A serine recombinase system for site-specific gene editing

Abstract

The present disclosure provides engineered large serine recombinases and systems for gene editing containing the recombinases.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 304,565, filed January 28, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy created on January 26, 2023 is named 025297.WO043.xml and is 361,227 bytes in size.

[0003] Large serine recombinases (LSRs) are a family of enzymes encoded by lysogenic phages. These enzymes can precisely cleave and recombine DNA in a site-specific manner, thereby moving DNA elements in and out of bacterial chromosomes between short DNA attachment sites of the phage (attP) and the host bacteria (attB) (Duyne and Rutherford, Crit Rev Biochem Mol Biol. (2013) 48(5):476-91; Smith, Microbiol Spectr. (2015) 3(4):doi10.1128 / microbiolspec). The highly directed and controlled process of DNA recombination mediated by LSRs makes them promising tools in genetic engineering and gene therapy, where site-specific DNA integration, excision, inversion, or cassette exchange in the genome is desired.

[0004] Bxb1 recombinase, also known as Bxb1 integrase, is an LSR encoded by the Bxb1 phage that facilitates integration of phage DNA into the genome of Mycobacterium smegmatis (Russell et al., Biotechniques (2018) 40(4):doi.org / 10.2144 / 000112150). The recombinase contains an N-terminal catalytic domain similar to that of smaller resolvases / invertases, and a larger C-terminal domain responsible for coordinating the activity specific to the LSR. This C-terminal domain is further divided into a recombinase domain (RD) and a zinc ribbon domain (ZD) that contains a coiled-coil (CC) motif (Rutherford et al., Nucleic Acids Res. (2013) 41(17):8341-56). In a recombination event, the ZD and RD domains of a Bxb1 recombinase dimer bind to an attachment site (attB or attP) on a double-stranded DNA segment, while the ZD and RD domains of another Bxb1 recombinase dimer bind to the other attachment site (attP or attB) on another double-stranded DNA segment. The complex formation of the two dimers then leads to the formation of a Bxb1 recombinase tetramer, which brings together the attP and attB sites on the two DNA fragments. The serine residues in the active sites of the tetramer form a covalent bond with their central dinucleotide base pair and cleave the double-stranded DNA. This cleavage leaves the central dinucleotide as a 3' overhang. The resulting protein / DNA complex rotates and the above-dissociated DNA molecules or segments are ligated if the central dinucleotides of the attB and attP sequences are identical. This process creates attachment R (attR) and attachment L (attL) sites that are no longer substrates for the recombinase without the presence of additional cofactors.

[0005] Despite the precision of gene editing provided by large serine recombinases, their usefulness as genetic engineering and therapeutic tools is limited by the rarity of their binding sites in the human genome, including therapeutically relevant genes.Therefore, there is still a need for novel recombinases that can recognize various DNA attachment sites and realize the therapeutic potential of this category of enzymes. Summary of the Invention

[0006] The present disclosure provides large serine recombinase variants with altered DNA target specificity compared to their wild-type counterparts. In one aspect, the present disclosure provides non-naturally occurring variants of Bxb1 recombinase with altered DNA target specificity compared to wild-type Bxb1 recombinase (e.g., SEQ ID NO: 1) that include one or more amino acid mutations (e.g., substitutions, deletions, or insertions) within the ZD or RD.

[0007] In some embodiments, the one or more amino acid mutations are present at one or more of positions 147, 148, 149, 154, 155, 156, 158, 197, 198, 230, 231, 232, 233, 237, 257, 309, 312, 314, 315, 316, 318, 323, 324, 325, 326, and 335 (numbered according to SEQ ID NO:1). In a further embodiment, the one or more amino acid mutations are F314A, F314C, F314D, F314E, F314G, F314H, F314I, F314L, F314N, F314Q, F314S, F314T, F314V, F314W, F314Y, A315F, A315G, A315H, A315I, A315M, A315N, A315S, A315T ...L, A314N, A315G, A315G, A315G, A315G, A315G, A315G 5W, A315Y, G316A, G316C, G316D, G316E, G316F, G316H, G316I, G316K, G316L, G316M, G316P, G316Q, G 316R, G316S, G316T, G316V, G316W, G316Y, G318I, G318K, G318R, G318W, R323G, R323K, R325D, R325E , R325K, R325L, R325M, R325N, R325Q, R325S, R325W, F147A, F147K, F147R, N148Q, N148T, L158D, L15 8N, L158S, L158T, L158W, P197H, P197R, P197T, S231F, S231G, S231H, S231K, S231R, S231V, S231Y, T 233F, T233H, T233K, T233R, T233W, T233Y, R237A, R237C, R237D, R237E, R237F, R237G, R237H, R237I, R237K, R237L, R237M, R237N, R237P, R237Q, R237S, R237T, R237V, R237W, R237Y, and D257K.

[0008] In some embodiments, the Bxb1 recombinase variant provides increased transgene insertion at the endogenous target site in the eukaryotic genome compared to wild-type Bxb1 recombinase.

[0009] In other aspects, the disclosure provides non-naturally occurring variants of φC31 integrase, Pa557 recombinase, or Pa570 recombinase that contain one or more amino acid mutations (e.g., substitutions, deletions, or insertions) that result in altered DNA targeting specificity compared to the wild-type counterpart.

[0010] In some embodiments, the φC31 integrase variant comprises one or more amino acid mutations at one or more of positions 273, 275, 279, 375, 376, 377, 379, and 386 (numbered according to SEQ ID NO: 77). In further embodiments, the one or more amino acid mutations are selected from D273, A275, R279, K375, R376, G377, E379, and R386.

[0011] In some embodiments, the Pa557 recombinase variant comprises one or more amino acid mutations at one or more of positions 236, 238, 242, 327, 328, 329, 331, and 338 (numbered according to SEQ ID NO: 78). In further embodiments, the one or more amino acid mutations are selected from G236, A238, A242, R327, T328, G329, G331, and R338.

[0012] In some embodiments, the Pa570 recombinase variant comprises one or more amino acid mutations at one or more of positions 243, 245, 249, 333, 334, 335, 337, and 344 (numbered according to SEQ ID NO: 79). In further embodiments, the one or more amino acid mutations are selected from E243, S245, K249, I333, N334, P335, I337, and Q344.

[0013] In some embodiments, the DNA target is specific for a particular allele of a gene.

[0014] In some embodiments, the DNA target is present in a cell, eg, a eukaryotic cell, eg, a mammalian cell (eg, a human cell).

[0015] Also provided herein are nucleic acid molecules encoding the recombinase variants herein, and expression vectors comprising the coding sequences. The vectors can be, for example, plasmids or viral vectors (e.g., adeno-associated virus vectors, adenovirus vectors, or lentivirus vectors).

[0016] The present disclosure also provides a system for editing DNA in a cell, comprising a recombinase variant, a nucleic acid molecule, or a vector herein. In some embodiments, the system further comprises a donor DNA, e.g., a circularized DNA or a linear DNA. In further embodiments, the donor DNA comprises a sequence selected from SEQ ID NOs: 80-81 and 134-136, and sequences found in Tables 3, 4, and 5. The donor DNA can be delivered, for example, by a plasmid or a viral vector.

[0017] Editing by the system herein can include integration of DNA into the genome of the cell, excision or inversion of DNA in the genome of the cell, or chromosomal translocation in the genome of the cell. In some embodiments, editing occurs in a genomic region that includes a sequence selected from SEQ ID NOs: 80-81 and 134-136, and the sequences found in Tables 3, 4, and 5.

[0018] In another aspect, the present disclosure provides a method for editing the genome of a cell, comprising providing a cell with a gene editing system as described herein. In some embodiments, two or more genomic regions are edited. The editing method may result in excision of DNA from the genome, inversion of DNA in the genome, chromosomal exchange, recombinase-mediated cassette exchange (RMCE), and / or integration of donor DNA into the genome.

[0019] Also provided herein is a cell comprising the system, a cell edited by the methods of the invention, or a progeny thereof. The cell may be a eukaryotic cell (e.g., a mammalian cell, such as a human cell).

[0020] In another aspect, the disclosure provides a method of treating a disease in a subject in need thereof comprising administering an edited cell to the subject. Also included are cells for use in treating a disease in a subject in need thereof, and use of the cells in the manufacture of a medicament for treating a disease in a subject in need thereof.

[0021] Other features, objects, and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that the detailed description, while illustrating embodiments and aspects of the present invention, is provided for illustrative purposes only and is not intended to be limiting. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0022] [Figure 1] A shows a schematic diagram depicting the structure of Bxb1 recombinase. B shows a schematic diagram depicting the mechanism of action of Bxb1 recombinase as a tetramer at bound attB and attP sites (B: NTD: N-terminal catalytic domain (NTD); αE: conserved α-helix; CTD: C-terminal domain; RD: recombinase domain; CC: coiled-coil motif; and ZD: zinc ribbon domain (ZD)). Numbers below the protein structure in (A) indicate the approximate distances from the N-terminus of the protein to the C-terminal boundary of the NTD (approximately 150 amino acids), the N-terminal boundary of the CTD (300 amino acids), and the C-terminus of the CTD (600 amino acids). [Diagram 2] Schematic diagram showing the nucleotide numbering system in the attB and attP sequences, including the nucleotides bound by the ZD and RD domains of the Bxb1 recombinase. The attP sequence shown is the same as the wild-type sequence (SEQ ID NO:3), but the attB sequence has been edited to be more symmetrical compared to the wild-type attB site (SEQ ID NO:80). [Diagram 3]A shows a schematic diagram illustrating recombinase-mediated integration. B shows a schematic diagram illustrating recombinase-mediated excision. C shows a schematic diagram illustrating recombinase-mediated inversion. D shows a schematic diagram illustrating recombinase-mediated chromosomal exchange. E shows a schematic diagram illustrating recombinase-mediated cassette exchange. In recombinase-mediated cassette exchange (RMCE), the donor DNA can be circular (shown) or linear (not shown). [Figure 4] FIG. 1 shows a schematic diagram depicting a plasmid-based recombination assay used in conjunction with NGS to determine the DNA sequence specificity of mutant Bxb1 recombinase variants. One plasmid is shown in light grey and the other in black. White and dark grey boxes indicate attB and attP sites, respectively. P1 and P2 refer to primer binding positions. Products resulting from successful integration events are shown at the bottom. [Diagram 5] A schematic diagram showing a chromosomal recombination assay in human K562 cells is shown. Bxb1 promotes targeted integration (TI) of a donor plasmid into the chromosomal pseudoattB site of Bxb1 (an endogenous human sequence with some homology to the natural attB target site of Bxb1). White and dark grey boxes indicate the attB and attP sites, respectively. F-primer and R-primer refer to the binding positions of the primers used for the PCR-based NGS assay to quantify TI events. Products resulting from successful TI events are shown at the bottom. [Figure 6]A-C show the change in target site preference of Bxb1 S231F variant. (A) Target preference change in Bxb1 S231F variant obtained by the plasmid-based experimental system shown in FIG. 4. S231F shows improved targeting of both C and T at position 10 compared to wild-type (WT) Bxb1. (B) Human endogenous target sites of Bxb1 s5-8 (SEQ ID NO: 141 and 264), s5-1 (SEQ ID NO: 137 and 265), s5-11 (SEQ ID NO: 143 and 266), and s1-41 (SEQ ID NO: 161 and 267) (both strands of DNA are shown, the central dinucleotide is shaded in grey, the RD motif is underlined, and the base at position 10 of both the left and right half sites is shown in bold). The change from the A base preferred by WT Bxb1 is indicated by the arrow. (C) Targeted integration (TI) values ​​(percent of endogenous alleles with targeted integration) at these endogenous sites in human cells for either a 50% mixture of S231F Bxb1 variant and WT Bxb1 or 100% WT Bxb1. Any changes in the 10th position of the target site compared to the preferred target of WT Bxb1 are shown in the second column. Multiple replicates of WT Bxb1 were performed. Data for WT Bxb1 are the mean of all replicates + / - standard deviation. The last column shows the ratio of TI at S231F variant to TI at WT Bxb1. The increased TI activity of S231F variant at endogenous sites s5-1, s5-11, and s1-41 is consistent with both the changes in target selectivity observed in the plasmid-based experimental system and the targeting rules shown in Table 1. [Figure 7]List of Bxb1 variants that improve targeted integration (TI) activity at endogenous human target sites compared to wild-type (WT) Bxb1, consistent with the DNA target sequence changes shown in Table 1. Variants that showed TI values ​​at least 3 standard deviations above the mean for WT Bxb1 for the same targets in parallel experiments were considered improved compared to WT Bxb1. TI and variant / WT data are presented as in FIG. 6C. Sequence changes at these sites consistent with changes in Table 1 are shown in columns 2, 3, and 4, with column 2 indicating the corresponding position of the change in the target site, column 3 indicating the change at a given position in the left half site of the target site listed in column 1, and column 4 indicating the change at the corresponding position in the right half site of the target site. If the base at the indicated position in the indicated half site matches the target selectivity of WT Bxb1 (e.g., A at position 10, C at position 9, or A at position 7), the base is indicated as "WT Bxb1." [Figure 8]A-C show the change in target site selectivity of the Bxb1 F314G variant and the Bxb1 G316Y variant. (A) Change in target selectivity specificity at position 19 of the Bxb1 F314G variant (left panel) and at position 21 of the Bxb1 G316Y variant (right panel) obtained by the plasmid-based experimental system shown in Figure 4. The bases are numbered according to the scheme shown in Figure 2. Note that these endogenous human target sites all resemble attB sites. The attP site has five bases inserted compared to the attB sequence, and those inserted bases, if present, are at positions 13-17, so that the base labeled as position 18 is adjacent to the base labeled as position 12. (B) The symmetric attB site (SEQ ID NOs: 268 and 269) used in the plasmid-based experimental system shown in FIG. 4 compared to the endogenous human target sites for Bxb1, s5-16 (SEQ ID NOs: 146 and 270) and s3-28 (SEQ ID NOs: 163 and 271). The central dinucleotide is shaded grey and the ZD motif in each half-site is underlined. Note that the ZD motif in the left half-site of site s5-16 deviates so much from the ZD motif used in the plasmid-based system that it is likely a different sequence motif and cannot be treated as the same ZD sequence motif as characterized in the plasmid-based system. Thus, it likely does not follow the same rules of target selectivity as shown in Table 1. Site s5-16 has a ZD motif in the right half-site that is similar to the ZD motif in the plasmid-based system. The T at position 19 in the right half-site of this site is shown in bold and pointed to by an arrow. Both half sites of s3-28 contain a ZD motif similar to the one used in the plasmid-based system, therefore the ZD motif in both half sites is underlined. The G at position 21 in the right half site is shown in bold and pointed to by an arrow.(C) Comparison of targeted integration (TI) data at the endogenous human target site s5-16 in human cells with a 50% mixture of Bxb1 F314G variant and wild type (WT) Bxb1 versus 100% WT Bxb1 (upper panel), and comparison of TI data at the endogenous human target site s3-28 in human cells with a 50% mixture of Bxb1 G316Y variant and WT Bxb1 versus 100% WT Bxb1 (lower panel). Target shifts and TI data are presented as in Figure 6C. [Figure 9] A list of Bxb1 variants that improve targeted integration (TI) activity at the indicated endogenous human target sites compared to wild-type (WT) Bxb1 is shown. Bases that match the targeting preference of WT Bxb1 (e.g., G at position 19) are indicated as "WT Bxb1." Half-sites with ZD domains that deviate sufficiently from the ZD motif characterized in the plasmid system to likely result in a different sequence motif are indicated by a blank in the entry for the relevant half-site. Data are presented as in Figures 6C, 7, and 8C. [Figure 10] List of Bxb1 variants that have changes at positions 21, 22, 23, or 24 in the ZD motif of one or both half-sites that improve targeted integration (TI) activity at the indicated endogenous human target sites compared to wild-type (WT) Bxb1. Target site changes and data are presented as in FIG. 9. Target sequence preference of WT Bxb1 is T at positions 21 and 22, and G at positions 23 and 24. [Figure 11]Data are presented demonstrating the improvement of targeted integration (TI) activity at various different endogenous human target sites by D257K Bxb1 variant. D257K, both as a 50% mixture containing WT Bxb1 and as a 100% D257K variant without any WT Bxb1, improved TI activity compared to wild-type (WT) Bxb1. In these examples, 100% D257K variant has higher activity compared to 50% D257K or 100% WT Bxb1. Thus, D257K appears to be a Bxb1 variant that can increase activity at most or all endogenous target sites, regardless of the change in its exact target site compared to the preferred target site of WT Bxb1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present disclosure provides large serine recombinase variants, such as Bxb1 recombinase variants and orthologous recombinase variants, and systems for gene editing that include the variants. These recombinase variants have target DNA sequences that are altered compared to their wild-type counterparts, and can be used to target endogenous DNA sequences in the genome of organisms of interest, including humans. These enzymes can integrate donor DNA into genomes, or excise or invert target genome sequences. Thus, they can be used to integrate therapeutic genes into genomes, or repair or remove pathogenic genes from genomes to achieve therapeutic effects.

[0024] The gene editing system of the present invention comprising recombinase variants has several important advantages over other gene editing systems. First, the system prevents undesired modifications of the genome. The most widely used gene editing systems are the CRISPR / Cas (clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas)) system, the zinc finger nuclease (ZFN) system, and the transcription activator-like effector nuclease (TALEN) system. These systems rely on the activity of nucleases and DNA repair mechanisms, such as homologous recombination and non-homologous end joining, which are prone to errors and can introduce insertions or deletions (indels) and / or translocations. By having both cleavage and ligation functions, the recombinase of the present invention cleaves DNA in a very precise site-specific manner and ligates the breaks, thereby preventing the introduction of harmful indels into the genome. Second, there is no inherent size restriction on the DNA that can be integrated into the host genome using the editing system of the present invention. Third, recombinations generated by the system of the invention are not easily reversible without accessory proteins termed recombination directivity factors (RDFs), and thus the gene edits are stable and heritable. In summary, the recombinase variants of the invention can be used to stably and precisely remove DNA from a host genome at sites specifically recognized by the recombinase variants of the invention, to integrate large synthetic and / or exogenous donor DNA into a host genome, or to invert segments of a host genome.

[0025] I. Variant serine recombinase system In the present disclosure, a system for gene editing is provided, comprising a large serine recombinase variant, for example, a Bxb1 recombinase variant or an orthologous recombinase variant, and optionally a donor DNA to be integrated into the host genome at a target site. Also provided are expression constructs for delivering any of the above components, and methods for gene editing using one or more of the above components. Each component of the gene editing system of the present invention is described in further detail below.

[0026] A. Large serine recombinase variants with altered target DNA sequences As used herein, the term "recombinase" refers to a protein that catalyzes recombination. The term "recombination" refers to DNA excision, inversion, integration, chromosomal exchange, RMCE, or transposition in a target DNA sequence, e.g., a host genome. The terms "large serine recombinase" and "serine recombinase" refer to a family of recombinase proteins that induce double-stranded DNA breaks and use serine residues in their active sites to join the separated DNA fragments during recombination.

[0027] As used herein, the term "Bxb1 recombinase" refers to a serine recombinase having the exemplary amino acid sequence shown below. Wild type Bxb1 10 20 30 MRALVVIRLS RVTDATTSPE RQLESCQQLC 40 50 AQRGWDVVGV AEDLDVSGAV 60 70 80 DPFDRKRRPN LARWLAFEEQ PFDVIVAYRV 90 100 DRLTRSIRHL QQLVHWAEDH 110 120 130 KKLVVSATEA HFDTTTPFAA VVIALMGTVA 140 150 QMELEAIKER NRSAAHFNIR 160 170 180 AGKYRGSLPP WGYLPTRVDG EWRLVPDPVQ 190 200 RERILEVYHR VVDNHEPLHL 210 220 230 VAHDLNRRGV LSPKDYFAQL QGREPQGREW 240 250 SATALKRSMI SEAMLGYATL 260 270 280 NGKTVRDDDG APLVRAEPIL TREQLEALRA 290 300 ELVKTSRAKP AVSTPSLLLR 310 320 330 VLFCAVCGEP AYKFAGGGRK HPRYRCRSMG 340 350 FPKHCGNGTV AMAEWDAFCE 360 370 380 EQVLDLLGDA ERLEKVWVAG SDSAVELAEV 390 400 NAELVDLTSL IGSPAYRAGS 410 420 430 PQREALDARI AALAARQEEL EGLEARPSGW 440 450 EWRETGQRFG DWWREQDTAA 460 470 480 KNTWLRSMNV RLTFDVRGGL TRTIDFGDLQ 490 500 EYEQHLRLGS VVERLHTGMS (SEQ ID NO:1)

[0028] The domain structure of Bxb1 recombinase and its mechanism of action are shown in Figure 1. The following sequences are the attB and attP sites recognized by wild-type Bxb1 recombinase, in which the central dinucleotide is in italics and bold, the single underlined region is recognized by the ZD domain, and the double underlined region is recognized by the RD domain. [Table 1]

[0029] To simplify the search for Bxb1 recombinase variants with altered target DNA specificity, a more symmetric version of the attB target site can be used, having the exemplary nucleotide sequence shown below (SEQ ID NO:80; see also FIG. 2 ): The central dinucleotide is italicized and bold, the single underlined region is recognized by the ZD domain, and the double underlined region is recognized by the RD binding domain. Symmetric attB [Table 2] However, variants identified by this attB sequence will not be limited to recognition of symmetric attB sequences.

[0030] The present disclosure provides variants of SEQ ID NO: 1 above having modified DNA recognition sequences. As used herein, the terms "Bxb1 recombinase variant(s)", "variant Bxb1 recombinase(s)", "Bxb1 variant(s)", or "variant Bxb1" refer to Bxb1 recombinases having one or more amino acid mutations (e.g., substitutions, insertions, and / or deletions) that alter the DNA targeting specificity of the variants compared to wild-type (WT) Bxb1 recombinase. As used herein, the terms "orthologous recombinase variant(s)" or "orthologous serine recombinase variant(s)" refer to orthologous serine recombinases having one or more amino acid mutations that alter the DNA targeting specificity of the variants compared to their WT counterparts.

[0031] The serine recombinase, including the recombinase variants of the present invention, comprises a catalytic NTD and a CTD involved in sequence-specific DNA recognition. The CTD is further divided into an RD and a ZD containing a CC motif (FIG. 1). The NTD domain may generally correspond to amino acids 1-145 (numbering according to SEQ ID NO: 1) in wild-type Bxb1 recombinase, or a functionally similar sequence in a Bxb1 recombinase variant, or a functionally similar sequence in an orthologous serine recombinase(s) and variants thereof. The term "functionally similar sequence" refers to a sequence of amino acid residues or a protein domain having the same or substantially the same biological function. The RD domain may generally correspond to amino acids 140-287 (numbering according to SEQ ID NO: 1) in wild-type Bxb1 recombinase, or a functionally similar sequence in a Bxb1 recombinase variant, or a functionally similar sequence in an orthologous serine recombinase(s) and variants thereof. The ZD domain may generally correspond to amino acids 302-500 (numbering according to SEQ ID NO: 1) in wild-type Bxb1 recombinase, or a functionally similar sequence in a Bxb1 recombinase variant, or a functionally similar sequence(s) in orthologous serine recombinase(s) and variants thereof. The coiled-coil (CC) motif may generally correspond to that found within the ZD domain of wild-type Bxb1 recombinase, or a functionally similar sequence in a Bxb1 recombinase variant, or a functionally similar sequence(s) in orthologous serine recombinase(s) and variants thereof.

[0032] In some embodiments, the recombinase variants provided may comprise the C-terminal amino acid sequence GSGSGSHHHHHHGSGPKKKRKV (SEQ ID NO: 249), or a similar linker, His tag, and / or nuclear localization sequence. In some embodiments, the recombinase variants provided comprise a nuclear localization signal at the N-terminus or C-terminus.

[0033] In some embodiments, the Bxb1 recombinase variants of the invention may contain mutations in the amino acid sequence of the ZD domain or the RD domain. For example, substitutions in the ZD domain may be made at one or more of amino acids 311-335. Substitutions in the RD domain may be made at one or more of amino acids 137-160, 195-201, 229-239, 249-252, and 284-287. The region between the ZD and RD is believed to function as a linker between these domains, and therefore substitutions in this linker region may be made at one or more of amino acids 288-301. In some embodiments, recombinase variants of the invention may contain one or more mutations at the following amino acid positions: 147, 148, 149, 154, 155, 156, 158, 197, 198, 230, 231, 232, 233, 237, 257, 309, 312, 314, 315, 316, 318, 323, 324, 325, 326, and 335. Unless otherwise stated, the amino acid positions in the Bxb1 recombinase of the present disclosure are numbered according to SEQ ID NO:1.

[0034] In further embodiments, the ZD domain of the Bxb1 variant may comprise one or more of the following amino acid substitutions: F314: substitution with A, C, D, E, G, H, I, L, N, Q, S, T, V, W, or Y; A315: Substitution for F, G, H, I, M, N, S, T, W, or Y; G316: Substitution with A, C, D, E, F, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; G318: substitution with I, K, R, or W; R323: substitution with G or K; and R325: Substitution with D, E, K, L, M, N, Q, S, or W.

[0035] In further embodiments, the Bxb1 variant RD domain may comprise any one or more of the following amino acid mutations: F147: mutation to A, K, or R; N148: mutation to Q or T; L158: mutation to D, N, S, T, or W; P197: mutation to H, R, or T; S231: mutation to F, G, H, K, R, V, or Y; T233: mutation to F, H, K, R, W, or Y; and R237: Mutation to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y.

[0036] In some embodiments, the recombinase variants of the invention may contain mutations within the amino acid sequence of the NTD domain or the CC coiled motif.

[0037] The effect of exemplary single amino acid substitutions of Bxb1 recombinase (SEQ ID NO:1) on the recognition sites of the recombinase is shown in Tables 1 and 2 below. In these tables, "WT Nt" refers to the nucleotide at the wild-type attB or attP site (e.g., SEQ ID NOs:2 and 3). Table 1 shows the results of a saturation mutagenesis experiment at amino acids 147, 148, 149, 154, 155, 156, 158, 197, 198, 230, 231, 232, 233, 237, 257, 309, 312, 314, 315, 316, 318, 323, 324, 325, 326, and 335 (see Example 3 below). In the table, "new Nt" refers to a nucleotide at which mutation of Bxb1 recombinase results in at least a 3-fold change in the nucleotide selectivity of wild-type Bxb1 recombinase or changes the preferred nucleotide of wild-type Bxb1 recombinase to a different nucleotide. For example, if the Bxb1 recombinase variant has a F147A, N148T, L158W, T233F, or T233Y substitution, at nucleotide 6 of the WT attB or attP site, the Bxb1 recombinase variant has at least a 3-fold higher binding affinity for nucleotide A compared to wild-type Bxb1 recombinase. [Table 3] TIFF2025504019000004.tif223159TIFF2025504019000005.tif30159

[0038] In some embodiments, the Bxb1 recombinase variant may contain two or more substitutions, such as those shown in Table 1 above. Such variants may have higher binding affinity for target sequences with two or more nucleotide differences from the WT attB or attP recognition sequence. For example, a Bxb1 recombinase variant containing an R237K and a T233R mutation will have higher binding affinity for a target sequence containing an A at position 9 and a G at position 10. Some of the single substitutions result in increased binding affinity at multiple nucleotide positions. As an example, a Bxb1 recombinase variant containing an F147R mutation will have higher binding affinity for a target sequence containing a G at position 6, a C at position 7, a G at position 9, and a C at position 10. Some of the substitutions result in increased binding affinity for multiple nucleotides at a single position. For example, a Bxb1 recombinase variant containing an R237V mutation will have higher binding affinity for a target sequence containing an A, G, or T at position 9.

[0039] In certain embodiments, Bxb1 recombinase variants may contain one or more substitutions, such as those shown in Table 2 below. Table 2 shows two Bxb1 recombinase mutations that result in the greatest increase in binding affinity for A, C, G, or T at the indicated nucleotide positions (position numbering according to FIG. 2) in the targeted DNA binding sequence. For example, F147A, N148T, L158W, T233F, and T233Y mutations result in Bxb1 recombinase variants with increased binding affinity for target sequences containing an A at position 6, while T233F and N148T result in the greatest increase in affinity. The underlined mutations result in Bxb1 recombinase variants that recognize target sequences containing a novel, preferred nucleotide at the indicated position. For example, a Bxb1 recombinase variant carrying a T233R mutation preferentially recognizes sequences with a G at position 10, whereas wild-type Bxb1 recombinase preferentially recognizes sequences with an A at position 10. [Table 4] TIFF2025504019000007.tif100159

[0040] In some aspects of the disclosure, recombinases orthologous to Bxb1 recombinase may also be used. In some embodiments, the orthologous serine recombinase is φC31 integrase, which includes the following exemplary amino acid sequence: Wild type φC31 10 20 30 MDTYAGAYDR QSRERENSSA ASPATQRSAN 40 50 EDKAADLQRE VERDGGRFRF 60 70 80 VGHFSEAPGT SAFGTAERPE FERILNECRA 90 100 GRLNMIIVYD VSRFSRLKVM 110 120 130 DAIPIVSELL ALGVTIVSTQ EGVFRQGNVM 140 150 DLIHLIMRLD ASHKESSLKS 160 170 180 AKILDTKNLQ RELGGYVGGK APYGFELVSE 190 200 TKEITRNGRM VNVVINKLAH 210 220 230 STTPLTGPFE FEPDVIRWWW REIKTHKHLP 240 250 FKPGSQAAIH PGSITGLCKR 260 270 280 MDADAVPTRG ETIGKKTASS AWDPATVMRI 290 300 LRDPRIAGFA AEVIYKKKPD 310 320 330 GTPTTKIEGY RIQRDPITLR PVELDCGPII 340 350 EPAEWYELQA WLDGRGRGKG 360 370 380 LSRGQAILSA MDKLYCECGA VMTSKRGEES 390 400 IKDSYRCRRR KVVDPSAPGQ 410 420 430 HEGTCNVSMA ALDKFVAERI FNKIRHAEGD 440 450 EETLALLWEA ARRFGKLTEA 460 470 480 PEKSGERANL VAERADALNA LEELYEDRAA 490 500 GAYDGPVGRK HFRKQQAALT 510 520 530 LRQQGAEERL AELEAAEAPK LPLDQWFPED 540 550 ADADPTGPKS WWGRASVDDK 560 570 580 RVFVGLFVDK IVVTKSTTGR GQGTPIEKRA 590 600 SITWAKPPTD DDEDDAQDGT EDVAA (SEQ ID NO: 77)

[0041] In some embodiments, the orthologous serine recombinase is a Pa557 recombinase comprising the following exemplary amino acid sequence: Wild type Pa557 10 20 30 MNMHSPTVTT RAALYLRVST ARQAEHDISI 40 50 PDQKRQGEAY CEQRGFQLVE 60 70 80 TYVEPGATAT NDKRPEFQRM IEAGTSKPAP 90 100 FDIVVVHSFSRFFRDHFEME 110 120 130 FYVRKLAKNG VKLVSITQEM GDDPMHQMMR 140 150 QIMALFDEYQ SKENAKHVLR 160 170 180 AMNENARQGF WNGARPPIGY RIVAAEQRGS 190 200 KTKKKLEIDP LHADTVRLIY 210 220 230 RLFLEGDGTR GAMGVKAIAT YLNERRFFTR 240 250 DGGRWGLAQI HAILTRTTYI 260 270 280 GEHRFNTRSH KDREKKPESE IAIMAVPPLI 290 300 EREIYDAVQA RLKSRNPMVT 310 320 330 PARVSSGPTL LTGICFCAKC GGAMTLRTGQ 340 350 GSTGATYRYY TCSTKARQGK 360 370 380 TGCKGRTIPM DKLDHLVADH IGDRLLQPKR 390 400 LETVLASVID RRQERAERRR 410 420 430 EHLAELNRRI TEADQRLGRL FDAIEAGMVD 440 450 KDDAMAKERM VSLKALRDQA 460 470 480 AADAERTQLA LDSSGNQGVS PDMLKGFARK 490 500 ARERIRLDDG GYRRDHLRAL 510 520 530 AQRVEVADDE VRIMGSKSEL LRTLVAASSV 540 550 ETAAFGVQSS VLKWRTQEDS NLRPLGS (SEQ ID NO: 78)

[0042] In some embodiments, the orthologous serine recombinase is a Pa570 recombinase comprising the following exemplary amino acid sequence: Wild type Pa570 10 20 30 MARLISYLRM STSEQLRGFS LERQRKLIAD 40 50 FAAKNGLSVE ENTLEDIGR 60 70 80 SSFSDDAQQK ELTRFFENLN AGKYEPGDVF 90 100 ALENIDRLTR RGPVDAILKV 110 120 130 NQIISKGLKL AIISGNEQRI IEDVNDVFTI 140 150 INLSIDASRA NKESKNKSDK 160 170 180 GLSNWQEKRN LASTYKIAMT AQAPAWLDTE 190 200 IFYIFDEEKK KNTKRRKYVL 210 220 230 NEEKAEAVRL IFDLYSNGNG ALKIKNILNE 240 250 RNIPTFKGAP YWEPSIITKI 260 270 280 LKNPATFGLY QPKKQGTGKR DLIAAGEPIN 290 300 DYFPPVITRD LFEQCEHIRE 310 320 330 GNSTRKGRKG KLFTNLFTGL LTCSKCGGPV 340 350 HLINPGIDKR NKVQKSIYYL 360 370 380 VCKRAKFTKE CTTKRVRYDD FEIALLKAIQ 390 400 INVITED NNPLEILVKK 410 420 430 QRSKETEINK KRKLIENFQR QFLENDGDLP 440 450 SFMISQAKDA EISIKELEED 460 470 480 QREIASEIAQ LNIYNSNVDN AIEELKENAD 490 500 YGTRSKINLL FHEIIKNISL 510 520 530 DTENQFYTVR FKNGVMRVIT AAGFIATTEE 540 550 QTQADINAIL QSIEGPRIPR 560 570 EIATDAEKLI EYLKAREVIE (SEQ ID NO: 79)

[0043] In some embodiments, the orthologous serine recombinase is a LI integrase comprising the following exemplary amino acid sequence: Wild type LI 10 20 30 MKAAIYIRVS TQEQVENYSI QAQTEKLTAL 40 50 CRSKDWDVYD TFIDGGYSGS 60 70 80 NMNRPALNEM LSKLHEIDAV VVYRLDRLSR 90 100 SQKDTITLIE EYFLKNNVEF 110 120 130 VSLSETLDTS SPFGRAMIGI LSVFAQLERE 140 150 TIRDRMVMGK IKRIEAGLPL 160 170 180 TTAKGRTFGY DVIDTKLYIN EEEAKQLRLI 190 200 YDIFEEEQSI TFLQKRLKKL 210 220 230 GFKVRTYNRY NNWLTNDLYC GYVSYKDKVH 240 250 VKGIHEPIIS EEQFYRVQEI 260 270 280 FSRMGKNPNM NKESASLLNN LVVCSKCGLG 290 300 FVHRRKDTVS RGKKYHYRYY 310 320 330 SCKTYKHTHE LEKCGNKIWR ADKLEELIID 340 350 RVNNYSFASR NIDKEDELDS 360 370 380 LNEKLKIEHA KKKRLFDLYI NGSYEVSELD 390 400 SMMNDIDAQI NYYEAQIEAN 410 420 430 EELKKNKKIQ ENLADLATVD FNSLEFREKQ 440 450 LYLKSLINKI YIDGEQVTIE WL (SEQ ID NO: 131)

[0044] In some embodiments, the orthologous serine recombinase is A118 integrase, which comprises the following exemplary amino acid sequence: Wild type A118 10 20 30 MKAAIYIRVS TQEQVENYSI QAQTEKLTAL 40 50 CRSKDWDVYD IFIDGGYSGS 60 70 80 NMNRPALNEM LSKLHEIDAV VVYRLDRLSR 90 100 SQRDTITLIE EYFLKNNVEF 110 120 130 VSLSETLDTS SPFGRAMIGI LSVFAQLERE 140 150 TIRDRMVMGK IKRIEAGLPL 160 170 180 TTAKGRTFGY DVIDTKLYIN EEEAKQLQLI 190 200 YDIFEEEQSI TFLQKRLKKL 210 220 230 GFKVRTYNRY NNWLTNDLYC GYVSYKDKVH 240 250 VKGIHEPIIS EEQFYRVQEI 260 270 280 FTRMGKNPNM NRDSASLLNN LVVCSKCGLG 290 300 FVHRRKDTMS RGKKYHYRYY 310 320 330 SCKTYKHTHE LEKCGNKIWR ADKLEELIIN 340 350 RVNNYSFASR NVDKEDELDS 360 370 380 LNEKLKIEHA KKKRLFDLYI NGSYEVSELD 390 400 SMMNDIDAQI NYYESQIEAN 410 420 430 EELKKNKKIQ ENLADLATVD FDSLEFREKQ 440 450 LYLKSLINKI YIDGEQVTIE WL (SEQ ID NO: 132)

[0045] In some embodiments, the orthologous serine recombinase is a TP901 recombinase comprising the following exemplary amino acid sequence: Wild type TP901 10 20 30 <h2 style=";text-align:left;direction:ltr">MTKKVAIYTR VSTTNQAEEG FSIDEQIDRL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 40 50<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TKYAEAMGWQ VSDTYTDAGF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 60 70 80<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SGAKLERPAM QRLINDIENK AFDTVLVYKL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 90 100<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DRLSRSVRDT LYLVKDVFTK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 110 120 130<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NKIDFISLNE SIDTSSAMGS LFLTILSAIN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 140 150<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EFERENIKER MTMGKLGRAK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 160 170 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SGKSMMWTKT AFGYYHNRKT GILEIVPLQA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 190 200<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TIVEQIFTDY LSGISLTKLR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 210 220 230<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DKLNESGHIG KDIPWSYRTL RQTLDNPVYC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 240 250<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GYIKFKDSLF EGMHKPIIPY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 260 270 280<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ETYLKVQKEL EERQQQTYER NNNPRPFQAK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 290 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YMLSGMARCG YCGAPLKIVL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 310 320 330<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GHKRKDGSRT MKYHCANRFP RKTKGITVYN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 340 350<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DNKKCDSGTY DLSNLENTVI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 360 370 380<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DNLIGFQENN DSLLKIINGN NQPILDTSSF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 390 400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KKQISQIDKK IQKNSDLYLN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 410 420 430<h2 style=";text-align:left;direction:ltr"> DFITMDELKD RTDSLQAEKK LLKAKISENK 440 450 FNDSTDVFEL VKTQLGSIPI 460 470 480 NELSYDNKKK IVNNLVSKVD VTADNVDIIF KFQLA (SEQ ID NO: 133)

[0046] An alignment of some of the orthologous recombinases listed above can be found in Figure 3 of Rutherford et al., supra.

[0047] In some embodiments, the orthologous recombinase variants may include mutations in the ZD or RD. In some embodiments, the orthologous recombinase variants may include mutations in the amino acid sequence of the NTD domain or the CC coiled motif. In some embodiments, the orthologous recombinase may include one or more mutations at positions corresponding to residues S231, T233, R237, F314, A315, G316, G318, and R325 of the Bxb1 recombinase. For example, a φC31 integrase variant may include one or more mutations at amino acid positions D273, A275, R279, K375, R376, G377, E379, and R386 (numbering according to SEQ ID NO: 77), a Pa557 recombinase variant may include one or more mutations at amino acid positions G236, A238, A242, R327, T328, G329, G331, and R338 (numbering according to SEQ ID NO: 78), and a Pa570 recombinase variant may include one or more mutations at amino acid positions E243, S245, K249, I333, N334, P335, I337, and Q344 (numbering according to SEQ ID NO: 79).

[0048] In some embodiments, the system comprises a mixture of different Bxb1 recombinase variants and / or other orthologous serine recombinase variants, and / or the nucleic acids encoding them, as well as any associated donor DNA molecules.

[0049] In some embodiments, the Bxb1 recombinase variants result in increased transgene insertion at endogenous target sites in eukaryotic genomes compared to wild-type Bxb1 recombinase, hi some embodiments, the Bxb1 recombinase variants increase transgene insertion by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold compared to wild-type Bxb1 recombinase.

[0050] B. Recombinase variant recognition sites The recombinase variants of the invention are overall more versatile than their WT counterparts in that the variants can bind a broader repertoire of DNA recognition sites. The following table (Table 3) shows attB-like sequences that can be bound by one or a mixture of the variants herein more efficiently than the wild-type Bxb1 recombinase. The sequences in the following table are SEQ ID NOs: 83-130 in order of appearance. In the table, the underlined nucleotide is the central dinucleotide, the "hg38 coordinate" refers to the position according to the Genome Reference Consortium Human Build 38 standard, and the bold and italicized nucleotides indicate mismatches to the following consensus attB sequence (for sites 1-14, this is SEQ ID NO: 82 (see Examples), for sites 15-24, this is SEQ ID NO: 134, for sites 25-36, this is SEQ ID NO: 135, and for sites 37-48, this is SEQ ID NO: 136). 5'-GGTTTGTNNACNACNGNNNNNNCNGTNGTNAGGATCNN (SEQ ID NO: 134) 5'-NNGATCCTGACGACNGNNNNNNCNGTNGTNNACAAACC (SEQ ID NO: 135) 5'-NNGATCCTGACGACNGNNNNNNCNGTNGTNAGGATCNN (SEQ ID NO: 136) [Table 5] TIFF2025504019000009.tif211159TIFF2025504019000010.tif189159

[0051] Wild-type Bxb1 recombinase or its orthologues can be engineered as described above, for example by applying the amino acid substitutions shown in Tables 1 and 2, such that the engineered protein is capable of binding with high affinity to their endogenous genomic target sites in human cells.

[0052] C. Donor DNA with engineered attB or attP sequences The provided recombinase variants can integrate donor DNA (e.g., circularized donor DNA) into a host genome when the donor DNA and the host DNA contain wild-type or variant attB and / or attP sequences. The disclosure provides nucleic acid molecules that contain variant attB and attP sequences recognized by the provided recombinase variants.

[0053] In some embodiments, an attB sequence described herein comprises any of SEQ ID NOs: 83-130 as shown in Table 3. In some embodiments, an attB sequence described herein comprises any of SEQ ID NOs: 4-40 as shown in Table 4. In some embodiments, an attP sequence described herein comprises any of SEQ ID NOs: 41-76 as shown in Table 5. In some embodiments, an attB or attP sequence may comprise a sequence selected from SEQ ID NOs: 80-82 and 134-136.

[0054] D. Recombination Events Bxb1 recombinase can generate various dimers that recognize and specifically bind to DNA recognition attB and attP sites. When attB and attP sites are present in the same cell, a pair of Bxb1 recombinase dimers that bind to the attB and attP sites form a tetramer, thereby assembling DNA fragments containing attB and attP sites and initiating DNA recombination. When the DNA fragments are present on the same DNA molecule (e.g., chromosome), this recombination event can cause DNA inversion or deletion. When the DNA fragments are present on different DNA molecules, this recombination event can cause DNA integration or cassette exchange. The recombinase variants of the present invention have mutations that allow them to catalyze recombination between variant attB and variant attP sequences, greatly expanding the repertoire of recognizable endogenous sites and improving the versatility of the enzyme.

[0055] The recombinase variants of the present disclosure can cause recombination events of interest, such as integration, inversion, excision, chromosomal exchange, and RMCE. Figure 3A shows integration mediated by the recombinase variants of the present disclosure. Integration requires the presence of a donor DNA with an attachment site compatible with the attachment site of the target DNA. Figure 3B shows excision mediated by the recombinase variants of the present disclosure. Excision requires two complementary attachment sites oriented in the same direction on the same DNA molecule. Figure 3C shows inversion mediated by the recombinase variants of the present disclosure. Inversion requires two complementary attachment sites oriented in opposite directions on the same DNA molecule. Figure 3D shows chromosomal exchange mediated by the recombinase variants of the present disclosure. Chromosomal exchange requires two complementary attachment sites oriented in the same direction on two separate linear DNA molecules. Figure 3E shows RMCE mediated by the recombinase variants of the present disclosure. RMCE requires a donor DNA molecule and a target DNA molecule, each containing two complementary attachment sites that are not transversely compatible. For example, the donor DNA molecule contains a gene X flanked by two different attB sites, and the target DNA molecule contains a gene Y flanked by two different attP sites, where the compatible attachment sites upstream of genes X and Y are complementary, and the compatible attachment sites downstream of genes X and Y are complementary, but the upstream and downstream sites are not transversely compatible. Transverse compatibility can be avoided, for example, by using different central dinucleotides for the upstream and downstream attachment sites. This system allows genes X and Y to be exchanged in the presence of a recombinase variant of the present disclosure. Only in the presence of the appropriate RDFs can the serine recombinase bind to the attR and attL sites and mediate the reverse recombination event.

[0056] II. Delivery of variant recombinase systems The Bxb1 recombinase variants or orthologous recombinase variants of the present disclosure can be introduced into target cells as proteins by various methods (e.g., electroporation, lipid nanoparticles, cationic or anionic liposomes, or nuclear localization signals (e.g., in combination with liposomes)). In some embodiments, the provided recombinase variants are introduced into target cells by a nucleic acid molecule encoding it, e.g., a DNA plasmid or mRNA. The nucleic acid molecule can be present in a nucleic acid expression vector, which can include expression control sequences, e.g., promoters, enhancers, transcription signal sequences, and transcription termination sequences, that allow expression of the coding sequence of the provided recombinase variants. As described herein, "delivery of a system" can refer to either delivery of a system comprising a Bxb1 recombinase variant or an orthologous recombinase variant as described herein, or delivery of a nucleic acid molecule encoding said system of the provided recombinase variants or a vector or expression construct comprising said nucleic acid molecule.

[0057] In some embodiments, the promoter on the vector for inducing expression of the provided recombinase variant is a constitutively active promoter or an inducible promoter. Suitable promoters include, but are not limited to, Rous Sarcoma Virus (RSV) long terminal repeat (LTR) promoter (optionally combined with an RSV enhancer), cytomegalovirus (CMV) promoter (optionally combined with a CMV enhancer), CMV immediate early promoter, Simian Virus 40 (SV40) promoter, dihydrofolate reductase (DHFR) promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, EF1α promoter, Moloney Murine Leukemia Virus (MoMLV) promoter, and the like. ), creatine kinase-based (CK6) promoter, transthyretin promoter (TTR), thymidine kinase (TK) promoter, tetracycline-responsive promoter (TRE), hepatitis B virus (HBV) promoter, human alpha 1-antitrypsin (hAAT) promoter, liver-specific chimeric promoter (LSP), E2 factor (E2F) promoter, human telomerase reverse transcriptase (hTERT) promoter, CMV enhancer / chicken beta-actin / rabbit beta-globin promoter (CAG promoter; Niwa et al., Gene (1991) 108(2):193-9), and RU-486-responsive promoter. In addition, the promoter may contain one or more autoregulatory elements to which the provided recombinase variant can bind and suppress its own expression level to a preset threshold value.

[0058] Any method of introducing nucleotide sequences into cells can be used, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, liposomes combined with nuclear localization signals, naturally occurring liposomes (e.g., exosomes), or viral transduction. In certain embodiments, the nucleotide sequence is in the form of mRNA and is delivered to cells by electroporation.

[0059] For in vivo delivery of expression vectors, viral transduction can be used. Various viral vectors known in the art, such as vaccinia vectors, adenovirus vectors, lentivirus vectors, poxvirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, and hybrid virus vectors, can be adopted by those skilled in the art for use in the present disclosure. In some embodiments, the viral vector used herein is a recombinant AAV (rAAV) vector. Any suitable AAV serotype can be used. For example, the AAV can be AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAV.PHP.B, AAV.PHP.eB, or AAVrhlO, or a new serotype of AAV or a pseudotype of AAV, such as AAV2 / 8, AAV2 / 5, AAV2 / 6, AAV2 / 9, or AAV2 / 6 / 9. In some embodiments, the expression vector is an AAV viral vector, and is introduced into target human cells by recombinant AAV viral particles, whose genome comprises a construct with AAV terminal inverted repeat (ITR) sequences at both ends to allow production of AAV viral particles in a production system, such as an insect cell / baculovirus production system or a mammalian cell production system. AAV can be genetically engineered so that its capsid protein has reduced immunogenicity or enhanced transduction capacity in humans. The viral vectors described herein can be produced using methods known in the art. Any suitable permissive or packaging cell type can be used to produce viral particles. For example, mammalian (e.g., 293) cells or insect (e.g., sf9) cells can be used as packaging cell lines.

[0060] Any type of cell can be targeted by the gene editing methods described herein. For example, the cell can be a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a mammalian (e.g., human) cell or a plant cell. Human cells can include, for example, T cells, natural killer (NK) cells, NK T cells, α-β T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, B cells, human embryonic stem cells, tumor infiltrating lymphocytes (TILs), or pluripotent stem cells (e.g., induced pluripotent stem cells (iPSCs)) from which lymphoid cells can be differentiated. In some embodiments, the system can be used to modify pluripotent stem cells before they are differentiated into multiple cell types. For example, lymphoid progenitor cells can be modified before differentiation into lymphoid cell types, such as regulatory T cells, effector T cells, natural killer cells, etc. In particular, the disclosed systems comprising two or more Bxb1 recombinase variants or orthologous recombinase variants can be used to prepare cells with multiple genes integrated, excised, or inverted at once, including pluripotent cells. In some embodiments, systems containing two or more provided recombinase variants can be used to prepare, for example, allogeneic T cells.

[0061] For agricultural applications, any method for the introduction of proteins or nucleic acid molecules into plant cells is contemplated, such as T-DNA delivery mediated by Agrobacterium tumefaciens.

[0062] III. Therapeutic applications The present disclosure provides a method of integrating a gene or DNA sequence into cellular DNA, excising a gene or DNA sequence in cellular DNA, or inverting a gene or DNA sequence in cellular DNA, comprising delivering a Bxb1 recombinase variant or orthologous recombinase variant system described herein to a cell (e.g., from a patient). The cell can be present in the patient (in vivo treatment), or the method described herein can be performed on a cell removed from the patient, and then the edited cell is delivered to the patient (ex vivo treatment). In some embodiments, the cell is further manipulated ex vivo before being used as a treatment. The term "treat" encompasses alleviating symptoms, preventing the onset of symptoms, slowing disease progression, improving quality of life, and prolonging survival. In some embodiments, the patient treated by the methods described herein is a mammal (e.g., human).

[0063] In some embodiments, the methods of the present disclosure are used to insert or excise disease-related genes or regulatory sequences to restore normal gene expression or activity. In some embodiments, the methods of the present disclosure can target specific alleles of genes, such as wild-type or mutant alleles. In certain embodiments, the alleles can be associated with cancer.

[0064] In some embodiments, the patient has cancer. In certain embodiments, cells from the patient are further modified before or after gene editing to confer resistance to chemotherapy drugs. The patient can then be treated with chemotherapy drugs, which in some embodiments can result in a higher survival rate of the edited cells compared to unedited cells.

[0065] In some embodiments, the patient has an autoimmune disorder.

[0066] In some embodiments, the patient has an autosomal dominant disease, for example, autosomal dominant polycystic kidney disease.

[0067] In some embodiments, the patient has a neurodevelopmental disorder.

[0068] In some embodiments, the patient has a mitochondrial disorder.

[0069] In some embodiments, the patient has sickle cell disease, hemophilia (e.g., hemophilia A, B, or C), cystic fibrosis, phenylketonuria, Tay-Sachs disease, prion disease, color blindness, lysosomal storage disease (e.g., Fabry disease), Friedreich's ataxia, prostate cancer, beta thalassemia, Huntington's disease, kidney transplant, inflammatory bowel disease, multiple sclerosis, amyotrophic lateral sclerosis, or frontotemporal dementia.

[0070] The present disclosure further provides pharmaceutical compositions comprising components of the gene editing system described herein, such as Bxb1 recombinase variants or orthologous recombinase variants, or nucleotide sequences encoding such components (e.g., in a viral or non-viral vector as described herein). The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, such as water, saline (e.g., phosphate-buffered saline), dextrose, glycerol, sucrose, lactose, gelatin, dextran, albumin, or pectin. In addition, the composition may contain auxiliary agents, such as wetting or emulsifying agents, pH buffers, stabilizers, or other agents that enhance the effectiveness of the pharmaceutical composition. The pharmaceutical composition may contain delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles.

[0071] In some embodiments, the recombinase variants provided herein may be used in the methods of treatment described herein, may be for use in the treatments described herein, or may be used in the manufacture of a medicament for the treatments described herein.

[0072] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the meanings commonly understood by those of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, exemplary methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In general, the technical terms used in connection with neurology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein and the techniques thereof are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms include plurals and plural terms include the singular. Throughout the specification and embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises", or "comprising", are understood to mean the inclusion of a specified integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not acknowledge that any of these documents constitute part of the general knowledge in the art. As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value that is on the same order of magnitude as the specified reference value. In certain embodiments, the term refers to a range of values ​​within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than a number) of the specified reference value, unless otherwise specified or otherwise clear from the context.

[0073] In order that this invention might be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as in any way limiting the scope of the invention. EXAMPLES

[0074] Example 1: attB and attP Plasmid Variants Below is the consensus target sequence for wild-type Bxb1 recombinase (the underlined sequence 5'-GGTTTGT is the ZD domain binding site, the underlined sequence 5'-ACNACNG is the RD domain binding site, and the position of the central dinucleotide is shown as a bold XX): [Table 6]

[0075] To simplify the search for Bxb1 recombinase variants with altered target DNA specificity, a more symmetric version of the Bxb1 attB target site was created (Table 4; see SEQ ID NO: 2 for the wild-type attB site). This facilitated the creation of symmetric mutations to both halves of the attB target site. Because the wild-type attP site is already palindromic at the ZD and RD domain binding sites (see SEQ ID NO: 3), it was not necessary to create a palindromic version. As with the attB plasmid variants, individual nucleotides in the binding domain were mutated to create constructs with each available nucleotide (Table 5), as shown below in bold for mutations at positions 9 and 10 (the underlined outer and inner sequences are bound by the ZD and RD domains, respectively; the central dinucleotide is in italics). "SEQ" in the table refers to the sequence number. Mutation at position 10: [Table 7] [Table 8] TIFF2025504019000014.tif216159TIFF2025504019000015.tif218159TIFF2025504019000016.tif222159 TIFF2025504019000017.tif223159TIFF2025504019000018.tif221159TIFF2025504019000019.tif193159 [Table 9] TIFF2025504019000021.tif218159TIFF2025504019000022.tif217159TIFF2025504019000023.tif151159

[0076] Example 2: Initial serine scanning screening Individual constructs with serine scanning mutations were made from a pVax vector expressing Bxb1 recombinase driven by a CMV promoter. The Bxb1 recombinase construct contained the amino acid sequence GSGSGSHHHHHHGSGPKKKRKV (SEQ ID NO: 249) at the C-terminus. Individual constructs corresponding to SEQ ID NOs: 2-76 were made in a non-expressing plasmid. Sets of constructs were pooled together to ensure equal concentrations of all four bases at a particular site (e.g., plasmids with attP of SEQ ID NOs: 3, 41, 42, and 43 were pooled together, plasmids with attP of SEQ ID NOs: 3, 44, 45, and 46 were pooled together, plasmids with attB of SEQ ID NOs: 4, 5, 6, and 7 were pooled together, and plasmids with attB of SEQ ID NOs: 4, 8, 9, and 10 were pooled together).

[0077] K562 cells were transfected according to the manufacturer's protocol. 2E5 cells were transfected with 200 ng of Bxb1 expression plasmid, 20 ng total of attB plasmid, and 1600 ng total of attP plasmid. The attB plasmids had 5 ng of each pooled nucleotide variant at a particular site. For example, when testing base 24 (Figure 2), SEQ ID NOs: 4, 5, 6, and 7 were pooled, so that 5 ng of each was delivered as a total of 20 ng of attB plasmid. In that same well, SEQ ID NOs: 3, 56, 57, and 58 were pooled, so that 400 ng of each was delivered as a total of 1600 ng of attP plasmid.

[0078] Across the library for all seven nucleotides thought to be recognized by the ZD domain, M244S, L245S, G246S, Y247S, A248S, L250S, N251S, G252S, K253S, T254S, V255S, R256S, D257S, D258S, D259S, G260S, A262S, K284S, T285S, S286A, R287S, A288S, K289S, P290S, A291S, V292S, S293A, T294S, P295S, S296A, L297S, L298S, L299S, R300S, V301S, A302S, and A303S were significantly higher than those of the control. The individual expression plasmids harboring the 11S, Y312S, K313S, F314S, A315S, G316S, G317S, G318S, R319S, K320S, H321S, P322S, R323S, Y324S, R325S, C326S, R327S, S328A, M329S, G330S, F331S, P332S, K333S, H334S, C335S, E445S, Q446S, D447S, A449S, A450S, K451S, Y452S, T453S, W454S, L455S, R456S, M458S, N459S, and V460S mutations were transfected.

[0079] Across the nucleotide mutant library spanning the five bases thought to be recognized by the RD domain (see Figure 2 ), the following mutants were identified: I137S, K138S, E139S, R140S, N141S, R142S, S143A, A144S, A145S, H146S, F147S, N148S, I149S, R150S, A151S, G152S, K153S, Y154S, R155S, G156S, Y157S, R158S, and G159S. 6S, S157A, L158S, P159S, P160S, H195S, E196S, P197S, L198S, H199S, L200S, V201S, E229S, W230S, S231A, A232S, T233S, A234S, L235S, K236S, R237S, S238A, M239S, M244S, L245S, G246S, Y247S, A248S, T249S, L250 S, N251S, G252S, K253S, T254S, V255S, R256S, D257S, D258S, D259S, G260S, A262S, K284S, T285S, S286A, R 287S, A288S, K289S, P290S, A291S, V292S, S293A, T294S, P295S, S296A, L297S, L298S, L299S, R300S, V301S , A311S, Y312S, K313S, F314S, A315S, G316S, G317S, G318S, R319S, K320S, H321S, P322S, R323S, Y324S, R325S, C326S, R327S, S328A, M329S, G330S, F331S, P332S, K333S, H334S, and C335S mutations were transfected into the individual expression plasmids.

[0080] Transfected cells were harvested after 3 days of incubation at 37° C. using Lucigen's QuickExtract™ (Cat. No. QE09050) according to the manufacturer's protocol. Polymerase chain reaction (PCR) was performed on the extracted DNA using Invitrogen's Accuprimer™ Taq DNA Polymerase, High Fidelity (Cat. No. 12346094) as shown in Table 6 below. [Table 10]

[0081] PCR was carried out using the primers shown in Table 7 below, where P1 and P2 refer to primers 1 and 2, respectively. [Table 11]

[0082] PCR was performed using the temperature cycling protocol in Table 8 below, times are in minutes:seconds. [Table 12]

[0083] The product of PCR1 for next generation sequencing (NGS) was used as a template for PCR2 for NGS as shown in Table 9 below. [Table 13]

[0084] PCR was performed using the temperature cycling protocol in Table 10 below, times are in minutes:seconds. [Table 14]

[0085] The primers used were universal primers that added the DNA sequence required for Illumina sequencing. NGS PCR2 products were purified and sequenced on an Illumina sequencing instrument.

[0086] Sequences were analyzed for changes in specificity at the nucleotide sites pooled together. For example, in samples tested at nucleotide 21, integration events were examined and recorded that differed from the percentage observed with the wild-type Bxb1 recombinase (Figure 4).

[0087] Example 3: Saturation mutagenesis of identified residues Saturation mutagenesis was performed on amino acids that showed different incorporation profiles in the serine scanning of Example 2. These differences varied by eliminating incorporation, relaxing specificity, enhancing specificity, or changing specificity. The sites selected for saturation mutagenesis included F147, N148, I149, Y154, R155, G156, L158, P197, L198, W230, S231, A232, T233, R237, D257, E309, Y312, F314, A315, G316, G318, R323, Y324, R325, C326, and C335. Transfection and NGS analysis of K562 were performed in the same manner as described in Example 2 (Figure 4).

[0088] A summary of the results of saturation mutagenesis in the RD domain of Bxb1 recombinase is shown in Table 11 below. "Specificity change" refers to the largest change in the direction of the non-WT nucleotide for a given mutant at a given position. For example, if WT Bxb1 recombinase has a preference of 4.5% A, 85.6% C, 1.0% G, and 8.9% T at position 9, and the R237A mutant has a preference of 23.6% A, 1.9% C, 17.1% G, and 57.4% T at position 9, then the largest change in the direction of the non-WT nucleotide is a change to T (57.4%-8.9%=48.5% change), and R237A has a specificity change of 48.5% at position 9. "Average % specificity change" is the average of the specificity change for all mutants at a given amino acid position. "%TI" refers to the percentage of successful targeted integration, which is calculated as the percentage of sequence reads that had a sequence consistent with the integration of the donor sequence relative to the total number of sequence reads. The %TI for a mutant class (e.g., R237X) is the average of the %TI for all members of the class (e.g., the average %TI for R237A, R237C, R237D, etc.). The WT is the average of four replicates. In Tables 11 and 12 below, the "%TI" for a mutant class (e.g., R237X) is the average of the %TI for all members of the class (e.g., the average %TI for R237A, R237C, R237D, etc.), and the WT is the average of four replicates. [Table 15]

[0089] A summary of the results of saturation mutagenesis in the ZD domain of Bxb1 recombinase is shown in Table 12 below. [Table 16] Detailed results from side-by-side experiments for S231F and WT Bxb1 at position 10 are shown in FIG. 6A, and detailed results for F314G and G316Y at positions 19 and 21, respectively, are shown in FIG. 8A.

[0090] Example 4: Targeted integration mediated by Bxb1 at endogenous sites in human cells The performance of Bxb1 variants was characterized at a panel of endogenous Bxb1 sites (Fig. 5 ) that enable detectable integration activity of wild-type Bxb1.

[0091] The pseudo attB target sequence of the Bxb1 recombinase in the human genome is shown in Table 13 below. The pseudo attB site is flanked by 5 nucleotides at the 5' and 3' ends. The position of the central dinucleotide is shown in bold. [Table 17] TIFF2025504019000032.tif88159

[0092] Individual donor constructs were made by cloning the target site-specific sequences shown in Table 14 below into a standard plasmid DNA backbone. Each donor DNA sequence consists of a Bxb1 attP sequence with a target site matching the central dinucleotide and a binding site for a target site-specific primer. The attP sequence and primer binding site are flanked by additional genomic sequences that are partially randomized to form tag sequences for detection of targeted integration events by next generation sequencing. [Table 18] TIFF2025504019000034.tif216159TIFF2025504019000035.tif206159TIFF2025504019000036.tif93159

[0093] K562 cells were transfected according to the manufacturer's protocol. 2E5 cells were transfected with a total of 200 ng of Bxb1 expression plasmid (200 ng of one Bxb1 expression plasmid, or 100 ng of Bxb1 variant expression plasmid mixed with 100 ng of WT Bxb1 expression plasmid) and a total of 1600 ng of target site-specific attP donor plasmid. In most experiments, a mixture of 50% Bxb1 variant expression plasmid and 50% WT Bxb1 expression plasmid was used to obtain TI activity data for Bxb1 variants. One exception was the data shown in the left panel of Figure 11, where 100% (200 ng) Bxb1 D257K expression plasmid or 100% WT Bxb1 expression plasmid was used. Furthermore, a 50% mixture of Bxb1 variant and WT Bxb1 was used except for the experiment involving the D257K Bxb1 variant. This is because Bxb1 variants with altered target selectivity will generally only show improved targeting to the half-sites with the changes shown in Table 1 and may target the donor attP site and / or other half-sites poorly compared to WT Bxb1. A 50% mixture of variant Bxb1 and WT Bxb1 should allow the Bxb1 variant to bind to its preferred half-site and allow WT Bxb1 to bind to half-sites not preferred by the donor and Bxb1 variant.

[0094] Transfected cells were harvested after 3 days of incubation at 37° C. using Lucigen's QuickExtract™ (Cat. No. QE09050) according to the manufacturer's protocol. Polymerase chain reaction (PCR) was performed on the extracted DNA using Invitrogen's Accuprimer™ Taq DNA Polymerase, High Fidelity (Cat. No. 12346094) as shown in Table 6.

[0095] Target site-specific PCR was performed using the forward primers shown in Table 15. [Table 19] TIFF2025504019000038.tif89159 Target site-specific PCR was performed using the reverse primers shown in Table 16 below. [Table 20] TIFF2025504019000040.tif182159

[0096] PCR was performed using the temperature cycling protocol in Table 8. Time format is minutes:seconds. The product of PCR1 for next generation sequencing (NGS) was used as template for PCR2 for NGS as shown in Table 9. PCR was performed using the temperature cycling protocol in Table 10. Time format is minutes:seconds. The primers used were universal primers that add DNA sequences required for Illumina sequencing instruments. PCR2 for NGS products were purified and sequenced on an Illumina sequencing instrument. See also Miller et al., Nat Biotechnol. (2019) 37(8):945-52 for further description of this sequencing-based assay.

[0097] In one exemplary experiment, the change in target selectivity at position 10 of the Bxb1 S231F variant was measured. The S231F variant demonstrated improved targeting of C and T bases compared to WT Bxb1 recombinase (Figure 6A), which resulted in improved targeted integration at endogenous human target sites s5-1, s5-11, and s1-41 (Figure 6B-C). The change in target selectivity at positions binding to the RD domain (e.g., positions 7, 9, and 10) of different Bxb1 variants is shown in Figure 7.

[0098] In another exemplary experiment, the change in target selectivity of the Bxb1 F314G variant and the Bxb1 G316Y variant at positions 19 and 21, respectively, was measured. The F314G variant demonstrated improved targeting of T at position 19 compared to WT Bxb1, and the G316Y variant demonstrated improved targeting of G at position 21 compared to WT Bxb1 (Figure 8A). It was found that the F314G and G316Y variants improved targeted integration at endogenous human target sites s5-16 and s3-28, respectively (Figures 8B-C). The change in target selectivity at positions binding to the ZD domain (e.g., positions 19, 21, 22, 23, and 24) of various Bxb1 variants is shown in Figures 9 and 10.

[0099] The Bxb1 variants described herein also demonstrated improved targeted integration at a variety of different endogenous human target sites compared to WT Bxb1. For example, the Bxb1 D257K variant demonstrated improved targeted integration at human target sites, including s1-10, s1-39, s3-28, s3-41, s5-10, s5-11, s5-14, s5-15, s5-16, and s5-17 (Figure 11).

[0100] List of Arrays The sequences disclosed herein are listed in the following table (SEQ: sequence number): [Table 21]

Claims

1. A non-naturally occurring variant of Bxb1 recombinase that has altered DNA target specificity compared to wild-type Bxb1 recombinase, comprising one or more amino acid mutations within the zinc ribbon domain (ZD) or recombinase domain (RD).

2. 2. The Bxb1 recombinase variant of claim 1, wherein the one or more amino acid mutations are present at one or more of positions 231, 233, 237, 314, 318, 323, 325, 147, 148, 149, 154, 155, 156, 158, 197, 198, 230, 232, 257, 309, 312, 315, 316, 324, 326, and 335 (numbered according to SEQ ID NO: 1).

3. The one or more amino acid mutations are S231Y, T233S, T233W, R237Q, R237C, F314L, G316R, G316W, G318K, R323G, R325K, R325Q, F314A, F314C, F314D, F314E, F314G, F314H, F314I, F314N, F314Q, F314S, F314T, F314V, F314W, F314Y, A3 15F, A315G, A315H, A315I, A315M, A315N, A315S, A315T, A315W, A315Y, G316A, G316C, G316D, G316E, G316F , G316H, G316I, G316K, G316L, G316M, G316P, G316Q, G316S, G316T, G316V, G316Y, G318I, G318R, G318W, R3 23K, R325D, R325E, R325L, R325M, R325N, R325S, R325W, F147A, F147K, F147R, N148Q, N148T, L158D, L158 N, L158S, L158T, L158W, P197H, P197R, P197T, S231F, S231G, S231H, S231K, S231R, S231V, T233F, T233H, T 2. The Bxb1 recombinase variant of claim 1, selected from 233K, T233R, T233Y, R237A, R237D, R237E, R237F, R237G, R237H, R237I, R237K, R237L, R237M, R237N, R237P, R237S, R237T, R237V, R237W, R237Y, and D257K (numbering according to SEQ ID NO: 1).

4. A Bxb1 recombinase variant described in any one of claims 1 to 3, wherein the DNA target is specific to a particular allele of a gene.

5. A Bxb1 recombinase variant described in any one of claims 1 to 4, wherein the DNA target is present within a cell.

6. The Bxb1 recombinase variant of claim 5, wherein the cell is a eukaryotic cell, optionally a mammalian cell, and further optionally a human cell.

7. A nucleic acid molecule encoding the Bxb1 recombinase variant according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid molecule of claim 7, which may be a plasmid or a viral vector selected from an adeno-associated viral vector, an adenoviral vector, or a lentiviral vector.

9. A system for editing DNA in a cell, comprising the Bxb1 recombinase variant according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the vector according to claim 8.

10. 10. The system of claim 9, further comprising donor DNA, optionally wherein the donor DNA is circularized or linear, wherein the donor DNA may comprise a sequence selected from SEQ ID NOs: 80-81 and 134-136, and sequences found in Tables 3, 4, and 5; the donor DNA may be delivered by a plasmid or viral vector; the editing may comprise integration of DNA into the genome of the cell; the editing may comprise excision or inversion of DNA in the genome of the cell; the editing may comprise a chromosomal translocation in the genome of the cell; and the editing may occur in a genomic region comprising a sequence selected from SEQ ID NOs: 80-81 and 134-136, and sequences found in Tables 3, 4, and 5.

11. 11. A method for editing the genome of a cell, the method comprising providing the cell with a system according to claim 9 or 10.

12. A cell or its descendants comprising the system described in claim 9 or 10 inserted into the genome of the cell.

13. A cell or its progeny edited by the method of claim 11.

14. 14. The cell or progeny of claim 12 or 13, wherein the cell is a eukaryotic cell, optionally a mammalian cell, further optionally a human cell, for use in a method of treating a disease in a subject in need thereof, said use comprising administering the cell or progeny to the subject.

15. 12. The method of claim 11, wherein the editing results in increased transgene insertion at the endogenous target site in the eukaryotic genome compared to wild-type Bxb1 recombinase.

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