplicate (n = 3). Comparison with YR9 wt was done with a t-test, and the p-value was adjusted for multiple comparisons using the Bonferroni method. Significance: (ns) p ＞ 0.05, (*) p ≦ 0.05, (**) p≦ 0.01, (***) p ≦ 0.001, (****) p ≦ 0.0001. Figure 7B , Mutation analysis of YR9 improved strains (single letter code). The amino acid sequence of wt YR9 recombinase is shown as a reference. Letters from top to bottom represent the changes found in strains YR9.10, YR9.2 and YR9.1. The predicted C-terminal catalytic domain of the DNA breakage-rejoining superfamily is marked. Figure 7C , Activity of the most active strain YR9.10 in mammalian cells. FACS analysis of wt YR9 and YR9.10 recombinases is shown. Dark grey histograms depict control samples in which a non-recombinant reporter plasmid was co-transfected with an "empty" expression plasmid, while light grey histograms show samples transfected with the corresponding recombinase.

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TW202507002A_113114269_SEQL.xml

Claims (15)

A method for generating site-specific DNA recombination, the method comprising the steps of: a) contacting a nucleic acid comprising at least a first recognition site and a second recognition site that are substantially identical or substantially reverse complementary to each other with a protein having recombinase activity, and b) allowing the protein having recombinase activity to generate site-specific DNA recombination. wherein the recognition sites comprise a first half site, a spacer and a second half site, and wherein substantially identical or substantially reverse complementary to each other means that the nucleotide sequences of the first half site and the second half site in the first recognition site may differ from the nucleotide sequences of the first half site and the second half site in the second recognition site by at most two nucleotides, wherein (i) the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO:7, and wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:16 or a functional variant thereof or a nucleic acid sequence reverse complementary to SEQ ID NO:16 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO:16; or (ii) the protein having recombinase activity comprises an amino acid sequence having at least 80% identity with SEQ ID NO:7 NO:3 has an amino acid sequence with at least 80% identity, and wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:12 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:12 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence with at least 60% sequence identity to SEQ ID NO:12; or (iii) the protein having recombinase activity comprises an amino acid sequence with at least 80% identity to SEQ ID NO:1, and wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:10 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence with at least 60% sequence identity to SEQ ID NO:10; or (iv) the protein having recombinase activity comprises an amino acid sequence with at least 80% identity to SEQ ID NO:10, and wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:10 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence with at least 60% sequence identity to SEQ ID NO:10; NO:2, wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:11 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:11 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:11; or (v) the protein having recombinase activity comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:4, wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:13 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:13 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:13; or (vi) the protein having recombinase activity comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:4, wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:13 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:13 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:13; NO:5, wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:14 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:14 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:14; or (vii) the protein having recombinase activity comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:6, wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:15 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:15; or (viii) the protein having recombinase activity comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:6, wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:15 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:15; NO:8 has an amino acid sequence with at least 80% identity, and wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:17 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:17 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence with at least 60% sequence identity to SEQ ID NO:17; or (ix) the protein having recombinase activity comprises an amino acid sequence with at least 80% identity to SEQ ID NO:9, and wherein the at least two recognition sites comprise a nucleic acid sequence according to SEQ ID NO:15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:15 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence with at least 60% sequence identity to SEQ ID NO:15. The method of claim 1, wherein the protein having recombinase activity comprises at least two protein monomers. The method of claim 1 or 2, wherein the recombinant nucleic acid sequence is present in a cell, preferably further comprising the step of introducing a nucleic acid encoding the protein having recombinase activity into the cell, or wherein the cell comprises a nucleic acid encoding the protein having recombinase activity. The method of claim 3, wherein 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, and/or wherein the cell is a eukaryotic cell or a bacterial cell. 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 use in generating site-specific DNA recombination. A protein having recombinase activity for catalyzing site-specific DNA recombination at substantially identical or substantially mutually reverse complementary recognition sites, wherein the recognition sites comprise a first half site, a spacer and a second half site, and wherein substantially identical or substantially mutually reverse complementary means that the nucleotide sequence of the first half site and the second half site in the first recognition site may differ from the nucleotide sequence of the first half site and the second half site in the second recognition site by at most two nucleotides, wherein: (i) the protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and wherein at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO: 16 or a functional variant thereof or a nucleic acid sequence reverse complementary to SEQ ID NO: 16 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 16; or (ii) the protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and wherein at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO: 16 or a functional variant thereof or a nucleic acid sequence reverse complementary to SEQ ID NO: 16 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 16; or NO:3, wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO:12 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:12 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:12; or (iii) the protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:1, wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO:10 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:10; or (iv) the protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO:10 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO:10 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO:10. NO: 11 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 11 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that has at least 60% sequence identity with SEQ ID NO: 11; or (v) the protein comprises an amino acid sequence that has at least 80% identity with SEQ ID NO: 4, and wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO: 13 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 13 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence that has at least 60% sequence identity with SEQ ID NO: 13; or (vi) the protein comprises an amino acid sequence that has at least 80% identity with SEQ ID NO: 5, and wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO: 14 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 14 or a functional variant thereof NO: 14 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 14; or (vii) the protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 6, and wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15; or (viii) the protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 8, and wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO: 17 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 17 or a functional variant thereof, wherein the functional variant comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 18. NO: 17 has at least 60% sequence identity; or (ix) the protein comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and wherein the at least one recognition site comprises a nucleic acid sequence according to SEQ ID NO: 15 or a functional variant thereof or a nucleic acid sequence that is reverse complementary to SEQ ID NO: 15 or a functional variant thereof, wherein the functional variant comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15. A nucleic acid having a length of no more than 40 base pairs and comprising: (i) a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 16 or a nucleic acid sequence that is the reverse complement thereof; or (ii) a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 12 or a nucleic acid sequence that is the reverse complement thereof; or (iii) a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 10 or a nucleic acid sequence that is the reverse complement thereof; or (iv) a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 11 or a nucleic acid sequence that is the reverse complement thereof; or (v) a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 13 or a nucleic acid sequence that is the reverse complement thereof; or (vi) a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 14 or a nucleic acid sequence that is the reverse complement thereof; or (vii) a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO: 15 or a nucleic acid sequence that is the reverse complement thereof; NO: 15 has at least 60% sequence identity or a nucleic acid sequence that is reverse complementary to it; or (viii) a nucleic acid sequence that has at least 60% sequence identity with SEQ ID NO: 17 or a nucleic acid sequence that is reverse complementary to it. A vector comprising at least one, and preferably at least two substantially identical or substantially reverse complementary nucleic acids as claimed in claim 7, wherein the DNA fragment is preferably flanked by two substantially identical or substantially reverse complementary nucleic acids, and the vector preferably 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 with any one of SEQ ID NOs: 1 to 9. 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 with any one of SEQ ID NOs: 1 to 9. A use of a carrier as claimed in any one of claims 8 or 9 in a method as claimed in any one of claims 1 to 4. 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 as claimed in claim 8 or 9, for use in medicine, preferably for treating a genetic disease or disorder in an individual, more preferably wherein the genetic disease or disorder is characterized by modification of the genome of the individual. An isolated host cell comprising the following recombinant DNA fragments: (i) at least one, and preferably at least two, nucleic acids as described in claim 7; and/or (ii) a vector as described in any one of claims 8 or 9. The isolated host cell of claim 12, further comprising: (i) a nucleic acid encoding a protein having recombinase activity, wherein the protein comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 1 to 9; or (ii) a vector of any one of claims 8 or 9. A non-human host organism comprising: (i) at least one, and preferably at least two, nucleic acids as in claim 7; or (ii) a vector as in any one of claims 8 or 9. A pharmaceutical composition comprising a protein having at least 80% identity with 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, a nucleic acid as in claim 7, a vector as in any one of claim 8 or 9, an isolated host cell as in claim 12 or 13, or a non-human host organism as in claim 14, and optionally a pharmaceutically acceptable excipient.