Large serine recombinases and systems and uses thereof

Abstract

Disclosed herein are engineered LSR sequences, compositions, and uses including in genome-editing systems and therapeutic compositions. The LSRs can facilitate nucleic acid recombination at particular attachment sites, using suitable donor sequences. The disclosed LSRs may be used to site-specifically recombine or integrate nucleic acid sequences for a variety of purposes, including treatment of human diseases.

Description

Attorney Docket No: 16290.0011-0304 LARGE SERINE RECOMBINASES AND SYSTEMS AND USES THEREOF FIELD OF THE DISCLOSURE

[0001] This disclosure relates to large serine recombinases (LSRs), genome-editing systems comprising LSRs, and methods of modifying or editing nucleic acids using LSRs. BACKGROUND

[0002] Large serine recombinases (LSRs) are a class of enzymes within the DNA integrase family that precisely catalyze the recombination of DNA in a controllable way. Generally found in phage and bacteria, these enzymes recognize pairs of distinct attachment sites and facilitate the integration of genetic elements into bacterial genomes via recombination at these cognate attachment sites. Historically, the attachment site within the bacterial genome is called AttB, while the cognate phage attachment site is called AttP. Recombinant attachment sites for LSRs are also referred to as a donor attachment site AttD and an acceptor attachment site AttA. Upon insertion of the genetic elements, parts of the attachment sites are incorporated at the boundaries of the integrated nucleic acids, forming sequences referred to as AttL and AttR. Unlike other classes of integrases that permit bidirectional integration, LSRs typically drive unidirectional integration of nucleic acids between the cognate attachment sites and require additional factors to reverse the reaction.

[0003] In recent years, a number of proteins that can modify DNA, such as TALENs, Cas proteins, transposases, and integrases, have been explored as genome editing tools with many scientific applications. Genome editing is particularly desirable as a therapeutic, as it has the potential to treat and even cure disease at the genetic level, as opposed to merely managing symptoms. Initial research on several LSR systems has been reported for genome modification purposes, but a number of limitations have emerged, including inefficient integration, gene-editing by-products like indel formation, lack of specificity, and the need to separately engineer a “landing pad” into human cells that can act as an appropriate attachment site for the LSR. Accordingly, there remains an unmet need for LSRs with improved properties for genome editing applications, including for use in simplified systems containing fewer components for applications such as therapeutics. SUMMARY

[0004] In some aspects, the present disclosure provides LSR variants and genome-editing systems comprising the LSR variants disclosed herein for recombination and recombination resulting in the integration of nucleic acids. The present disclosure also provides mutationsAttorney Docket No: 16290.0011-0304 for improving the genome integration activity and / or integration specificity given a wild-type LSR sequence.

[0005] In some embodiments, the LSR variant comprises an amino acid sequence having one or more mutations as compared to the LSR of SEQ ID NO: 2, wherein the LSR variant has at least 80% identity to SEQ ID NO: 2 and wherein the LSR variant has increased genome integration activity and / or integration specificity compared to the LSR of SEQ ID NO: 2.

[0006] In some embodiments, the one or more mutations increases genome integration activity by at least 5%, 10%, 15%, or 20% at an integration site of interest compared to the LSR of SEQ ID NO: 2. In some embodiments, the integration site of interest is GS010. In some embodiments, the one or more mutations comprise G10L, N29R, the combination of N29R and V66I, L31I, A36K, L43V, E57D, K58G, H74K, L116I, S117G, T153R, T153S, A156K, M160G, L163I, V184I, S213R, the combination of S213K and Q214R, Q214K, Q214H, the combination of Q214Y and P217C, K216N, P217D, S231I, S231V, T241V, N243K, N243R, S244A, Q260K, T266D, T266S, the combination of S284V and K286A, the combination of S284R and K286A, G285K, G285H, G285R, G285Y, A317K, K321G, K348E, C353R, T366L, V375H, G391R, G391K, S399A, N413S, P436I, P436L, P436Q, G452S, F462L, Q484S, S486K, K488S, and / or Y494K.

[0007] In some embodiments, the LSR variant comprises one or more mutations that increase the integration specificity compared to the LSR of SEQ ID NO: 2, wherein the one or more mutations are in the alphaE-beta6 linker, alphaG-alphaH linker, RD-ZD linker, or at the putative dimer interface in the coiled coil domain. In some embodiments, the LSR variant has a greater specificity ratio for the integration site of interest compared to the LSR of SEQ ID NO: 2. In some embodiments, the LSR variant has a greater specificity ratio for the GS010 integration site compared to the LSR of SEQ ID NO: 2; and wherein the specificity ratio is determined with any one or more secondary integration sites selected from GS011, GS012, GS013, and GS018. In some embodiments, the specificity ratio of the LSR variant is 1.5x greater than the specificity ratio of the LSR of SEQ ID NO: 2. In some embodiments, the one or more mutations comprise L5K, Y7K, L43V, the combination of S47G and L49R, the combination of S47A and L49R, the combination of L49R and I51F, L49R, I51R, the combination of K55W and E57N, the combination of K55S and K58S, K58N, L116I, P inserted at position 116, N119G, the combination of N119G and F145L, R139N, the combination of G142E and F145K, F145R, F145K, E150L, a substitution of the amino acids 155-164 with GKWNGGFAPYG, the combination of A156K and P161V, theAttorney Docket No: 16290.0011-0304 combination of Y157M and R160Q, the combination of W157K and A161F (starting from the loop swap variant; see Table 4), the combination of A159N and R160M, the combination of A159N and R160A, the combination of A159N and R160G, A159T, P161A, L163F, K166T, V168D, H170N, P173K, R204E, the combination of G209D and K216I, the combination of K211C and S213L, the combination of S213W and K216N, the combination of S213W and P217M, Q214K, Q214F, the combination of Q214Y and P217C, the combination of Q214Y and G215E, the combination of K216N and F218M, F218M, E219S, E224R, the combination of N243K, T245S, and K251A, S244 deletion, S244D, S244E, R247T, K249R, D250L, E253D, the combination of P262K and I265T, I264F, I264L, the combination of Y281S and K286A, the combination of Y281I and S284K, the combination of P283L and K286A, the combination of S284V and K286A, PK inserted at position 284, the combination of G285T and K286A, G285F, G285H, G285R, G285Y, the combination of G285Y and K286A, the combination of G285K and K286A, K286A, K286G, the combination of K286A and R287K, the combination of A286H and T291M, the combination of K286C and T291G, M314P, the combination of Y328W and G329K, the combination of Y328W and G329F, the combination of Y328V and G329Q, the combination of G329R and K334M, the combination of G329Q and H331F, P338S, Y365E, G391R, K392R, S399A, H423E, and / or D433Q.

[0008] In some embodiments, the LSR variant comprises an amino acid sequence having one or more mutations as compared to the LSR of SEQ ID NO: 1, wherein the LSR variant has at least 80% identity to SEQ ID NO: 2; and wherein the LSR variant has increased genome integration activity and / or integration specificity compared to the LSR of SEQ ID NO: 1.

[0009] In some embodiments, the one or more mutations increase genome integration activity by at least 5%, 10%, 15%, 20% at an integration site of interest compared to the LSR of SEQ ID NO: 1. In some embodiments, the integration site of interest is GS010. In some embodiments, the one or more mutations comprise M2 deletion, N29R, T36K, N119G, T153R, M175V, S213K, Q214H, S231V, Q261H, S262E, A317K, S326Q, V354L, T366L, I373A, G391K, S399A, N445E, S459N, and / or F462V.

[0010] In some embodiments, the LSR variant comprises one or more mutations that increase the integration specificity compared to the LSR of SEQ ID NO: 1, wherein the one or more mutations are in the alphaE-beta6 linker, alphaG-alphaH linker, RD-ZD linker, or at the putative dimer interface in the coiled coil domain.^In some embodiments, the LSR variant has a greater specificity ratio for the integration site of interest compared to the LSR of SEQAttorney Docket No: 16290.0011-0304 ID NO: 1. In some embodiments, the LSR variant has a greater specificity ratio for the GS010 integration site compared to the LSR of SEQ ID NO: 1; and wherein the specificity ratio is determined with any with one or more secondary integration sites selected from GS011, GS012, GS013, and GS018. In some embodiments, the specificity ratio of the LSR variant is 1.5x greater than the specificity ratio the LSR of SEQ ID NO: 1. In some embodiments, the one or more mutations comprise N119G.

[0011] In some embodiments, the LSR variant comprises the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394- 404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763- 767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR variant comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593. In some embodiments, the LSR variant is capable of genome integration activity atAttorney Docket No: 16290.0011-0304 the GS010 integration site. In some embodiments, the LSR variant further comprises a nuclear localization signal.

[0012] In some aspects, the present disclosure provides a fusion protein (referred to herein as an “LSR-fusion protein”) comprising the LSR variant and a fusion domain. In some embodiments, the fusion domain comprises a polypeptide, protein domain, or protein with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a DNA-binding domain, a cell-penetrating peptide, an arginine-rich peptide, an arginine-rich dipeptide repeat protein, or a combination thereof. In some embodiments, the fusion domain comprises a DNA-binding domain comprising a Cas polypeptide, a zinc finger polypeptide (ZNF), or a transcription activator-like effector nuclease (TALEN).

[0013] In some embodiments, the LSR variant or LSR-fusion protein disclosed herein is encoded in an isolated nucleic acid.

[0014] In some aspects, the present disclosure provides a genome-editing system. In some embodiments, the genome-editing system comprises (i) an LSR variant, an LSR-fusion protein, or an isolated nucleic acid encoding an LSR or LSR-fusion protein disclosed herein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the AttD site comprises the nucleic acid sequence of an AttD from any one of SEQ ID NOs: 885-887, 1131-1145, or a nucleic acid sequence having at least 90%, or at least 95% identity to the nucleic acid sequence of an AttD from any one of SEQ ID NOs: 885-887 or 1131-1145. In some embodiments, the genome-editing system is capable of integrating a sequence from the nucleic acid comprising an AttD site at the GS010 integration site.

[0015] In some embodiments, the LSR-fusion protein of the genome-editing system comprises a DNA-binding domain. In some embodiments, the DNA-binding domain comprises a Cas polypeptide, a zinc finger polypeptide (ZNF), or a transcription activator- like effector nuclease (TALEN).

[0016] In some embodiments, the genome-editing system comprises an LSR variant disclosed herein and a nucleic acid comprising an AttD site comprising any one of SEQ ID NOs: 885-887, 1131-1145, or an AttD site having at least 90%, or at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 885-887 or 1131-1145, wherein the genome-editing system is capable of integrating a sequence from the nucleic acid comprising an AttD site at the GS010 integration site.

[0017] In some embodiments, the nucleic acid encodes a CAR amino acid sequence. In some embodiments, the genome-editing system is capable of integrating the CAR sequence at the GS010 integration site in a cell.Attorney Docket No: 16290.0011-0304

[0018] In some aspects, the present disclosure provides a method for nucleic acid recombination. In some embodiments, the method for nucleic acid recombination comprises contacting one or two nucleic acids comprising a pair of cognate AttA and AttD sites with an LSR variant, LSR-fusion protein, nucleic acid encoding an LSR, or a genome-editing system disclosed herein. In some embodiments, the nucleic acid is synthetic DNA. In some embodiments, the nucleic acid is genomic DNA. In some embodiments, the nucleic acid is human DNA. In some embodiments, the nucleic acid is non-human DNA.

[0019] In some aspects, the present disclosure provides a method of integrating a nucleic acid sequence comprising an AttD into a cellular genome. In some embodiments, the method of integrating a nucleic acid sequence comprising an AttD into a cellular genome comprises contacting a cell with the genome-editing system disclosed herein such that the sequence from the nucleic acid comprising an AttD is integrated into the cellular genome. In some embodiments, the LSR variant of the genome-editing system comprises the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70- 229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351- 364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528- 544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748- 756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047- 1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322- 326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428- 441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569- 573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923- 1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274,Attorney Docket No: 16290.0011-0304 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, wherein the LSR variant exhibits genome integration activity in a mammalian cell. In some embodiments, the LSR variant comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593.

[0020] In some aspects, the present disclosure provides a method of editing a genome at the GS010 integration site in a human cell. In some embodiments, the method of editing a genome at the GS010 integration site in a human cell comprises contacting the human cell with the genome-editing system disclosed herein such that a sequence from the nucleic acid comprising an AttD is integrated into the cellular genome, wherein the LSR variant comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233- 235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878- 882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the AttD site comprises a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NOs: 885-887 or 1131-1145. In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593. In some embodiments, the method is an in vitro or ex vivo method. In some embodiments, the method is an in vivo method. In some embodiments, the cell is a T cell. In some embodiments, the sequence from the nucleic acid comprising an AttD further encodes a CAR amino acid sequence.

[0021] In some aspects, the present disclosure provides an engineered cell. In some embodiments, the engineered cell is made by a process comprising (i) isolating a cell and (ii)Attorney Docket No: 16290.0011-0304 contacting the isolated cell with an effective amount of the genome-editing system disclosed herein, wherein the genome-editing system integrates a sequence from the nucleic acid comprising an AttD of the genome-editing system into the isolated cell’s genome. In some embodiments, the cell is a T cell.

[0022] In some aspects, the present disclosure provides a method of treating a disease in a subject. In some embodiments, the method of treating a disease in a subject comprises administering to the subject an effective amount of the genome-editing system disclosed herein, wherein the method comprises integrating a sequence from the nucleic acid comprising an AttD of the genome-editing system into the subject’s genome, thereby treating the disease. In some embodiments, the method of treating a disease in a subject comprises (i) contacting isolated cells with an effective amount of the genome-editing system disclosed herein, wherein the method comprises integrating a sequence from the nucleic acid comprising an AttD of the genome-editing system into the cells to yield engineered cells and (ii) administering the engineered cells to the subject, thereby treating the disease. In some embodiments, the subject is human. In some embodiments, the disease is caused by a genetic mutation. In some embodiments, the sequence from the nucleic acid comprising an AttD further encodes a CAR amino acid sequence. In some embodiments, the cells are T cells.

[0023] In some embodiments, genome editing systems disclosed herein can be used in treating a disease in a subject, wherein the use comprises administering an effective amount of the genome-editing system to the subject or contacting isolated cells with an effective amount of the genome-editing system to yield engineered cells and administering the engineered cells to the subject. In some embodiments, the genome editing systems disclosed herein can be used in the manufacture of a medicament for a disease. In some embodiments, the genome editing system disclosed herein can be used in the manufacture of a host cell to produce a biologic. BRIEF DESCRIPTION OF THE FIGURES

[0024] FIG 1 shows mutagenesis of the wild-type LSRs of SEQ ID NOs: 1 and 2 improves genome integration activity at the GS010 integration site in HEK393FT cells.

[0025] FIGS 2A-BG show the results of ddPCR assays for variant LSRs on 5 integration sites in human HEK293FT cells.

[0026] FIG 3 shows the wild-type LSR of SEQ ID NO: 2 and the activity-enhanced LSR variant of SEQ ID NO: 304 can insert clinically relevant cargos at the GS010 integration site.Attorney Docket No: 16290.0011-0304

[0027] FIGS 4A-E show mutagenesis of the wild-type LSRs improves the specificity to the GS010 integration site. Specificity is shown as a ratio of percent integration at sites GS010 / GS011 (FIG 4A), GS010 / GS012 (FIG 4B), GS010 / GS013 (FIG 4C), and GS010 / GS018 (FIG 4D). Larger ratios corresponded to LSRs that integrate payload more often at GS010 than at a secondary integration site. Integration values at GS010 are shown in FIG 4E and correspond to the percent integration reported in FIGS 2A-BG.

[0028] FIG 5 shows identification of integration sites and relative measurement of genome integration activities for exemplar LSR variants (referred to herein as “loop swap” variants) as assessed by a genome integration assay (GIA).

[0029] FIGS 6A-B show bacterial directed evolution screens in LSR loop swap variants. FIG 6A shows the workflow for identifying improved LSR variants through library design, positive selection, negative selection, and DNA sequencing. FIG 6B shows enrichment due to negative selection (low genome integration activity at secondary sites) versus enrichment due to positive selection (genome integration activity at primary site) as determined by NGS read counts.

[0030] FIG 7 shows LSRs derived from bacterial directed evolution screening with improved integration specificity in a human cell line (HEK293FT).

[0031] FIGS 8A-B show an activity-enhanced LSR variant integrates clinically relevant cargo (a CAR sequence) into primary human T cells.

[0032] FIG 9 shows identification of integration sites and relative measurement of genome integration activities for LSR variant (SEQ ID NO: 775) as assessed by a genome integration assay (GIA).

[0033] FIGS 10A-E show ex vivo engineered T cells with LSR variant (SEQ ID NO: 775) achieve high CAR+ cells by flow cytometry (FIG 10A) and percent integration by ddPCR at the GS010 site (FIG 10B) as compared to lentivirus. A population shift and higher MFI was observed by flow cytometry with LSR-based integration (FIG 10C) as compared to lentivirus (FIG 10D) or mock control (FIG 10E).

[0034] FIGS 11A-C show that ex vivo engineered T cells with LSR variant (SEQ ID NO: 775) have stable integration of the CAR in the GS010 site as evaluated by ddPCR when cultured in homeostatic cell culture conditions (FIG 11A) or when co-cultured with CD19+ Nalm6 tumor cells (FIG 11B). FIG 11C shows that the proportion of CAR+ T cells was maintained under homeostatic cell culture conditions while the proportion of CAR+ T cells increased in response to repeat antigen exposure via repeated Nalm6 tumor cell exposure.Attorney Docket No: 16290.0011-0304

[0035] FIG 12 shows ex vivo engineered T cells with LSR variant (SEQ ID NO: 775) are functional for tumor killing in vitro after co-culture for 24 hours with Nalm6-luciferase tumor cells.

[0036] FIGS 13A-D show ex vivo engineered T cells with LSR variant (SEQ ID NO: 775) have a superior cytokine production as compared to lentiviral-engineered cells as measured by ELISA of IFN-γ (FIG 13A), IL-2 (FIG 13B), TNF-α (FIG 13C), and Granzyme B (FIG 13D).

[0037] FIGS 14A-B show ex vivo engineered T cells with LSR variant (SEQ ID NO: 775) show anti-tumor activity and expand in vivo in an anti-tumor NGS mouse model. Tumor burden was measured after IV injection of 5 million (“5M”) or 1 million (“1M”) CAR+ LSR- engineered T cells or CAR+ lentivirus-engineered T cells (FIG 14A), and CAR+ T cell expansion was assessed by flow cytometry over time (FIG 14B).

[0038] FIG 15 shows engineered T cells with LSR variants (SEQ ID NOs: 775, 830, 4278, 4390, 4394, 4398, 4402, 4406) express the anti-CD19 CAR as assessed by flow cytometry. DETAILED DESCRIPTION

[0039] Disclosed herein are LSR variants. In some instances, the LSR variants have one or more mutations compared to a wild-type LSR, or are LSRs containing sequences from more than one or more LSR variants or wild-type LSRs. In addition, disclosed herein are nucleic acids encoding the LSRs variants, and genome-editing systems comprising an LSR. The LSRs and genome-editing systems disclosed herein are capable of recombining nucleic acids and as such can perform genome-editing. This recombination activity of the disclosed LSRs does not require insertion of a landing pad or other genomic elements to direct LSR attachment. Also, disclosed herein are methods for using the LSRs and associated genome- editing systems for recombination of nucleic acids, recombination of a genome, and recombination in which a nucleic acid sequence is integrated into the genome. The genome editing systems disclosed herein may include a nucleic acid that comprises a donor attachment site (AttD) recognized by the LSR. The disclosure further provides methods for treating a disease by integrating a nucleic acid sequence (e.g., a gene) in a genome of a subject, or generating an engineered cell, or a cell that expresses a new protein or has a new function through the introduction of a nucleic acid sequence (e.g., gene), that is administeredAttorney Docket No: 16290.0011-0304 to a subject, thereby treating the disease using the LSRs, LSR-fusion proteins, and genome- editing systems disclosed herein. I. DEFINITIONS

[0040] Unless stated otherwise, the following terms and phrases have the meanings described below. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present disclosure.

[0041] AttA site: As used herein, the term “AttA site” (or “AttA”) refers to the attachment site within an acceptor nucleic acid to which an LSR binds. An AttA site may exist in the genome of a non-phage and non-bacterial organism, e.g., a human.

[0042] Attachment site: As used herein, the term “attachment site” refers to a nucleic acid sequence to which an LSR binds and uses for facilitating recombination of nucleic acid sequences.

[0043] AttD site: As used herein, the term “AttD site” (or “AttD”) refers to an attachment site in a nucleic acid to which an LSR binds. AttD sites may be engineered into a nucleic acid to facilitate recombination of the nucleic acid at a cognate AttA site.

[0044] Loop swap: As used herein, the term “loop swap” refers to exchanging or substituting a loop in the recombinase domain of an LSR with a loop from the recombinase domain from another LSR.

[0045] Identity: As used herein, the term “identity” in the context of sequence comparisons refers to the number of exact matches between two different sequences in a sequence alignment. Sequence alignment techniques and software include Basic Local Alignment Search Tool (BLAST, which includes e.g., BLASTP for protein sequences and BLASTN for nucleic acid sequences), the Needleman-Wunsch algorithm for pairwise alignment, and ClustalOmega, MUSCLE, and MAFFT for multiple-sequence alignment. Preferably, the sequence alignment is performed using BLAST.

[0046] Integration Activity: As used herein, the term “integration activity” or “genome integration activity” refers to the amount of integration (e.g., the percent integration) of a nucleic acid sequence at an integration site (or sites) in the genome of a cell contacted with a genome-editing system comprising an LSR and a nucleic acid comprising an AttD. Integration activity can be measured by determining the percent integration by nucleic acid quantification (e.g., ddPCR) or nucleic acid sequencing (e.g., NGS read counts) at a particular integration site (or sites) in the genome of a cell contacted with the genome-editing system. A variant LSR can exhibit increased (or improved or enhanced) integration activityAttorney Docket No: 16290.0011-0304 compared to the wild-type LSR by comparing the percent integration of the LSR variant and the wild-type LSR at the same integration site (or across sites in the genome). Integration activity may be determined by measuring the total or sum of integration across sites in the genome (e.g., by protein expression of the integrated nucleic acid by flow cytometry).

[0047] Specificity ratio: As used herein, the term “specificity ratio” refers to the percent integration at an integration site of interest for an LSR compared to the percent integration at one or more selected secondary integration sites. II. LARGE SERINE RECOMBINASES

[0048] The present disclosure provides variants of large serine recombinases (LSRs), also called engineered LSRs. As used herein, “LSR” or “LSR variant” refers to a variant of a wild-type LSR (also referred to as a native or naturally occurring LSR) having one or more mutations in the amino acid sequence as compared to the wild-type LSR. Such mutations may be additions, or deletions, or substitutions, and include LSRs containing sequences from more than one wild-type LSR. The variant LSRs may also comprise conjugates, chemical modifications, LSR-fusions with other proteins or peptides, truncations and the like. The disclosed LSRs are capable of recombining nucleic acids. In some embodiments, the disclosed LSRs are capable of recombining nucleic acids with greater genome integration activity and / or integration specificity compared to the wild-type LSR. In some embodiments, the recombination occurs between a donor and acceptor nucleic acid, resulting in integration of a sequence from the nucleic acid into an acceptor nucleic acid at an integration site. In certain embodiments, the LSRs disclosed herein are capable of integrating a sequence from a nucleic acid into a chromosomal location, also called an integration site.

[0049] In some embodiments, the LSR exhibits recombination activity. Assays for determining the recombination activity of an LSR can be performed using various methods. For example, an assay involving the recombination of plasmids by an LSR to introduce a reporter molecule into a cell (e.g., to induce fluorescence in a cell) can demonstrate if an LSR has activity with a pair of attachment sites. These recombination events can be evaluated by e.g., flow cytometry or fluorescent microscopy, both techniques known in the art. Flow cytometry instruments include Attune (Thermo Fisher Scientific), ID7000 spectral cell analyzer (Sony), BD FACSymphony (BD Biosciences), and CytoFLEX (Beckman Coulter). Fluorescent microscopes include EVOS fluorescent microscope (Thermo Fisher Scientific), Eclipse fluorescent microscope (Nikon), and Axiovert fluorescent microscope (Zeiss). Recombination activity can be evaluated by comparing to negative control conditions, e.g.,Attorney Docket No: 16290.0011-0304 without addition of the LSR. See Example 1 herein for exemplary methods for evaluating recombination activity.

[0050] One approach for assessing LSR recombination activity includes providing an LSR and a nucleic acid comprising an AttD site to a cell and determining if a sequence from the nucleic acid comprising an AttD is integrated into the genome.

[0051] In some embodiments, the LSR exhibits recombination activity resulting in integration of a nucleic acid sequence from a nucleic acid comprising an AttD site. In some embodiments, the integrated sequence comprises the entire nucleic acid. In some embodiments, the integrated sequence consists of a portion of the nucleic acid. As used herein, recombination or integration of a “nucleic acid sequence” or a “sequence from a nucleic acid” or a “donor sequence” or “donor nucleic acid sequence” are used interchangeably and may refer to the entire nucleic acid or may refer to a portion of the nucleic acid. For example, a nucleic acid from a genome-editing system may be integrated into a cellular genome and once integrated, no longer comprises the intact AttD (as the sequences of the AttD recombine with an AttA to form an AttL and an AttR). Integration of “a nucleic acid”, as used herein, is meant to include such scenario.

[0052] In some embodiments, the LSR exhibits genome integration activity. The resulting integration can be measured by e.g., nucleic acid quantification or nucleic acid sequencing. Nucleic acid quantification can be performed using droplet digital PCR (ddPCR) and suitable nucleic acid quantification kits or instruments. Non-limiting examples include Qubit BR dsDNA assay (Thermo Fisher Scientific), Qubit HS dsDNA assay (Thermo Fisher Scientific), Nanodrop spectrophotometer (Thermo Fischer Scientific), Stunner spectrophotometer (Unchained Labs), and Lunatic spectrophotometer (Unchained Labs). Nucleic acid sequencing can be performed using NextSeq (Illumina), GridION (Oxford Nanopore Technologies), Revio System (Pacific Biosciences), and the like. Integration activity may also be measured using any one of multiple assays known in the art, including phenotypic assays, flow cytometry, and others. In some embodiments, genome integration activity is measured by nucleic acid quantification (e.g., ddPCR) by measuring the percent integration at a particular integration site in the genome of a cell that has been contacted with a LSR genome-editing system. In some embodiments, integration activity is measured by nucleic acid sequencing (e.g., NGS read count). In some embodiments, the LSR is capable of integrating a nucleic acid in a mammalian cell, e.g., as measured by nucleic acid quantification or nucleic acid sequencing, as described herein and known in the art. In some embodiments, the mammalian cell is a non-human mammalian cell (e.g., a mouse, rat, orAttorney Docket No: 16290.0011-0304 non-human primate). In some embodiments, the mammalian cell is a human cell. In general, any cell to which an LSR system disclosed herein can be delivered is suitable for editing by the LSR. In some embodiments, the human cell is an immune cell. In some embodiments, the human cell is a T cell. In some embodiments, the human cell is a primary T cell.

[0053] In some embodiments, the LSR is capable of integrating a nucleic acid sequence into the chromosomal location GS010 (also referred to herein as the GS010 integration site). In some embodiments, GS010 is the integration site of interest. As shown herein, the GS010 site is particularly suitable for integration in a human cell. For example, the GS010 site permits durable protein expression of the integrated nucleic acid sequence, shows stable integration over time, and supports functional CAR-T generation, expansion, and anti-tumor activity in vitro and in vivo.

[0054] In some embodiments, the integration site of interest is an integration site other than the GS010 integration site. Table 1 shows the chromosomal location of the integration site GS010 and exemplar secondary sites GS011, GS012, GS013, and GS018. In some embodiments, the LSR has higher integration activity at the integration site GS010 than at the integration site GS011. In some embodiments, the LSR has higher integration activity at the integration site GS010 than at the integration site GS012. In some embodiments, the LSR has higher integration activity at the integration site GS010 than at the integration site GS013. In some embodiments, the LSR has higher integration activity at the integration site GS010 than at the integration site GS018. In some embodiments, the LSR has higher integration activity at the integration site GS010 than at any integration site. Table 1: Exemplary Genome Integration Sites ID Genome Chromosomal Location GS010 Hg38 chr10:69637352-69637354 GS011 Hg38 chr3:56776199-56776201 GS012 Hg38 chr3:9946175-9946177 GS013 Hg38 chr14:20413080-20413082 GS018 Hg38 chr16:54631484-54631486

[0055] The LSRs of the present disclosure comprise variants of the wild-type LSRs of SEQ ID NO: 1 or SEQ ID NO: 2. Tables 2-5 disclose exemplary mutations that can be used to generate variants of SEQ ID NOs: 1 or 2. Additional mutations at any of the listed sites may be incorporated into an LSR variant and analyzed using the methods described herein.Attorney Docket No: 16290.0011-0304 Table 2: Point Mutations for Variants of SEQ ID NO: 2PositionMutation(s)153 E, M, R, S 2 K, Deletion 156 K, M, W 5 K 157 L, Y, I, A, F, G, V, E, C, K, N, M 7 K, R 158 Q, Y, C, H, A, N, T, V 9 I, V 159 T, N, S, T, P, W, N 10 L 160 R, M, W, A, G, Q, G, V, Deletion 20 L, Q 161 P, F, M, C, Q, S, T, A, V, H, D 22 Y 162 S, M, Deletion 28 K, L 163 F, L, T, I, F, M, S, H, V, Y 29 R 166 T 31 I 168 D 36 T, K, A 170 N, G 41 N 173 K, E, G, Q, R, T 43 V, P, S, V 174 F, I, L, M, V, Y 47 A, G, K, R 177 V 49 R 182 K 51 K, F, Y, R 184 I 53 V 185 S 55 H, Q, S 190 L 57 D, S, Y 192 A 58 N, S, G, N, R, Deletion 196 M, K 66 I 198 Y 67 K, R 199 A, D, G, I, K, N, R, S, T 69 I 200 A 70 A, E, Q 204 E 71 K, Q 209 H, S, Q, D, Deletion 74 K 210 V, Y, P 75 K 211 C, W, R, G 76 F 212 S, P 82 W 213 K, R, H, L, F, W, S 104 E, K 214 H, R, G, S, C, K, F, Y, L, V 105 C, F, L, Y, Deletion 215 N, W, E 116 I 216 N, V, H, R, S, I, N 117 G R, Q, I, L, E, F, C, D, M, A, K, S, 119 G, D, S 217 T, Y 121 A, D, S, T, Y 218 M, V, R 127 Q, S 219 S, Q, R 139 N, Q 220 T, L, G 142 E, Q, T 221 S, A, I, Deletion 145 L, I, K, M, R 222 G, R, L, W, K, Q, Deletion 150 L 223 DeletionAttorney Docket No: 16290.0011-0304 R, A, C, N, Q A, F, G, H, I, K, N, Q, R, S, T, V, F, Q 288 Y D, L, M, T 289 N, T G, A, T 290 E K 291 G, Q, M L, M 292 G, Y V, A, I, K 295 M, I, P, V I, A 314 P, S, A, N, T F 317 K, T V 318 S S 321 G R, T, K, Q, C, A 326 Q A, D, E, R, Deletion 328 W, V, T G, S, K, H, Deletion 329 K, A, F, Q, R G, L, H, A, D, S, Y 331 N, F T, E 333 A L, Y 334 M R F, G, Y 336 K L, P 337 T, R Q 338 S, A, G, K, N, R, T S, E, M 342 K, N, R D, N 345 K, I E, L 348 E K, Q 349 A H, F, I 353 Y, A, D, E, I, K, L, Q, R, S, T, V E, K 354 L P 355 E L, F 357 I L, T 361 L S, N, D 365 E F 366 L K 373 A E 375 H R 379 I S 390 K, R, Y R 391 M, K, R, S T, Q, F, H, R, M, S, I 392 R H, K 393 L A, L 394 K Y, R, K, T, I, E, M, V 396 V K, P, F, C, E, I, T, V, A, Y, H, R 397 R A, H, C, S, Q, G 399 A K 401 L, A, D, I, Q 411 RAttorney Docket No: 16290.0011-0304 12 R 452 S 13 K, R, S 458 N, D, E 22 E 459 N, D 23 E 462 L, V 33 Q 464 A, Y 34 N 470 L 36 A, E, I, K, L, Q, S, V 472 R 37 H 476 R 39 P 479 I 42 E, K 483 K 44 V 484 S 45 E 486 K 46 L, W 488 S 47 M 492 H, N, R 50 I 494 K 51 Q, R Table 3: Insertions for Variants of SEQ ID NO: 2 Position Inserted Sequence(s) 159 N 116 P 242 RR 251 RR 252 R 284 PK Table 4: Substitution for Variants of SEQ ID NO: 2 (also referred to herein as the “loop swap” variant) Position Substitution 155-164 GKWNGGFAPYG Table 5: Point Mutations for Variants of SEQ ID NO: 1 Position Mutation(s) 119 G 2 Deletion 139 N 29 R 145 L 36 K 153 R 49 R 159 T 66 I 175 VAttorney Docket No: 16290.0011-0304 196 M 354 L 213 K 357 I 214 H, R 366 L 231 V 373 A 261 H 391 K 262 E, P 399 A 314 S 401 L 317 K 445 E 326 Q 458 N 336 K 459 N 353 A 462 V

[0056] In some aspects, the LSRs comprise one or more mutations. In some embodiments, the one or more mutations increase genome integration activity. In some embodiments, the one or more mutations increase integration specificity.

[0057] Integration specificity can be considered as the integration at an integration site of interest as compared to the integration at one or more secondary integration sites. In some embodiments, increased (or improved) integration specificity can be assessed by comparing a specificity ratio for a LSR variant to the specificity ratio of the wild-type LSR using the same sites for determining the ratio. It is understood that the specificity ratio for an LSR is measured in the context of a genome editing system comprising the LSR and a nucleic acid comprising an AttD as described herein. In determining a specificity ratio for an LSR, one or more secondary integration sites are selected based on integration sites identified for such LSR using techniques such as Next Generation Sequencing assays. For example, a genome integration assay (GIA) can be used to identify secondary sites having signals, for example unique read count, greater than or equal to 0.1x the signal of the integration site of interest. The percent integration of a nucleic acid comprising an AttD at the integration site of interest and a selected secondary site may be measured using ddPCR.

[0058] In some embodiments, a specificity ratio of the LSR variant is greater than a specificity ratio of the wild-type LSR. In some embodiments, the quotient of a specificity ratio of LSR variant divided by a specificity ratio of the wild-type LSR is greater than 1.5 when using any one or more of the secondary integration sites for that particular LSR. In such embodiments, the LSR variant has improved integration specificity compared to the wild- type LSR. In some embodiments, the LSR variant has improved integration specificity compared to the wild-type LSR or another LSR variant at the GS010 site. For example,Attorney Docket No: 16290.0011-0304 Figures 4A, 4B, 4C and 4D depict specificity ratios using GS010 as the integration site of interest and GS011, GS012, GS013, and GS018 sites as secondary sites, respectively, for an exemplary selection of LSRs. Comparisons of specificity ratios for wild-type LSR, SEQ ID NO: 2, to variant LSRs SEQ ID NOs: 194, 242, and 661, using a nucleic acid comprising the AttD of SEQ ID NO: 885, are shown above the black brackets. At each of the four selected secondary integration sites, SEQ ID NOs: 242 and 664, have specificity ratios relative to wild-type LSR greater than 1.5, ranging from greater than 2.3 to >500, and the amino acid changes in SEQ ID NOs: 242 and 664 are considered to improve specificity.

[0059] The mutations for activity and specificity can be used in combination to generate an LSR variant with improved overall integration properties compared to a wild-type LSR.

[0060] In some embodiments, the LSR variant of the LSR of SEQ ID NO: 2 has increased genome integration activity compared to the LSR of SEQ ID NO: 2. In some embodiments, the increase in genome integration activity is at the GS010 integration site. In some embodiments, the increased genome integration activity is at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increase in integration at integration site of interest (e.g., the GS010 integration site) by the LSR variant compared to the LSR of SEQ ID NO: 2. In some embodiments, the increased genome integration activity is at least a 5%, 10%, 15%, or 20% increase in integration at the GS010 integration site by the LSR variant compared to the LSR of SEQ ID NO: 2. In some embodiments, the increased genome integration activity is at least a 10% increase in integration at the GS010 integration site by the LSR variant compared to the LSR of SEQ ID NO: 2. In some embodiments, the LSR variant comprises one or more of the following mutations: G10L, N29R, the combination of N29R and V66I, L31I, A36K, L43V, E57D, K58G, H74K, L116I, S117G, T153R, T153S, A156K, M160G, L163I, V184I, S213R, the combination of S213K and Q214R, Q214K, Q214H, the combination of Q214Y and P217C, K216N, P217D, S231I, S231V, T241V, N243K, N243R, S244A, Q260K, T266D, T266S, the combination of S284V and K286A, the combination of S284R and K286A, G285K, G285H, G285R, G285Y, A317K, K321G, K348E, C353R, T366L, V375H, G391R, G391K, S399A, N413S, P436I, P436L, P436Q, G452S, F462L, Q484S, S486K, K488S, and Y494K. Exemplary LSR variants of the LSR of SEQ ID NO: 2 with increased genome integration activity comprise any one of the amino acid sequences of SEQ ID NOs: 6, 10, 11, 13, 19, 37, 43, 44, 56, 80, 91, 102, 108, 112, 120, 122, 130, 131, 132, 133, 185, 186, 194, 199, 205, 215, 218, 219, 314, 315, 346, 358, 379, 394, 404, 407, 409, 410, 437, 443, 444, 476, 477, 590-594, 596-598, 600, 601, 603, 604, 609, 720, 756, 776, 876,Attorney Docket No: 16290.0011-0304 925, 942, 1050, 4398, 4523, 4551, 4558, 4559, 4573, 4577, 4579, 4584, 4588, 4593, and 4595.

[0061] In some embodiments, the LSR variant of the LSR of SEQ ID NO: 2 has increased integration specificity at an integration site of interest. In some embodiments, the increased integration specificity is at the GS010 integration site as compared to the LSR of SEQ ID NO: 2. In some embodiments, the LSR variant comprises one or more mutations that increase integration specificity, wherein the one or more specificity mutations are in the alphaE-beta6 linker, alphaG-alphaH linker, RD-ZD linker, or at the putative dimer interface in the coiled coil domain. In some embodiments, the one or more mutations are in the alphaE-beta6 linker at one or more amino acid positions at or within T153-L163. In some embodiments, the one or more mutations are in the alphaG-alphaH linker at one or more amino acid positions at or within G209-E219. In some embodiments, the one or more mutations are in the RD-ZD linker at one or more amino acid positions at or within T280-Y292. In some embodiments, the one or more mutations are in the putative dimer interface in the coiled coil domain at one or more amino acid positions at or within I396-L407.

[0062] In some embodiments, the LSR variant has a greater specificity ratio at the GS010 integration site as compared to the LSR of SEQ ID NO: 2. In some embodiments, the specificity ratio at the GS010 integration site is determined with one or more secondary integration sites selected from GS011, GS012, GS013, and GS018. In some embodiments, a specificity ratio of the LSR variant is greater than 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, or 500x a specificity ratio for the LSR of SEQ ID NO: 2. In some embodiments, the LSR variant comprises one or more of the following mutations: L5K, Y7K, L43V, the combination of S47G and L49R, the combination of S47A and L49R, the combination of L49R and I51F, L49R, I51R, the combination of K55W and E57N, the combination of K55S and K58S, K58N, L116I, P inserted at position 116, N119G, the combination of N119G and F145L, R139N, the combination of G142E and F145K, F145R, F145K, E150L, a substitution of the amino acids 155-164 with GKWNGGFAPYG, the combination of A156K and P161V, the combination of Y157M and R160Q, the combination of W157K and A161F (starting from the loop swap variant; see Table 4), the combination of A159N and R160M, the combination of A159N and R160A, the combination of A159N and R160G, A159T, P161A, L163F, K166T, V168D, H170N, P173K, R204E, the combination of G209D and K216I, the combination of K211C and S213L, the combination of S213W and K216N, the combination of S213W and P217M, Q214K, Q214F, the combination of Q214Y and P217C, theAttorney Docket No: 16290.0011-0304 combination of Q214Y and G215E, the combination of K216N and F218M, F218M, E219S, E224R, the combination of N243K, T245S, and K251A, S244 deletion, S244D, S244E, R247T, K249R, D250L, E253D, the combination of P262K and I265T, I264F, I264L, the combination of Y281S and K286A, the combination of Y281I and S284K, the combination of P283L and K286A, the combination of S284V and K286A, PK inserted at position 284, G285H, G285R, G285Y, the combination of G285T and K286A, G285F, the combination of G285Y and K286A, the combination of G285K and K286A, K286A, K286G, the combination of K286A and R287K, the combination of A286H and T291M, the combination of K286C and T291G, M314P, the combination of Y328W and G329K, the combination of Y328W and G329F, the combination of Y328V and G329Q, the combination of G329R and K334M, the combination of G329Q and H331F, P338S, Y365E, G391R, K392R, S399A, H423E, and D433Q. Exemplary LSR variants of the LSR of SEQ ID NO: 2 with improved integration specificity comprise any one of the amino acid sequences of SEQ ID NOs: 4, 5, 13, 21, 29, 30, 31, 33, 41, 59, 81, 87, 99, 106, 112, 114, 115, 188, 191, 192, 195, 219, 242, 314, 324, 334.337, 339, 352, 360, 381, 385, 391, 394, 395, 415, 416, 426, 440, 441, 454, 455, 458, 476, 575, 584, 587, 595, 597, 599, 602, 606, 608-610, 613, 614, 625, 632, 643, 653, 654, 685, 690, 696, 703, 704, 721, 722, 725, 731, 734, 738, 741, 744, 749, 774, 775, 776, 777, 806, 810, 876, 881, 925, 942, 985, 986, 992, 999, 1000, 1003, 1004, 1009, 1010, 1011, 1012, 1013, 1015, 1017, 1018, 1020, 1022, 1024, 1025, 1037, 4563, 4564, 4573, 4577, 4579, and 4600.

[0063] In some embodiments, LSR variant of SEQ ID NO: 1 has increased genome integration activity compared to the LSR of SEQ ID NO: 1. In some embodiments, the increase in genome integration activity is at an integration site of interest. In some embodiments, the integration site of interest is the GS010 integration site. In some embodiments, the integration site of interest is at an integration site other than the GS010 integration site. In some embodiments, the increased genome integration activity is at least a 5%,10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%.90% increase in integration at the integration site of interest (e.g., the GS010 integration site) by the variant compared to the LSR of SEQ ID NO: 1. In some embodiments, the increased genome integration activity is at least a 10% increase in integration at the GS010 integration site by the LSR variant compared to the LSR of SEQ ID NO: 1. In some embodiments, the LSR variant comprises one or more of the following mutations: M2 deletion, N29R, T36K, N119G, T153R, M175V, S213K, Q214H, S231V, Q261H, S262E, A317K, S326Q, V354L, T366L, I373A, G391K, S399A, N445E, S459N, and F462V. Exemplary variants of the LSR comprising the amino acidAttorney Docket No: 16290.0011-0304 sequence of SEQ ID NO: 1 with increased genome integration activity comprise variants with the amino acid sequence of SEQ ID NOs: 139, 140, 141, 146, 149, 152-160, 162, 163, 171, 172, 174, 175, 180, and 181.

[0064] In some embodiments, an LSR variant of SEQ ID NO: 1 has increased specificity of genome integration at an integration site of interest. In some embodiments, the increased integration specificity is at the GS010 integration site as compared to the LSR of SEQ ID NO: 1. In some embodiments, the LSR variant comprises one or more mutations that increase integration specificity, wherein the one or more specificity mutations are in the alphaE-beta6 linker, alphaG-alphaH linker, RD-ZD linker, or at the putative dimer interface in the coiled coil domain (Rutherford K. et al., (2013) Nucleic Acids Res.41(17):8341–8356). In some embodiments, the one or more mutations are in the alphaE-beta6 linker at one or more amino acid positions at or within T153-L163. In some embodiments, the one or more mutations are in the alphaG-alphaH linker at one or more amino acid positions at or within G209-E219. In some embodiments, the one or more mutations are in the RD-ZD linker at one or more amino acid positions at or within T280-Y292. In some embodiments, the one or more mutations are in the putative dimer interface in the coiled coil domain at one or more amino acid positions at or within I396-L407.

[0065] In some embodiments, the LSR variant has a greater specificity ratio at the GS010 integration site as compared to the LSR of SEQ ID NO: 1. In some embodiments, a specificity ratio at the GS010 integration site is determined with one or more secondary integration sites selected from GS011, GS012, GS013, and GS018. In some embodiments, a specificity ratio of the LSR variant is greater 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, or 500x a specificity ratio for the LSR of SEQ ID NO: 1. In some embodiments, the LSR variant of SEQ ID NO: 1 comprises N119G. Exemplary variants of the LSR comprising the amino acid sequence of SEQ ID NO: 1 with improved specificity of genome integration at a particular chromosomal location comprise variants with the amino acid sequence SEQ ID NO: 146.

[0066] In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593 or an amino acid sequenceAttorney Docket No: 16290.0011-0304 having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto provided that the LSR is not a naturally occurring LSR. In some embodiments, the LSR comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence having at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR consists of the amino acid sequence of any one of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274,Attorney Docket No: 16290.0011-0304 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593.

[0067] In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394- 404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763- 767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto. In some embodiments, the LSR comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346- 349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452- 526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744- 746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NOs3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560,Attorney Docket No: 16290.0011-0304 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence having at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331- 343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584- 741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR consists of the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53,Attorney Docket No: 16290.0011-0304 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593.

[0068] The LSRs disclosed herein may be encoded by a nucleic acid construct, such as an mRNA or cDNA.

[0069] Each LSR disclosed herein is associated with one or more associated pairs of cognate attachment sites (an AttD site and an AttA site). In particular, a given LSR may recombine a nucleic acid comprising an associated AttD site with an acceptor nucleic acid comprising a cognate AttA site. In some instances, the recombination of the nucleic acid and acceptor nucleic acid may result in the integration of a sequence from the nucleic acid comprising an AttD into the AttA site within the acceptor nucleic acid. In some embodiments, the LSR comprises an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331- 343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584- 741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410,Attorney Docket No: 16290.0011-0304 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid into a chromosomal location comprising an AttA site associated with said LSR. In certain circumstances, these AttA site sequences are found in one or more locations in a mammalian or human genome. In some embodiments, the AttA site is the integration site of interest. In some embodiments, the LSR is capable of integrating a sequence from a nucleic acid comprising an AttD into one or more chromosomal locations. In some embodiments, the one or more chromosomal locations are one or more human chromosomal locations. In some embodiments, the LSR is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010.

[0070] In some embodiments, the LSR comprises an amino acid sequence (or a nucleic acid such as an mRNA or cDNA encoding such amino acid) where the amino acid sequence has at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23- 25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798- 801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368- 392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242,Attorney Docket No: 16290.0011-0304 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23- 25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798- 801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368- 392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises or consists of the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-Attorney Docket No: 16290.0011-0304 392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010.

[0071] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 304 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 660 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 661 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 664 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 677 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 741 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 775 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 822 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 825 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 830 and is capable of integrating a sequence from a nucleic acid comprising an AttD intoAttorney Docket No: 16290.0011-0304 GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 1010 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 1022 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 1024 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4278 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4326 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4390 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4394 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4523 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 14577 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4588 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD into GS010.

[0072] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 304. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 660. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 661. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 664. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 677. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 741. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 775. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 822. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 825. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 830. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 1010. InAttorney Docket No: 16290.0011-0304 some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 1022. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 1024. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4278. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4326. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4390. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4394. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4523. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4577. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4588. In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4593.

[0073] In some embodiments, an LSR-fusion protein is provided. In some embodiments, the LSR-fusion protein comprises an LSR disclosed herein and a fusion domain. In some embodiments, the fusion domain of the LSR-fusion protein comprises a polypeptide, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein domain, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a DNA-binding domain. In some embodiments, the DNA-binding domain comprises a catalytically inactive Cas polypeptide (dCas). In some embodiments, the dCas comprises dCas9 or dCas12. For fusions involving Cas proteins, a guide RNA sequence may also be provided that directs the Cas protein to a specific genomic location. In some embodiments, the DNA-binding domain comprises a catalytically inactive zine finger polypeptide (ZNF). In some embodiments, the DNA-binding domain comprises a catalytically inactive transcription activator-like effector nuclease (TALEN). In some embodiments, the LSR-fusion protein comprises a linker between the LSR and fusion domain. In some embodiments, the LSR-fusion protein does not comprise a linker between the LSR and fusion domain.

[0074] In some embodiments, the fusion domain comprises a cell-penetrating peptide. Suitable cell-penetrating peptides are well known in the literature. In some embodiments, the fusion domain comprises a combination of a DNA-binding domain, a cell-penetrating peptide, an arginine-rich peptide, and an arginine-rich dipeptide repeat protein.

[0075] In some embodiments, the LSR further comprises a nuclear localization signal. In some embodiments, the nuclear localization signal is not native to the LSR. In some embodiments, the LSR comprises more than one NLS. In some embodiments, the LSR comprises two NLSs. In some embodiments, the LSR comprises 3 NLSs. In someAttorney Docket No: 16290.0011-0304 embodiments, the LSR comprises more than 3 NLSs. In some embodiments, the NLS is from SV40. In some embodiments, the NLS is from myc. In some embodiments, the NLS is SEQ ID NOs: 1409, 1411, or 1412. In some embodiments, the NLS is from p53. Suitable nuclear localization signals are known in the literature. See e.g., Lu et al., (2021) Cell Commun. Signal.19(60).

[0076] In some embodiments, the LSR further comprises a FLAG tag (SEQ ID NO: 1410).

[0077] In some embodiments, the LSR further comprises a stabilization tag. In some embodiments, the stabilization tag comprises Exin21 (SEQ ID NO: 1414), dStab (SEQ ID NO: 1415), or Stab4 (SEQ ID NO: 1416).

[0078] In some embodiments, the NLS or stabilization tag is attached to the N-terminal or C-terminal end of the LSR. In some embodiments, the NLS or stabilization tag is inserted within the first 5 amino acids of the LSR. IV. GENOME-EDITING SYSTEMS

[0079] In some aspects, a genome-editing system comprising an LSR or LSR-fusion protein of this disclosure is provided. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the nucleic acid comprising an AttD disclosed herein is DNA. In some embodiments, the nucleic acid comprising an AttD is double-stranded DNA. In some embodiments, the nucleic acid comprising an AttD is single-stranded DNA. In some embodiments, the nucleic acid comprising an AttD is circular double-stranded DNA. In some embodiments, the nucleic acid comprising an AttD is circular single-stranded DNA. In some embodiments, the nucleic acid comprising an AttD is Z form DNA (Z-DNA). In some embodiments, the nucleic acid comprising an AttD is D form DNA (D-DNA).

[0080] In some embodiments, the AttD site is comprised within the nucleic acid sequence of any one of SEQ ID NOs: 885-889, 1131-1146, or a nucleic acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto. In some embodiments, the AttD site comprises a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of any one of SEQ IDAttorney Docket No: 16290.0011-0304 NOs: 885-889 or 1131-1146. In some embodiments, the AttD site comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments, the AttD site comprises a nucleic acid sequence having at least 99% identity to the nucleic acid sequence of any one of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments, the AttD site comprises the nucleic acid sequence of any one of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments, the AttD site comprises the nucleic acid sequence of any one of SEQ ID NOs: 885-889 or 1131-1146, wherein the dinucleotide core is modified and may be any of AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, and GG. In some embodiments, the AttD site consists of a nucleic acid sequence of any one of SEQ ID NOs: 885-889 or 1131-1146.

[0081] In some embodiments, an LSR disclosed herein binds or is capable of binding to an attachment site comprising 80 nucleotides (nt). In some embodiments, the LSR is capable of binding cognate attachment sites of different lengths. In some embodiments, at least one attachment site comprises a sequence less than 80 nt. In some embodiments, the cognate attachment sites each comprise 80 nt. In some embodiments, the cognate attachment sites each comprise less than 80 nt. In some embodiments, at least one of the attachment sites comprises a 52 nt sequence. In some embodiments, at least one of the attachment sites comprises a 48 nt sequence (Ghosh, et. al., (2003) Mol. Cell 12(5):1101-11). In some embodiments, at least one of the attachment sites comprises a 39 nt sequence (Groth, et. al., (2000) Proc. Natl. Acad. Sci. U.S.A.97(11):5995-6000). In some embodiments, at least one of the attachment sites comprises a 36 nt sequence (Ghosh, et. al., (2003) Mol. Cell 12(5):1101-11). In some embodiments, at least one of the attachment sites comprises a 34 nt sequence (Groth, et. al., (2000) Proc. Natl. Acad. Sci. U.S.A.97(11):5995-6000). In some embodiments, at least one of the attachment sites comprises a 26 nt sequence (Durrant, et. al., (2022) Nat. Biotechnol.41(4):488-99). In some embodiments, the AttD site comprises fewer nucleotides than the AttA site. In some embodiments, the AttD site comprises greater nucleotides than the AttA site. In some embodiments, the AttD site comprises at least 26, 34, 36, 39, 48, or 52 nucleotides of any of one of SEQ ID NOS: 885-889 or 1131-1146, wherein the nucleotides comprise a dinucleotide core and an even number of nucleotides directly adjacent on either side of the dinucleotide core. In some embodiments, the AttD site comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of 26, 34, 36, 39, 48, or 52 nucleotides of any of one of SEQ ID NOS: 885-889 or 1131-1146, wherein the nucleotides comprise a dinucleotide core and an even number of nucleotides directly adjacent on either side of the dinucleotide core.Attorney Docket No: 16290.0011-0304

[0082] In some embodiments, the AttD site comprises an AttP site. In some embodiments, the AttD site comprises a modified AttP site. In some embodiments, the modified AttP site has been optimized for a mammalian genome. In some embodiments, the mammalian genome is a non-human genome (e.g., a mouse, rat, or non-human primate genome). In some embodiments, the mammalian genome is a human genome. In some embodiments, the modified AttP site comprises a modified dinucleotide core. In some embodiments, the modified AttP sites comprises a modification outside of the dinucleotide core. In some embodiments, the modified AttP sites comprise modifications to both the dinucleotide core and the sequence outside of the dinucleotide core.

[0083] In some embodiments, the AttD site comprises an AttB site. In some embodiments, the AttD site comprises a modified AttB site. In some embodiments, the modified AttB site has been optimized for a mammalian genome. In some embodiments, the mammalian genome is a non-human genome (e.g., a mouse, rate, or non-human primate genome). In some embodiments, the mammalian genome is a human genome. In some embodiments, the modified AttB site comprises a modified dinucleotide nucleotide core. In some embodiments, the modified AttB site comprises a modification outside of the dinucleotide core. In some embodiments, the modified AttB site comprises modifications to both the dinucleotide core and the sequence outside of the dinucleotide core.

[0084] In some embodiments, the genome-editing system comprises an LSR with at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to one of the amino acid sequences of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 (or a nucleic acid such as an mRNA or cDNA encoding such LSR) and a nucleic acid comprising an AttD site with at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the nucleic acid sequence of SEQ ID NOs: 885-889 or 1131-1146.

[0085] In some embodiments, the genome-editing system comprises an LSR with at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to one of the amino acid sequences of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326,Attorney Docket No: 16290.0011-0304 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798- 801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and a nucleic acid comprising an AttD site with at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146.

[0086] In some embodiments, the genome-editing system comprises an LSR with at least 90% identity to one of the amino acid sequences of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and a nucleic acid comprising an AttD site with at least 90% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments, the genome-editing system comprises an LSR with at least 95% identity to one of the amino acid sequences of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322- 326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428- 441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569- 573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-Attorney Docket No: 16290.0011-0304 1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and a nucleic acid comprising an AttD site with at least 95% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments, the genome-editing system comprises an LSR with at least 99% identity to one of the amino acid sequences of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and a nucleic acid comprising an AttD site with at least 99% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments, the genome- editing system comprises an LSR with one of the amino acid sequences of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301- 320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917- 921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and aAttorney Docket No: 16290.0011-0304 nucleic acid comprising an AttD site with one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments the genome-editing system comprises an LSR with one of the amino acid sequences of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593 and a nucleic acid comprising an AttD site with one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146.

[0087] In some embodiments, the genome-editing system is capable of recombination that results in the integration of a sequence from the nucleic acid. The location of the recombination and integration is determined by the presence of one or more AttA sites in a genome, where each AttA site together with an AttD site forms cognate attachment sites.

[0088] In some embodiments, the genome-editing system is capable of integrating a sequence from the nucleic acid at an AttA site that is comprised within SEQ ID NO 890, or a nucleic acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto. In some embodiments, the AttA site comprises a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 890. In some embodiments, the AttA site comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO 890. In some embodiments, the AttA site comprises a nucleic acid sequence having at least 99% identity to the nucleic acid sequence of SEQ ID NO: 890. In some embodiments, the AttA site comprises the nucleic acid sequence of SEQ ID NO: 890. In some embodiments, the AttA site consists of the nucleic acid sequence of SEQ ID NO: 890. In some embodiments, the AttA site comprises a sequence within SEQ ID NO: 890. In some embodiments, the AttA site sequence is between 28 and 80 nucleotides. In some embodiments, the AttA site sequence is between 40 and 80 nucleotides. In some embodiments, the AttA site sequence is between 52 and 80 nucleotides. In some embodiments, the AttA site sequence is between 60 and 80 nucleotides. In some embodiments, the AttA site sequence is less than 80 nucleotides. In some embodiments, the genome editing system integrates a nucleic acid into a site within SEQ ID NO: 890.

[0089] In some embodiments, the genome-editing system comprises an LSR with at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to one of the amino acid sequences of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573,Attorney Docket No: 16290.0011-0304 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798- 801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 and is capable of integrating a sequence from a nucleic acid comprising an AttD at an AttA site comprising a nucleic acid sequence having at least 90% 95%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 890.

[0090] In some embodiments, the genome-editing system comprising an LSR with at least 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798- 801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 is capable of integrating a sequence from the nucleic acid comprising an AttD at GS010.

[0091] In some embodiments, the genome-editing system comprising an LSR with at least 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798- 801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213,Attorney Docket No: 16290.0011-0304 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593 is capable of integrating a sequence from the nucleic acid comprising an AttD at an integration site of interest.

[0092] In some embodiments, the genome-editing system comprises an LSR with at least 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 10244278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 304. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 660. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 661. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 664. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 677. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 741. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 775. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 822. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 825. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 830. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 1010. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 1022. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 4278. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 4326. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 4390. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 4394. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 4523. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 4577. In some embodiments, the genome editing system comprises an LSR withAttorney Docket No: 16290.0011-0304 the amino acid sequence of SEQ ID NO: 4588. In some embodiments, the genome editing system comprises an LSR with the amino acid sequence of SEQ ID NO: 4593.

[0093] In some embodiments, the LSR of the genome-editing system comprises a nuclear localization signal. In some embodiments, the nuclear localization signal is not native to the LSR. In some embodiments, the LSR comprises more than one NLS. In some embodiments, the LSR comprises two NLSs. In some embodiments, the LSR comprises 3 NLSs. In some embodiments, the LSR comprises more than 3 NLSs. In some embodiments, the NLS is from SV40. In some embodiments, the NLS is from myc. In some embodiments, the LSR comprises three copies of an NLS from myc. In some embodiments, the NLS is from p53. Suitable nuclear localization signals are known in the literature. See e.g., Lu et al., (2021) Cell Commun. Signal.19(60).

[0094] In some embodiments, the genome-editing system comprises an LSR-fusion protein. In some embodiments, the LSR-fusion protein comprises an LSR disclosed herein and a fusion domain. In some embodiments, the fusion domain comprises a polypeptide, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein domain, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a cell-penetrating peptide. In some embodiments, the fusion domain comprises an arginine-rich peptide. In some embodiments, the fusion domain comprises an arginine-rich dipeptide repeat protein. In some embodiments, the fusion domain comprises a combination of a DNA-binding domain, a cell-penetrating peptide, an arginine-rich peptide, and an arginine-rich dipeptide repeat protein. In some embodiments, the fusion domain comprises a DNA-binding domain. In some embodiments, the DNA-binding domain comprises a Cas polypeptide. In some embodiments, the Cas polypeptide comprises a catalytically inactive Cas polypeptide (dCas) that comprises dCas9 or dCas12. In some embodiments, the DNA-binding domain comprises a zinc finger polypeptide (ZNF) or a transcription activator-like effector nuclease (TALEN). In some embodiments, the DNA-binding domain comprises a catalytically inactive ZFN or TALEN polypeptide. In some embodiments, the LSR-fusion protein comprises a linker between the LSR and fusion domain. In some embodiments, the LSR-fusion protein does not comprise a linker between the LSR and fusion domain.

[0095] In some embodiments, the nucleic acid comprising an AttD comprises a sequence that encodes one or more gene products including but not limited to RNAs (e.g., such as tRNA, rRNA, microRNA, siRNA, and mRNA), proteins, or polypeptides. In someAttorney Docket No: 16290.0011-0304 embodiments, the nucleic acid comprises a non-coding sequence. In some embodiments, the nucleic acid comprises a sequence that encodes a transcription of translational control element (e.g., promoter elements, activator sequences, repressor sequences). In some embodiments the nucleic acid comprises a sequence that encodes a therapeutic protein. In some embodiments the nucleic acid comprises a sequence that encodes a therapeutic RNA. In some embodiments, the nucleic acid comprises one or more of a sequence that encodes for a transcriptional or translational control element, a sequence that encodes a therapeutic protein, and a sequence that encodes a therapeutic RNA. In some embodiments, the nucleic acid comprises a sequence for a gene, a gene variant, or a portion thereof.

[0096] In some embodiments, the nucleic acid comprising an AttD comprises a sequence that encodes a secreted protein. In some embodiments, the nucleic acid comprises a sequence that encodes a membrane-bound protein. In some embodiments, the nucleic acid comprises a sequence that encodes a clotting factor. In some embodiments, the nucleic acid comprises a sequence that encodes a cytokine. In some embodiments, the nucleic acid comprises a sequence that encodes a hormone. In some embodiments, the nucleic acid comprises a sequence that encodes an enzyme. In some embodiments, the nucleic acid comprises a sequence that encodes an antibody, or a portion thereof. In some embodiments, the nucleic acid comprises a sequence that encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR is a CD19 CAR. In some embodiments, the nucleic acid comprises a sequence that encodes a T cell receptor (TCR). In some embodiments, the nucleic acid comprises a sequence that encodes a B cell receptor (BCR). In some embodiments, the nucleic acid comprises a sequence that encodes an immune cell activation or inhibitory receptor. In some embodiments, the nucleic acid comprises a sequence that encodes a growth factor ligand. In some embodiments, the nucleic acid comprises a sequence that encodes a transcription factor. In some embodiments, the nucleic acid comprises a sequence that encodes a checkpoint inhibitor or agonist.

[0097] In some embodiments, the nucleic acid comprising an AttD comprises a sequence that encodes more than one gene product. In some embodiments, the nucleic acid comprises a sequence that encodes a CAR and at least one other gene product. In some embodiments, the nucleic acid comprises a sequence that encodes two CARs. In some embodiments, the nucleic acid comprises a sequence that encodes a CAR and an expansion factor (e.g., a T cell expansion factor). In some embodiments, the nucleic acid sequence comprises a sequence that encodes more than one CAR and an expansion factor (e.g., a T cell expansion factor). InAttorney Docket No: 16290.0011-0304 some embodiments, the nucleic acid comprises a sequence that encodes two CARs and an expansion factor (e.g., a T cell expansion factor).

[0098] In some embodiments, the nucleic acid comprising an AttD comprises a sequence that encodes a guide RNA. In some embodiments, the nucleic acid comprises a sequence that encodes a guide RNA for DNA-based Cas systems (e.g., Cas9). In some embodiments, the nucleic acid comprises a sequence that encodes a guide RNA for RNA-based Cas systems (e.g., Cas13). See e.g., Ding et al., (2024) Nature Commun.15:1572. In some embodiments, the nucleic acid comprises a sequence that encodes a micro RNA. In some embodiments, the nucleic acid comprises a sequence that encodes a tRNA. In some embodiments, the nucleic acid comprises a sequence that encodes a long non-coding RNA (lnc RNA). In some embodiments the nucleic acid comprises a sequence that encodes a circular RNA V. PRODUCTION OR ADMINISTRATION OF LARGE SERINE RECOMBINASES AND GENOME-EDITING SYSTEMS

[0099] The LSRs variants and genome-editing systems disclosed herein can be prepared using suitable methods known in the art. In some aspects, the present disclosure provides an LSR disclosed herein as a purified protein. In some embodiments, the present disclosure provides an LSR-fusion protein disclosed herein as a purified protein. In some aspects, the present disclosure provides a nucleic acid (e.g., an isolated nucleic acid) that encodes an LSR disclosed herein. In some aspects, the present disclosure provides a nucleic acid (e.g., an isolated nucleic acid) that encodes an LSR-fusion protein disclosed herein. In some embodiments, the nucleic acid encoding the LSR or LSR-fusion protein is DNA. In some embodiments, the DNA encoding the LSR or LSR-fusion protein is modified DNA. In some embodiments, the nucleic acid encoding the LSR or LSR-fusion protein is RNA. In some embodiments, the nucleic acid encoding the LSR or LSR-fusion protein is mRNA. In some embodiments, the nucleic acid encoding the LSR or LSR-fusion protein is a modified mRNA. In some embodiments the mRNA comprises one or more modified uridines. In some embodiments, the mRNA is modified with N1-methylpsuedouridine. In some embodiments, the mRNA comprises a 5’ cap structure, or a modified 5’ cap structure. In some embodiments, the mRNA comprises a 3’ polyadenylation sequence. In some embodiments, the mRNA comprises a 5’UTR. In some embodiments, the 5’ UTR comprises a 5’ UTR from alpha-globin, beta-globin, albumin, CYBA (cytochrome b-245 alpha chain), HSPA1A (Hsp70), or GAPDH. In some embodiments, the mRNA comprises a 3’ UTR. In some embodiments, the 5’ UTR comprises a 3’ UTR from alpha-globin, beta-globin, BNT162b2, or mRNA-1273. Suitable 5’ UTR and 3’ UTR sequences are known in the art (see, e.g.,Attorney Docket No: 16290.0011-0304 Reshentnikov, et al. (2024) Int. J. Mol. Sci.25(2):888; Ma, Q., et al. (2024) Sci Rep 14, 19845; Orlandini von Niessen, Alexandra G. et al. (2019) Molecular Therapy, 27(4):824 - 836; De Silva, D. et al., (2021) eLife 10; e75272; Kitte, R. et al., (2025) Int. J. Mol. Sci. 26(3), 965).

[0100] In some embodiments, the nucleic acid encoding the LSR or LSR-fusion protein is circular RNA. In some embodiments, the nucleic acid encoding the LSR may be codon optimized for expression. In some embodiments, the nucleic acid sequence encoding an LSR may be codon optimized for expression in a desired cell type.

[0101] In some embodiments, the nucleic acid encoding the LSR comprises cDNA. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1166- 1168. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1178-1180. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1190-1192. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1202-1204. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1214-1216. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1226-1228. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 238-1240. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1250-1252. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1262-1264. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1274- 1276. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1286-1288. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1298-1300. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1310-1312. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1322-1324. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1334-1336. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1346-1348. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1358-1360. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1370-1372. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1382- 1384. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1394-1396. In some embodiments, the cDNA comprises the nucleotide sequence of SEQ ID NOs: 1406-1408.Attorney Docket No: 16290.0011-0304

[0102] In some embodiments, the nucleic acid encoding the LSR comprises mRNA. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4680- 4682. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4692-4694. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4704-4706. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4716-4718. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4728-4730. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4740-4742. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4752-4754. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4764-4766. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4776-4778. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4788- 4790. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4800-4802. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4812-4813. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4824-4826. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4836-4838. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4848-4850. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4860-4862. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4872-4874. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4884-4886. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4896- 4898. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4908-4910. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NOs: 4920-4921.

[0103] In some embodiments, the LSRs or LSR-fusion proteins disclosed herein may be prepared by in vitro synthesis. In some embodiments, the in vitro synthesis comprises introducing an expression vector encoding an epitope tagged LSR into a suitable cell line and purifying the LSR via chromatography. Non-limiting examples of epitope tags include His, Ha, FLAG, Glutathione S-Transferase (GST), and maltose binding protein.

[0104] Suitable expression vectors include, but are not limited to, viral vectors. In certain embodiments, viral vectors may be used to express a nucleic acid or administer a nucleic acid to a patient. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available. Non-limiting examples of vectors include a plasmid, aAttorney Docket No: 16290.0011-0304 minicircle, a nanoplasmid, an adenovirus, an AAV, a lentivirus, a HSV, a retrovirus, a doggybone DNA (dbDNA)™, and exosome, and a fusosome. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods Enzymol., 153:516-544). The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include epitope tags (e.g., His, Ha, FLAG, Glutathione S-Transferase (GST), maltose binding protein, etc.). In some embodiments, the LSRs or LSR-fusion proteins may be prepared as mRNA by in vitro transcription of DNA.

[0105] Methods of introducing a nucleic acid into a host cell are known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include, include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)­mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0106] Lipid nanoparticle (LNP) delivery systems may be used to deliver nucleic acids to cells, and many are well known in the art, including for use in human therapeutic products such as siRNA products, vaccine products, and genome editing products. Generally, LNPs can be used to introduce nucleic acid cargo, such as siRNA or mRNA, into a cell. LNPs may be used to deliver and express an LSR or LSR-fusion protein in a cell ex vivo, or to an animal or human in vivo. In some embodiments, a nucleic acid encoding an LSR or LSR-fusion protein may be formulated in an LNP. Some embodiments include a composition comprising an mRNA encoding an LSR or LSR-fusion protein formulated in an LNP. Some embodiments include a composition comprising an LSR or LSR-fusion protein as an isolated protein, optionally formulated in an LNP. Some embodiments include a composition comprising a mixture of an LSR or LSR-fusion protein as an isolated protein and a nucleic acid comprising an AttD site, optionally formulated in an LNP. VI. METHODS

[0107] In some aspects, the present disclosure provides a method for nucleic acid recombination using (i) an LSR, (ii) LSR-fusion protein, (iii) nucleic acid encoding an LSR or LSR-fusion protein, or (iv) genome-editing systems provided herein. In some embodiments, the method comprises contacting one or two nucleic acids comprising a pair ofAttorney Docket No: 16290.0011-0304 cognate AttA and AttD sites with an LSR. In some embodiments, the method comprises contacting one or two nucleic acids comprising a pair of cognate AttA and AttD sites with an LSR-fusion protein. In some embodiments, the method comprises contacting one or two nucleic acids comprising a pair of cognate AttA and AttD sites with an isolated nucleic acid encoding an LSR. In some embodiments, the method comprises contacting one or two nucleic acids comprising a pair of cognate AttA and AttD sites with an isolated nucleic acid encoding an LSR-fusion protein. In some embodiments, the method comprises contacting one or two nucleic acids comprising a pair of cognate AttA and AttD sites with a genome-editing system disclosed herein. In some embodiments, the nucleic acid is DNA. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the method recombines synthetic DNA. In some embodiments, the synthetic DNA is a nanoplasmid. In some embodiments, the method recombines genomic DNA. In some embodiments, the method recombines human DNA. In some embodiments, the method recombines non-human DNA. In some embodiments, the method occurs in vitro. In some embodiments, the method occurs ex vivo.

[0108] In some aspects, the present disclosure provides a method for recombination in a genome using (i) an LSR, (ii) LSR-fusion protein, (iii) nucleic acid encoding an LSR or LSR- fusion protein, or (iv) genome-editing systems disclosed herein. In some embodiments, the method comprises contacting a cell with an LSR. In some embodiments, the method comprises contacting a cell with an LSR-fusion protein. In some embodiments, the method comprises contacting a cell with an isolated nucleic acid encoding an LSR. In some embodiments, the method comprises contacting a cell with an isolated nucleic acid encoding an LSR-fusion protein. In some embodiments, the method comprises contacting DNA with a genome-editing system disclosed herein. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acidAttorney Docket No: 16290.0011-0304 encoding LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the method is in vitro. In some embodiments, the method is ex vivo. In some embodiments, the method is in vivo.

[0109] In some embodiments, the method comprises contacting a cell with (i) an LSR, (ii) LSR-fusion protein, (iii) nucleic acid encoding an LSR or LSR-fusion protein, or (iv) genome-editing system disclosed herein. In some embodiments, the LSR exhibits recombination activity in a mammalian cell. In certain circumstances, the recombination activity results in integration of a sequence from a nucleic acid into the genome. In some embodiments, the recombination activity is measured by fluorescent microscopy. In some embodiments, the recombination activity is measured by nucleic acid quantification. In some embodiments, the recombination activity is measured by nucleic acid sequencing. In some embodiments, the mammalian cell is a non-human mammalian cell (e.g., a mouse, rat, or non-human primate cell). In some embodiments, the mammalian cell is a human cell. In some embodiments, the method comprises contacting a cell with an LSR comprising an amino acid sequence of any one of SEQ ID NOs: 3-884, 891-1130, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593 or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto. In some embodiments, the method comprises contacting a cell with an LSR comprising an amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394- 404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763- 767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577,Attorney Docket No: 16290.0011-0304 4588, 4593 or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto. In some embodiments, the LSR comprises an amino acid sequence with at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346- 349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452- 526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744- 746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence with at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence with at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815- 854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-Attorney Docket No: 16290.0011-0304 1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR consists of the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346- 349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452- 526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744- 746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593.

[0110] In some aspects, the present disclosure provides a method for integration of a sequence from a nucleic acid into a cellular genome using a genome-editing system comprising an LSR or LSR-fusion protein disclosed herein. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises an LSR or LSR fusion-protein that exhibits recombination activity resulting in integration of a nucleic acid sequence in a mammalian cell. In some embodiments, the mammalian cell is a non-human mammalian cell (e.g., a mouse, rat, or non-human primate cell). In some embodiments, the mammalian cell is a human cell. In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 3-902 or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identityAttorney Docket No: 16290.0011-0304 thereto. In some embodiments, the LSR comprises the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394- 404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763- 767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto. In some embodiments, the LSR comprises an amino acid sequence having 90% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856- 872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence having 95% identity to the amino acid sequence of any one of SEQ ID NOs: 3- 6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780- 789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135,Attorney Docket No: 16290.0011-0304 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR comprises an amino acid sequence having 99% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856- 872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the LSR consists of any one of the amino acid sequences of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65- 68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346- 349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452- 526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744- 746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593. In some embodiments, the method occurs in vitro. In some embodiments, the method occurs ex vivo. In some embodiments, the method occurs in vivo. In some embodiments, the integrated nucleic acid sequence is more than 5 kb. In some embodiments, the integrated nucleic acid sequence is less than 15 kb. In some embodiments, the integrated nucleic acid sequence is between 5 and 15 kb. In some embodiments, theAttorney Docket No: 16290.0011-0304 integrated nucleic acid sequence is between 5 and 6 kb. In some embodiments, the integrated nucleic acid sequence is between 6 and 7 kb. In some embodiments, the integrated nucleic acid sequence is between 7 and 8 kb. In some embodiments, the integrated nucleic acid sequence is between 8 and 9 kb. In some embodiments, the integrated nucleic acid sequence is between 9 and 10 kb. In some embodiments, the integrated nucleic acid sequence is between 10 and 11 kb. In some embodiments, the integrated nucleic acid sequence is between 11 and 12 kb. In some embodiments, the integrated nucleic acid sequence is between 12 and 13 kb. In some embodiments, the integrated nucleic acid sequence is between 13 and 14 kb. In some embodiments, the integrated nucleic acid sequences is 6.4 kb, 7.5 kb, 8.7 kb, 10.8 kb, or 13.5 kb.

[0111] The present disclosure provides, in part, a method of editing the GS010 integration site in a cell comprising contacting the human cell with a genome-editing system. Any genome editing system capable of specifically targeting the GS010 site may be used to insert a nucleic acid at such site, including for example, an LSR system as described herein, CRISPR-based genome editing systems, and other site-specific editing systems (e.g., TALENS, zinc fingers). Methods of editing the GS010 integration site results in a sequence from the nucleic acid of the genome-editing system being integrated into the genome. In some embodiments, the nucleic acid is stably integrated at the GS010 site for at least 1, 2, 3, or 4 weeks, or at least 1, 2, 3, 4, 5, or 6 months. Embodiments include methods of inserting a nucleic acid sequence of interest at the GS010 site by contacting a cell with a genome editing system targeted to the GS010 site and the nucleic acid of interest or a template for such nucleic acid. Additional embodiments include an engineered cell having an exogenous nucleic acid sequence at the GS010 site. In certain embodiments, the exogenous nucleic acid sequence is introduced by a genome editing system that specifically targets the GS010 site. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the chromosomal location is GS010. In some embodiments, the LSR comprises at least 90% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421,Attorney Docket No: 16290.0011-0304 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid comprises an AttD site comprising at least 90% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146, which through recombination can integrate a portion of the nucleic acid into GS010. In some embodiments, the LSR comprises at least 95% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid comprises an AttD site comprising at least 95% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146, which through recombination can integrate a portion of the nucleic acid into GS010. In some embodiments, the LSR comprises at least 99% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856- 872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164,Attorney Docket No: 16290.0011-0304 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid comprises an AttD site comprising at least 99% identity to one of the nucleic acid sequences of SEQ ID NOs: 885- 889 or 1131-1146, which through recombination can integrate a portion of the nucleic acid into GS010. In some embodiments, the LSR comprises the amino acid sequence of one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394- 404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763- 767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid sequence comprises an AttD site comprising the nucleic acid sequence of one of SEQ ID NOs: 885-889 or 1131-1146. In some embodiments, the method occurs in vitro. In some embodiments, the method occurs ex vivo. In some embodiments, the method occurs in vivo.

[0112] In some aspects, the present disclosure provides a method of integrating a nucleic acid sequence into the GS010 integration site in an immune cell using a genome-editing system, such that the protein encoded by the nucleic acid is expressed by the cell. In other embodiments, the immune cell is a lymphocyte (B cell, T cell), natural killer (NK) cell, or myeloid cell. In some embodiments, the immune cell is a T cell. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antigen receptor, for example a CAR. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the LSR comprises at least 90% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231,Attorney Docket No: 16290.0011-0304 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368- 392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid comprises an AttD site comprising at least 90% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146, which through recombination can integrate a portion of the nucleic acid into GS010. In some embodiments, the LSR comprises at least 95% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid comprises an AttD site comprising at least 95% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146, which through recombination can integrate a portion of the nucleic acid into GS010. In some embodiments, the LSR comprises at least 99% identity to the amino acid sequence of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028,Attorney Docket No: 16290.0011-0304 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid comprises an AttD site comprising at least 99% identity to one of the nucleic acid sequences of SEQ ID NOs: 885-889 or 1131-1146, which through recombination can integrate a portion of the nucleic acid into GS010. In some embodiments, the LSR comprises the amino acid sequence of one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233- 235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878- 882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the nucleic acid sequence comprises an AttD site comprising the nucleic acid sequence of one of SEQ ID NOs: 885-889 or 1131-1146.

[0113] In some embodiments, one or more proteins is expressed from a nucleic acid sequence integrated in the GS010 integration site. In some embodiments, the expression of protein from the nucleic acid sequence integrated in the GS010 integration site is durable. For example, the protein is expressed for at least 2, 3, or 4 weeks, or at least 1, 2, 3, 4, 5, or 6 months. In some embodiments, the expression of the nucleic acid sequence integrated in the GS010 integration site is durable in vivo. In some embodiments, the expression of the nucleic acid sequence integrated in the GS010 integration site is durable in vitro.

[0114] The present disclosure provides an engineered cell. In some embodiments, the engineered cell is made by a process comprising (i) isolating a cell and (ii) contacting the isolated cell with an effective amount of a genome-editing system disclosed herein, wherein the genome-editing system integrates a sequence from the nucleic acid of the genome-editing system into the isolated cell’s genome.Attorney Docket No: 16290.0011-0304

[0115] In some embodiments, the engineered cell has a nucleic acid sequence encoding an exogenous protein or peptide at the GS010 site. In certain embodiments, the exogenous protein is a therapeutic protein. In other embodiments, the exogenous protein is a wild-type protein introduced to a cell having a mutated version of such protein. In some embodiments, the engineered cell has a nucleic acid sequence encoding a secreted protein at the GS010 site. In some embodiments, the engineered cell has a nucleic acid sequence encoding a receptor at the GS010 site. In some embodiments, the engineered cell has a nucleic acid sequence encoding a CAR integrated at the GS010 integration site. In some embodiments, the engineered cell expresses a CAR. In some embodiments, the CAR is expressed in the cell in vivo. In some embodiments, the CAR is expressed in the cell in vitro, and such cell may be administered to a patient in need thereof. In some embodiments, a population of CAR- engineered cells expands upon exposure to a target antigen in vivo. In some embodiments, a population of CAR-engineered cells expands upon exposure to a target antigen in vitro. In some embodiments, the target antigen is a tumor antigen, for example CD19.

[0116] The present disclosure provides a method of treating a disease in a subject using the genome-editing systems disclosed herein. In some embodiments, the disease is caused by a genetic mutation. Non-limiting examples of mutations include polymorphisms such as single nucleotide polymorphisms (SNP), point mutations, frameshift mutations, genomic rearrangements, translocations, and inversions. In some embodiments, the disease is caused by a polymorphism. In some embodiments, the disease is caused by a SNP.

[0117] In some embodiments, the method comprises administering an effective amount of a genome-editing system to a subject, wherein a sequence from the nucleic acid of the genome-editing system integrates into the subject’s genome, thereby treating the subject’s disease. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome- editing system comprising (i) an isolated nucleic encoding an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the subject is a human. In some embodiments, the disease is caused by a genetic mutation. In some embodiments, the disease comprises cancer.

[0118] In some aspects, the present disclosure provides methods for treating a disease in a subject by administering a genome-editing system to a patient, thereby generating anAttorney Docket No: 16290.0011-0304 engineered cell within the patient. In some aspects, the present disclosure provides methods for treating a disease in a subject by generating an engineered cell using a genome-editing system and administering the engineered cell to the subject, thereby treating the disease. In some embodiments, a method of treating a disease in a subject, comprises (i) contacting isolated cells with an effective amount of a genome-editing system to yield engineered cells and (ii) administering the engineered cells to the subject, thereby treating the disease. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the method occurs in vitro. In some embodiments, the method occurs in vivo. In some embodiments, the method occurs ex vivo. In some embodiments, the subject is a human. In some embodiments, the disease is caused by a genetic mutation. In some embodiments, the disease comprises cancer.

[0119] Any suitable cell type may be used in the embodiments disclosed herein. In some embodiments, the cell or genome is selected from the group consisting of a T cell, a natural killer cell, an induced pluripotent stem cell, a hematopoietic stem cell, a liver cell, a muscle cell, a lung cell, a heart cell, a pancreatic cell, a spleen cell, a B cell, a fibroblast, a neuron, an astrocyte, and a microglial cell.

[0120] In some embodiments, the cell is a T cell. In some embodiments, the cell is a natural killer cell. In some embodiments, the cell is an induced pluripotent stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is a lung cell. In some embodiments, the cell is a heart cell. In some embodiments, the cell is a pancreatic cell. In some embodiments, the cell is a spleen cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a fibroblast. In some embodiments, the cell is a neuron. In some embodiments, the cell is an astrocyte. In some embodiments, the cell is a microglial cell.

[0121] The number of administrations of treatment to a subject may vary. Introducing the engineered cells into the subject may be a one-time event; but in certain situations, such treatment may elicit improvement for a limited period of time and require an ongoing series of repeated treatments. In other situations, multiple administrations of the engineered cellsAttorney Docket No: 16290.0011-0304 may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.

[0122] The present disclosure provides an LSR, an LSR-fusion protein, a nucleic acid encoding an LSR or LSR-fusion protein, and / or a genome-editing system for use in treating a disease in a subject, wherein the use comprises administering an effective amount of the genome-editing system to the subject or contacting isolated cells with an effective amount of the genome-editing system to yield engineered cells and administering the engineered cells to the subject. In some embodiments, the disease is caused by a genetic mutation. In some embodiments, the disease comprises cancer. In some embodiments, the use comprises administering an effective amount of the genome-editing system to the subject. In some embodiments, the use comprises contacting isolated cells with an effective amount of the genome-editing system to yield engineered cells and administering the engineered cells to the subject. In some embodiments, the genome-editing system comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome- editing system comprises (i) an isolated nucleic acid encoding an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site.

[0123] The present disclosure provides uses of an LSR or variants thereof, an LSR-fusion protein, a nucleic acid encoding an LSR or LSR-fusion protein, and / or a genome-editing system in the manufacture of a medicament. In some embodiments, the medicament is for treating a disease caused by a genetic mutation. In some embodiments, the genome-editing system for use in the manufacture of a medicament comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system for use in the manufacture of a medicament comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system for use in the manufacture of a medicament comprises (i) an isolated nucleic acid encoding LSR-fusion protein and (ii) a nucleic acid comprising an AttD site.

[0124] The present disclosure provides uses of an LSR or variant thereof, an LSR-fusion protein, a nucleic acid encoding an LSR or LSR-fusion protein, and / or a genome-editing system in the manufacture of a host cell to produce a biologic. In some embodiments, the hostAttorney Docket No: 16290.0011-0304 cell is a bacterial cell, an insect cell, or a human cell. In some embodiments, the biologic is an antibody . In some embodiments, the genome-editing system for use in the manufacture of a host cell comprises (i) an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system comprises (i) an LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system for use in the manufacture of a host cell comprises (i) an isolated nucleic acid encoding an LSR and (ii) a nucleic acid comprising an AttD site. In some embodiments, the genome-editing system for use in the manufacture of a host cell comprises (i) an isolated nucleic acid encoding LSR-fusion protein and (ii) a nucleic acid comprising an AttD site. VII. PHARMACEUTICAL COMPOSITIONS

[0125] The genome-editing systems of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers, diluents, or vehicles. Pharmaceutically acceptable vehicles may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. Such pharmaceutical vehicles can be lipids or LNPs, including suitable solvents, buffers, diluents, or stabilizers. In some embodiments, the genome-editing systems of the present disclosure can be delivered by viral vectors. As such, administration of an LSR, an LSR-fusion protein, a nucleic acid encoding an isolated LSR or LSR-fusion protein, or a genome-editing system disclosed herein can be achieved in various ways, including intravenous, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, or intraocular administration. The active agent may be systemic after administration or may be localized by the use of regional administration or targeted delivery mechanisms. General Considerations

[0126] At various places in the present disclosure, substituents, or properties of compounds of the present disclosure are disclosed in groups or in ranges. It is intended that the present disclosure comprise each and every individual or sub-combination of the members of such groups and ranges, and that such groups or ranges include the endpoints. By way of nonlimiting example, if a group or range is from about 1 to about 10, then the group or range includes both the value of about 1 and the value of about 10.

[0127] Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions thatAttorney Docket No: 16290.0011-0304 comprise "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure can include embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure can include embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[0128] The term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the terms “consisting of” and “consisting essentially of” are also encompassed and disclosed.

[0129] The abbreviation, “e.g.,” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.,” is synonymous with the term “for example.” The abbreviation, “i.e.,” is derived from the Latin id est, and is used herein to indicate a non-limiting rewording or clarification. Thus, the abbreviation “i.e.,” is synonymous with the term “that is.”

[0130] Any embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Any embodiment of the agents, methods, and / or compositions of the present disclosure can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0131] The present specification will control in instances where publications, patent applications, patents, and other references mentioned herein are incorporated by reference and are in conflict with the present specification.

[0132] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting. EXAMPLES Example 1: Mutagenesis improves integration activity at the GS010 integration site

[0133] New LSRs were engineered with the objective of improving properties of the wild- type LSRs of SEQ ID NO: 1 and SEQ ID NO: 2 to increase integration activity of payloads at the GS010 integration site.

[0134] Individual or double mutations were selected by consideration of sequence alignments with homologous LSRs, covariation analysis, protein language model predictions, proximity to DNA in homology models, and net charge of the protein. Mutations determined experimentally to be favorable to activity or specificity of the enzyme were tested in combination for further enhancements in the LSR.Attorney Docket No: 16290.0011-0304

[0135] New LSRs from engineering campaigns were evaluated for integration activity in HEK293FT cells by droplet digital PCR (ddPCR) to quantitatively measure genomic integration at the GS010 integration site.

[0136] In a 96-well plate, 50,000 cells were added to each well containing a mixture of Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) and the two plasmids, 50 ng of LSR expression plasmid and 150 ng of donor attachment (AttD) plasmid, that were prepared according to the vendor’s protocol for reverse transfection. The AttD donor plasmid was 4.8 kb.48 hours (unless noted) after transfection, genomic DNA (gDNA) was extracted using the DNAvdance genomic DNA extraction kit (Beckman Coulter) with RNase treatment. The gDNA was quantified using the Quant-iT dsDNA assay kit (Thermo Fisher Scientific) and Stunner nucleic acid quantification system (Unchained Labs).

[0137] Primers and probes were designed using PrimerQuest (IDT) with default settings and ordered from IDT. A gBlocks Gene Fragment (dsDNA from IDT) of each expected template sequence was ordered to validate the primer / probe design and measure the sensitivity of the assay.

[0138] ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (Bio- Rad) according to the manufacturer’s protocol, using a genome site at RPPH1 locus as the genome copy number reference. Data analysis was performed with QX Manager (Bio-Rad) to count positive and negative droplets for each probe. The integration rate was defined as specific probe droplet count divided by that of reference droplet count.

[0139] Results shown in Figure 1 demonstrate that mutagenesis of the wild-type LSRs of SEQ ID NOs: 1 and 2 can improve integration activity and specificity at the GS010 integration site. Over 1,000 variants were tested, and the highest activity gains were found when multiple beneficial mutations were combined. The results are shown in Figures 2A- BG. Example 2: Improvements in integration activity at GS010 are independent of payload size

[0140] The wild-type LSR of SEQ ID NO: 2 and LSR variants were evaluated for the ability to insert cargos with clinical relevance of increasing lengths from 6.4 kb to >13.5 kb.

[0141] A pUC57-based vector was constructed to carry AttD sequence SEQ ID NO: 885. Coding sequences of OTC (Ornithine Transcarbamylase), PAH (Phenylalanine Hydroxylase), TMC1 (Transmembrane Channel-like 1), F8-BDD (Factor VIII B-domain deleted), F8 (Factor VIII) were cloned into the above AttD vector, separately, and generated donor plasmids. These plasmids were tested for genome integration activity at the GS010 site, bothAttorney Docket No: 16290.0011-0304 left and right junctions, as described in Example 1, replacing the AttD donor plasmid with these larger donor plasmids.

[0142] Results are shown in Figure 3. There was no loss in integration activity as plasmid and cargo size increased to >13 kb for both the wild-type LSR (SEQ ID NO: 2) and an activity-enhanced variant (SEQ ID NO: 304). There was also no loss in integration fidelity, as the left and right post-integration junctions (AttL and AttR, respectively) were equivalent in all cases measured, indicating full-length plasmid integration. Example 3: Mutagenesis improves the integration specificity to the GS010 integration site and integration at secondary sites

[0143] In order to see if the LSR variants could have their integration specificity improved, loop swaps were performed alone and in combination with activity enhancing mutations. Specifically, DNA-contacting loops in the recombinase domain of a particular LSR were swapped with the corresponding loops of homologs from LSRs predicted to bind to AttP sequences similar to GS010. An altered AttD (SEQ ID NO: 887) was designed to match bases 3-8 from the dicore GS010, making the AttD more similar to GS010.

[0144] The percent integration of the donor nucleic acid sequence at GS010, GS011, GS012, GS013, and GS018 was evaluated by ddPCR (as described in Example 1). Specificity was measured by determining specificity ratios with higher ratios corresponding to greater specificity.

[0145] Results are shown in Figures 4A-E. The LSR variants of SEQ ID NO: 486 and 669 are examples of activity enhancing mutations and specificity enhancing mutations being combined to produce improved LSRs with both higher integration activity and specificity than the wild-type LSR.

[0146] The sites of integration in the human genome were then evaluated in a genome integration assay. The assay used two plasmids: 1) an LSR expression plasmid; and 2) a plasmid with a donor attachment site (AttD), carrying an mCherry ORF with a promoter. In a 96-well plate, 50,000 cells were added to each well to a mixture of Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) and the two plasmids (50 ng of LSR expression plasmid and 150 ng of donor attachment (AttD) plasmid) that were prepared according to the vendor’s protocol for reverse transfection.48 hours after transfection, genomic DNA (gDNA) was extracted using the DNAvdance genomic DNA extraction kit (Beckman Coulter) with RNase treatment. The gDNA was quantified using the Quant-iT dsDNA assay kit (Thermo Fisher Scientific) and Stunner nucleic acid quantification system (Unchained Labs).Attorney Docket No: 16290.0011-0304

[0147] The free plasmid was removed from the gDNA sample using suitable methods. Libraries were generated from the purified gDNA through tagmentation using preassembled Tn5 transposome (seqWell). After stopping the reaction and purifying the tagmented gDNA, the ends were repaired using acyclnucleotides and Therminator DNA polymerase (New England Biolabs). After bead purification, two rounds of PCR amplification were then performed on the DNA fragments followed by bead purification.

[0148] DNA product was quantified using Qubit HS dsDNA Assay (Thermo Fisher Scientific) and KAPA Library Quantification Kit (Roche). Size selection was performed using a 2% BluePippin (Sage Science) gel cassette to select for DNA molecules between 400 to 850 bp from libraries after pooling them. After a final bead purification, the resulting library was quantified with Qubit HS dsDNA Assay (Thermo Fisher Scientific) and had its size distribution assessed with HS D5000 Agilent TapeStation (Agilent). The library was then sequenced on an Illumina NextSeq 1000 platform. The reads were aligned to the donor plasmid and the human genome using the alignment software BWA. Aligned reads were then analyzed using a custom computational pipeline that identified precise integration sites within the human genome through inspection of the donor-genome integration junctions.

[0149] LSR loop swap mutations along with the altered AttD improved overall integration specificity as assessed by GIA. Results are shown in Figure 5. The LSR variant of SEQ ID NO: 477 with the altered AttD of SEQ ID NO: 887 showed improved genome specificity, with ~4x less secondary integration sites detected and with GS010 as the highest ranked site with a larger relative difference to other sites. Example 4: Bacterial directed evolution screens of LSR loop swap variants

[0150] Bacterial directed evolutions screens were conducted to identify LSR variants with activity at GS010 and no, or low, activity at secondary integration sites GS011, GS012, and GS013.

[0151] The approach used is depicted in Figure 6A. A plasmid library of 31,209 variants in the loop region was prepared. A first round of positive selection for activity at the GS010 site was conducted by assessing the capability of an LSR to excise a stop codon flanked by an AttP site and the nucleic acid sequence of the GS010 site (SEQ ID NO: 890) on a positive selection plasmid to express an ampicillin resistance gene. Cells were cultured in carbenicillin to select against cells with inactive LSR variants. The post-positive selection plasmids containing putative active LSR variants were then isolated and subjected to aAttorney Docket No: 16290.0011-0304 second round of negative selection for low / no activity at GS011, GS012, GS013. The isolated post-positive selection LSR plasmids were electroporated into E. coli cells containing the negative selection plasmid, which would express SacB if the LSR recombines an AttP site with any one of the attachment sites from an array with the nucleic acid sequences of GS011, GS012, and GS013. Cells were cultured in LB broth containing sucrose to select against cells with LSR variants that have undesired activity at the secondary sites. The post-negative selection plasmids containing putative specific LSR variants were then isolated. Enrichment of LSR variants was determined by amplifying the LSR sequences by PCR, followed by DNA sequencing with NGS.

[0152] Results are shown in Figure 6B. The wild-type LSR sequence showed activity but low specificity. Exemplary LSR variants show striking enhancements in specificity (enrichment in negative selection) with similar activity to the wild-type LSR of SEQ ID NO: 2 for the GS010 integration site (enrichment in positive selection).

[0153] Bacterial directed evolution hits improved specificity in the human cell line HEK293FT. Percent integration results at the genome integration sites by ddPCR are shown in Figure 7. The LSR variants of SEQ ID NOs: 725-740 derived from the directed evolution screen showed improved specificity over the wild-type LSR of SEQ ID NO: 2. A preferred LSR variant of SEQ ID NO: 725 was further improved by combining mutations from an activity enhanced LSR variant of SEQ ID NO: 519 to yield an engineered LSR of SEQ ID NO: 741. The additional activity enhancing mutations in the LSR of SEQ ID NO: 741 increased activity beyond the wild-type LSR of SEQ ID NO: 2 or the loop swap specificity variant LSR of SEQ ID NO: 242 while showing much lower activity at secondary integration sites.

[0154] An exemplary LSR variant (SEQ ID NO: 775) with a wildtype AttD (SEQ ID NO: 885) showed improved genome-wide specificity, with a >10X difference between GS010 and secondary sites as assessed by GIA (Figure 9). Example 5: Engineered LSRs integrate clinically relevant cargo in primary human T cells

[0155] Activity-enhanced LSR consisting of SEQ ID NO: 304 was further evaluated for the ability to integrate a CAR construct in primary human T cells.

[0156] Pan-T cells isolated from three different human PBMC donors (StemCells Inc) were thawed and stimulated using a 3:1 ratio of pre-washed human anti-CD3, anti-CD28 T activator Dynabeads (Invitrogen) to T cells in X-Vivo15 media (Lonza) supplemented with 2% human AB serum, 100IU IL2, 10ng / ml IL7 and 10ng / ml IL15. After 24 hours,Attorney Docket No: 16290.0011-0304 Dynabeads were removed using a magnetic separator and T cells were reconstituted in Lonza P3 buffer for electroporation using a Lonza 4D instrument. Electroporation was carried out with 6ug / million T cells of LSR mRNA and 2ug / million T cells of nanoplasmid DNA template expressing CD19-CAR in manufacturer-supplied 100ul format cuvettes using the program EO115. Negative controls included LSR mRNA but no template DNA or template DNA but no LSR mRNA. T cells were recovered post electroporation in the same media and re-stimulated with Dynabeads at a ratio of 3:1 beads:T cells. Cells were cultured for 7 days after electroporation and supplemented with extra media containing the IL-2, IL-7, and IL-15 cytokine cocktail every 2 days. After 7 days, cells were harvested, processed, and analyzed by flow cytometry and ddPCR.

[0157] Flow cytometry was performed using standard procedures. Data was recorded in BL1 channel for FITC to reveal percentage of cells expressing CAR relative to total live cells using FlowJo 10.10.0 software. Percent CAR positive cells from three independent T cell donors was plotted using GraphPad Prism version 10.2.3.

[0158] ddPCR was performed on a Biorad QX600 equipment using manufacturer recommended protocol. Briefly, genomic DNA (gDNA) was prepared using a Zymo kit and 60-80ng gDNA per sample was analyzed on ddPCR using primer-probe designs aimed at investigating integration activity at the genomic sites GS010, GS012, GS013, and GS018. The percent integration per haploid genome was determined by dividing the integration signal at each site by that of RPPH1 reference. For each integration site, the test was performed with both the target and reference primer / probe in duplicate. Percent integration / haploid genome for the 4 indicated genomic sites from three independent T cell donors was plotted using GraphPad Prism version 10.2.3

[0159] The flow cytometry results shown in Figure 8A demonstrate average CAR positive cell rates approaching 40% in human primary T cells. Results in Figure 8B show the percent integration at the GS010, GS012, GS013, and GS018 sites. Example 6: Ex vivo engineered T cells with an LSR variant achieve high CAR+ rates and are functional for tumor killing in vitro

[0160] Primary human T cells were engineered ex vivo with an LSR variant (SEQ ID NO: 775) and nanoplasmid DNA encoding a CD19 CAR transgene and compared with T cells engineered with lentivirus.

[0161] For engineering T cells with an LSR variant, cryopreserved human T cells were obtained from Stemcell Technologies. T cells were thawed in X-Vivo15 media (Lonza) supplemented with 2% human AB serum (Fisher), 100IU / mL IL-2, 10ng / mL IL-7, 10ng / mLAttorney Docket No: 16290.0011-0304 IL-15 (Stemcell Technologies) and stimulated using a 3:1 ratio of pre-washed human anti- CD3, anti-CD28 Human T-Activator Dynabeads (Invitrogen) to T cells. After 24 hours, Dynabeads were removed using a magnetic separator and T cells were reconstituted in Lonza P3 buffer for electroporation using a Lonza 4D instrument. Electroporation was carried out with 6µg / million T cells of LSR mRNA and 2µg / million T cells of nanoplasmid DNA template expressing CD19-CAR in manufacturer-supplied 100µl format cuvettes using the program EO115.3 million cells per cuvette for a total of 8 cuvettes were electroporated. After electroporation, cells were recovered in thaw media with a 3:1 ratio of Dynabeads to T cells in a 12-well plate. After 72 hours, T cells were then pooled, de-beaded, and moved to a well of a 6-well GREX in a total volume of 20 mL Thaw media as noted above. Cells were maintained for the next 7 days at a final volume of 40 mL, and cytokines were replenished every two days.10 days after electroporation, T cells were harvested for freezing, ddPCR and flow cytometry.

[0162] For non-transfected T cells (negative control), T cells were thawed, transferred to a 6-well GREX containing X-Vivo15 media (Lonza) supplemented with 2% human AB serum (Fisher), 100IU / mL IL-2, 10ng / mL IL-7, 10ng / mL IL-15 (Stemcell Technologies) (7 million cells per 7 ml of media per well) and stimulated using a 3:1 ratio of pre-washed human anti- CD3, anti-CD28 Human T-Activator Dynabeads (Invitrogen) to T cells. After 96 hours, cells were pooled, debeaded, and seeded into GREX vessels with 20 mL of the above media. Cells were maintained for the next 7 days at a final volume of 40 mL, and cytokines were replenished every two days.10 days after transduction, T cells were harvested for freezing, ddPCR and flow cytometry.

[0163] For lentiviral engineering of T cells, T cells were thawed, transferred to a 6-well GREX containing X-Vivo15 media (Lonza) supplemented with 2% human AB serum (Fisher), 100IU / mL IL-2, 10ng / mL IL-7, 10ng / mL IL-15 (Stemcell Technologies) (7 million cells per 7 ml of media per well) and stimulated using a 3:1 ratio of pre-washed human anti- CD3, anti-CD28 Human T-Activator Dynabeads (Invitrogen) to T cells. After 24 hours, T cells were transduced with lentivirus using LentiBoost. After 72 hours, cells were pooled, debeaded, and seeded into GREX vessels with 20 mL of the above media. Cells were maintained for the next 7 days at a final volume of 40 mL and cytokines were replenished every two days.10 days after transduction, T cells were harvested for freezing, ddPCR and flow cytometry.

[0164] Flow cytometry was performed using standard procedures. Cells were stained with anti-FMC63 antibody to detect CAR-expressing cells (FITC; ACRO), and LiveDead AquaAttorney Docket No: 16290.0011-0304 (ThermoFisher). Cells were analyzed with a Thermo Fisher Attune Flow cytometer. The percentage of cells expressing CAR relative to total live cells was determined using FlowJo 10.10.0 software. Percent CAR positive cells from three independent T cell donors was plotted using GraphPad Prism version 10.2.3.

[0165] ddPCR was performed on a Biorad QX600 equipment using manufacturer recommended protocol. Briefly, genomic DNA (gDNA) was prepared using a Zymo kit and 60-80ng gDNA per sample was analyzed on ddPCR using primer-probe designs aimed at investigating integration activity at the genomic sites GS010, GS011, GS012, GS013, and GS018. The percent integration per haploid genome was determined by dividing the integration signal at each site by that of RPPH1 reference. For each integration site, the test was performed with both the target and reference primer / probe in duplicate. Percent integration / haploid genome for the indicated genomic sites was plotted using GraphPad Prism version 10.2.3

[0166] On day 10, T cells were harvested and evaluated for % CAR+ cells by flow cytometry. LSR-engineered T cells achieved high CD19-CAR+ cells (Figure 10A) with a clear population shift and higher MFI when compared to lentivirus (Figures 10C-E). A high percent integration at the GS010 site was confirmed by ddPCR (Figure 10B).

[0167] Also on day 10, T cells were harvested and cultured for 19 days in either homeostatic cell culture conditions, where the media contained IL-2, IL-7, and IL-15, or antigen exposure conditions, where the LSR-engineered T cells were stimulated with 5 rounds of CD19+ Nalm6 tumor cells every 3 to 4 days. The fraction of total integration of the CAR at GS010 was assessed in cells under both culture conditions using ddPCR after 0 days, 11 days, and 19 days after being introduced to the homeostatic or repeated antigen- stimulation cell culture conditions. The integration of the CAR at GS010 was maintained in the LSR-engineered T cells after long-term culture, both in the homeostatic cell culture conditions (Figure 11A) and after repeated antigen exposure (Figure 11B). The LSR- engineered T cells were evaluated for %CAR+ cell by flow cytometry at 0 days, 4 days, 7 days, 11 days, 14 days, and 19 days after being introduced to the homeostatic or repeated antigen-stimulation cell culture conditions. The proportion of CD19-CAR+ cells was maintained over the 19 days in homeostatic cell culture conditions (Figure 11C), indicating that the integration of the CAR into the GS010 site was stable. In the repeated antigen simulation conditions, the percentage of CD19-CAR+ cells increased with repeat antigen exposure (Figure 11C) with no change in the proportion of integration events at the GS010Attorney Docket No: 16290.0011-0304 site. These results demonstrate that the GS010 site supports stable, durable, and functional CAR-T expression.

[0168] The CAR+ T cells generated ex vivo were shown to kill tumor cells in an in vitro tumor cell-killing assay. CAR+ T cells were co-cultured for 24 hours with Nalm6-luciferase tumor cells at the indicated effector to target (E:T) ratios (Figure 12) and cytotoxicity was measured by luciferase quantitation.

[0169] A cytotoxicity assay was set up to assess the function of LSR engineered and lentiviral engineered T cells using NALM6 cells constitutively expressing firefly luciferase (Nalm6-Luc2; Imanis Life Sciences, CL151). Briefly, vials of 106cells of non-transfected, lentivirus, or LSR engineered T cells were thawed in X-Vivo15 media (Lonza) supplemented with 2% human AB serum (Fisher) without cytokines.24 hours later, cells were counted and run on flow cytometer for determination CAR+ percentage of cells according to protocol above. Subsequently, 10,000 Nalm6-Luc2 cells in 100uL T cell media were plated a 96-well flat-bottom plate. Different populations of CAR-T cells were normalized to the same %CAR+ cells using non-engineered T cells. Serially diluted populations of T cells were added to Nalm6 cells to a total volume of 200ul to achieve 3:1, 1:1, 0.5:1 Effector (CAR-T cells) to Target (Nalm6-Luc2 cells) cell ratios. The assay plate was stored in 37C incubator overnight.24 hours later, cells were moved into a 96-well round-bottom plate and centrifuged at 500g for 5min.100µL of supernatant was transferred into a clean 96-well round-bottom plate and stored at -80C for cytokine analysis. Cells were replenished with 100µL T cell media and transferred into a 96-well black wall clear bottom plate. The cell suspension was equilibrated with 100µl of Luciferase buffer for 30 minutes at room temperature following manufacturer’s protocol (Promega, Bright-Glo(TM) Luciferase Assay System; E2610). Luciferase signal was quantified using the Luminex Spectramax.

[0170] Cytokine production was measured by ELISA. The LSR-engineered CAR+ T cells generated ex vivo showed superior cytokine production as compared to lentiviral-engineered cells (Figures 13A-D).

[0171] Cytokines (IFN-γ, IL-2, TNF-alpha & Granzyme-B) were measured from the cell free supernatants obtained from the cytotoxicity assay described above by employing a standard sandwich-ELISA format following manufacturer’s instructions (ACRO biosystems; RES-A028, CRS-A003, RES-A027, and CEA-B033 respectively). Briefly, 100µl supernatant was distributed equally between four assay micro-plates pre-coated with anti-cytokine antibodies. Samples were added along with standards, subsequently sandwiched with Biotin- anti-cytokine antibody to form an antibody-antigen-antibody complex. Finally, Streptavidin-Attorney Docket No: 16290.0011-0304 HRP was added followed by a substrate solution for HRP and incubated in the dark for 20 mins at RT. The colorimetric reaction was stopped, and the absorbance was recoded at 450nm and 630nm within 10 mins using a Luminex Spectramax machine. To reduce the background noise, the readings at 630nm were subtracted from the readings at 450nm. The mean absorbance was calculated for each standard, control, and sample with the average zero standard optical density (O.D.) subtracted. The standard curve was plotted with the standard concentration as x-axis and the calibrated absorbance value as y-axis. Four parameter logistic regression was used to draw the standard curve and calculate the sample concentration. Normal range of Standard curve was R2≥0.9900, and the detection range of these assays was 3.906 pg / mL-500 pg / mL. Example 7: CAR T cells ex vivo engineered with an LSR variant show anti-tumor activity in vivo

[0172] The ex vivo engineered T cells of Example 6 were tested in vivo for anti-tumor activity in an CD19+ human tumor model in NSG mice.

[0173] On Day -5, NSG mice were injected with tumor cells IV (1 million Nalm6- luciferase cells / mouse) through the tail vein. IVIS imaging was performed after 4 days (Day - 1), and the results were used to randomize the mice into 5 groups, ensuring an even tumor burden across all groups. After five days (Day 0), the LSR-engineered CAR T cells or lentivirus engineered CAR T cell or non-engineered T cells shown in Example 6 were injected into the tail vein of NSG mice at the amounts shown in Table 6 below. Using non- engineered T cells, the total T cell numbers were normalized to be the same across all groups. Table 6: Group Engineered method Dose (12.5 M total T cells) 1 Non-engineered 0 CAR+2 Lentivirus 5M CAR+3 LSR variant 5M CAR+4 Lentivirus 1M CAR+5 LSR variant 1M CAR+

[0174] The ex vivo engineered CAR T cells showed robust anti-tumor activity (Figure 14A) and expanded in vivo (Figure 14B).Attorney Docket No: 16290.0011-0304

[0175] Tumor burden was assessed by IVIS imaging on Day -1, 3, 7, 10, 14, 17 and 22. Mice were injected IP (150 mg / kg) with 15 mg / mL D-luciferin solution in D-PBS and maintained on 2.5% isoflurane via nose cones attached to the internal anesthesia manifold. Mice were placed on the heated (37oC) shelf of the imaging chamber of the AMI HTX Spectral Imaging (Spectral Instruments) system for ventral image acquisition. The bioluminescence signal was quantitated using Aura In Vivo Imaging software (Spectral Instruments) following the manufacturer’s instruction. Total flux (p / s) was used to indicate the intensity of luciferase signal.

[0176] Expansion of CAR+ T cells was assessed on day 7, 10, 14, 17 and 23 by flow cytometry. Whole blood was collected with EDTA tubes. No-wash protocol was used to ensure the accurate count of T cell numbers / µl blood. Briefly, 10 µl blood / mouse was transferred to a 96-well U shape plate, and 25 ul staining mixture which contained antibodies against CD3, CD4, CD8, FMC63 (CAR), CD45, CCR7, CD45RA was added to each well and incubated for 30 minutes at room temperature. Then 165 µl 1X red blood cell (RBC) lysis buffer was added to each well and incubated for 15 min at room temperature. Samples were then acquired on the Attune NxT flow cytometer and data were analyzed using Flowjo software. Example 8: CAR T cells engineered with an LSR variant show expression of the CAR

[0177] Pan-T cells isolated from human PBMC donor (ALLCELLS Inc) were thawed and stimulated using 10 µl human T Cell TransAct (Miltenyi Biotec) per million cells in TexMACS media (Miltenyi Biotec) supplemented with 5% human AB serum, 20ng / ml IL2, 10ng / ml IL7 and 5 ng / ml IL15. After 24 hours, T cells were reconstituted in Lonza P3 buffer for electroporation using a Lonza 4D instrument. Electroporation was carried out with 6 µg / million T cells of LSR mRNA and 2µg / million T cells of nanoplasmid DNA template expressing CD19-CAR in manufacturer-supplied 20 µl format cuvettes using the program EO115. Negative controls included template DNA but no LSR mRNA. T cells were recovered post electroporation in the same media and re-stimulated with 10 µl human T Cell TransAct (Miltenyi Biotec) per ml media. Cells were cultured for 7 days after electroporation and supplemented with extra media containing the IL2, IL7, and IL15 cytokine cocktail every 2 days. After 7 days, cells were harvested, processed, and analyzed by flow cytometry.

[0178] Flow cytometry was performed using standard procedures. Cells were stained with anti-FMC63 antibody to detect CAR-expressing cells (FITC; ACRO), and LiveDead Aqua (ThermoFisher). Cells were analyzed with a Thermo Fisher Attune Flow cytometer. TheAttorney Docket No: 16290.0011-0304 percentage of cells expressing CAR relative to total live cells was determined using FlowJo 10.10.0 software. Percent CAR positive cells from different LSRs were plotted using GraphPad Prism version 10.5.0.

[0179] The flow cytometry results shown in Figure 15 demonstrate average CAR positive cell rates with the LSR variants.

Claims

Attorney Docket No: 16290.0011-0304 CLAIMS 1. A large serine recombinase (LSR) variant comprising an amino acid sequence having one or more mutations as compared to the LSR of SEQ ID NO: 2; wherein the LSR variant has at least 80% identity to SEQ ID NO: 2; and wherein the LSR variant has increased genome integration activity and / or integration specificity compared to the LSR of SEQ ID NO:

2.

2. The LSR variant of claim 1, wherein the one or more mutations increase genome integration activity by at least 5%, 10%, 15%, or 20% at an integration site of interest compared to the LSR of SEQ ID NO:

2.

3. The LSR variant of claim 1 or claim 2, wherein the integration site of interest is GS010.

4. The LSR variant of any one of claims 1-3, wherein the one or more mutations comprise G10L, N29R, the combination of N29R and V66I, L31I, A36K, L43V, E57D, K58G, H74K, L116I, S117G, T153R, T153S, A156K, M160G, L163I, V184I, S213R, the combination of S213K and Q214R, Q214K, Q214H, the combination of Q214Y and P217C, K216N, P217D, S231I, S231V, T241V, N243K, N243R, S244A, Q260K, T266D, T266S, the combination of S284V and K286A, the combination of S284R and K286A, G285K, G285H, G285R, G285Y, A317K, K321G, K348E, C353R, T366L, V375H, G391R, G391K, S399A, N413S, P436I, P436L, P436Q, G452S, F462L, Q484S, S486K, K488S, and / or Y494K.

5. The LSR variant of any one of claims 1-4, wherein the LSR variant comprises one or more mutations that increase the integration specificity compared to the LSR of SEQ ID NO: 2, wherein the one or more mutations are in the alphaE-beta6 linker, alphaG-alphaH linker, RD- ZD linker, or at the putative dimer interface in the coiled coil domain.

6. The LSR variant of any one of claims 1-5, wherein the LSR variant has a greater specificity ratio for the integration site of interest compared to the LSR of SEQ ID NO:

2.

7. The LSR variant of any one of claims 1-6, wherein the LSR variant has a greater specificity ratio for the GS010 integration site compared to the LSR of SEQ ID NO: 2; and wherein the specificity ratio is determined with any one or more secondary integration sites selected from GS011, GS012, GS013, and GS018.

8. The LSR variant of any one of claims 1-7, wherein the specificity ratio of the LSR variant is 1.5x greater than the specificity ratio of the LSR of SEQ ID NO:

2.

9. The LSR variant of any one of claims 1-8, wherein the one or more mutations comprise L5K, Y7K, L43V, the combination of S47G and L49R, the combination of S47A and L49R, the combination of L49R and I51F, L49R, I51R, the combination of K55W and E57N, the combination of K55S and K58S, K58N, L116I, P inserted at position 116, N119G, the combination of N119G and F145L, R139N, the combination of G142E and F145K, F145R, F145K, E150L, a substitution of the amino acids 155-164 with GKWNGGFAPYG, the combination of A156K and P161V, the combination of Y157M and R160Q, the combination of W157K and A161F (starting from the loop swap variant; see Table 4), the combination of A159N and R160M, the combination of A159N and R160A, the combination of A159N and R160G, A159T, P161A, L163F, K166T, V168D, H170N, P173K, R204E, the combination of G209D and K216I, the combination of K211C and S213L, the combination of S213W and K216N, the combination of S213W and P217M, Q214K, Q214F, the combination of Q214Y and P217C, the combination of Q214Y and G215E, the combination of K216N and F218M, F218M, E219S, E224R, the combination of N243K, T245S, and K251A, S244 deletion,Attorney Docket No: 16290.0011-0304 S244D, S244E, R247T, K249R, D250L, E253D, the combination of P262K and I265T, I264F, I264L, the combination of Y281I and S284K, the combination of Y281S and K286A, the combination of P283L and K286A, the combination of S284V and K286A, PK inserted at position 284, G285H, G285R, G285Y, the combination of G285T and K286A, G285F, the combination of G285Y and K286A, the combination of G285K and K286A, K286A, K286G, the combination of K286A and R287K, the combination of A286H and T291M, the combination of K286C and T291G, M314P, the combination of Y328W and G329K, the combination of Y328W and G329F, the combination of Y328V and G329Q, the combination of G329R and K334M, the combination of G329Q and H331F, P338S, Y365E, G391R, K392R, S399A, H423E, and / or D433Q.

10. An LSR variant comprising the amino acid sequence having one or more mutations as compared to the LSR of SEQ ID NO: 1, wherein the LSR variant has at least 80% identity to SEQ ID NO: 1; and wherein the LSR variant has increased genome integration activity and / or integration specificity compared to the LSR of SEQ ID NO:

1.

11. The LSR variant of claim 10, wherein the one or more mutations increase genome integration activity by at least 5%, 10%, 15%, 20% at an integration site of interest compared to the LSR of SEQ ID NO:

1.

12. The LSR variant of claim 10 or claim 11, wherein the integration site of interest is GS010.

13. The LSR variant of any one of claims 10-12, wherein the one or more mutations comprise M2 deletion, N29R, T36K, N119G, T153R, M175V, S213K, Q214H, S231V, Q261H, S262E, A317K, S326Q, V354L, T366L, I373A, G391K, S399A, N445E, S459N, and / or F462V.

14. The LSR variant of any one of claims 10-13, wherein the LSR variant comprises one or more mutations that increase the integration specificity compared to the LSR of SEQ ID NO: 1, wherein the one or more mutations are in the alphaE-beta6 linker, alphaG-alphaH linker, RD-ZD linker, or at the putative dimer interface in the coiled coil domain.^ 15. The LSR variant of any one of claims 10-14, wherein the LSR variant has a greater specificity ratio for the integration site of interest compared to the LSR of SEQ ID NO:

1.

16. The LSR variant of any one of claims 10-15, wherein the LSR variant has a greater specificity ratio for the GS010 integration site compared to the LSR of SEQ ID NO: 1; and wherein the specificity ratio is determined with any with one or more secondary integration sites selected from GS011, GS012, GS013, and GS018.

17. The LSR variant of any one of claims 10-16, wherein the specificity ratio of the LSR variant is 1.5x greater than the specificity ratio the LSR of SEQ ID NO:

1.

18. The LSR variant of any one of claims 10-17, wherein the one or more mutations comprise N119G.

19. An LSR variant comprising the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301- 320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566,Attorney Docket No: 16290.0011-0304 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917- 921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70- 229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351- 364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528- 544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748- 756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047- 1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593.

20. The LSR variant of claim 19, wherein the LSR variant comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593.

21. The LSR variant of claim 19 or claim 20, wherein the LSR variant is capable of genome integration activity at the GS010 integration site.

22. The LSR variant of any one of claims 19-21, wherein the LSR variant further comprises a nuclear localization signal.

23. An isolated nucleic acid encoding the LSR variant of any one of claims 1-22.

24. The isolated nucleic acid of claim 23, wherein the nucleic acid is mRNA.

25. The isolated nucleic acid of claim 24, wherein the mRNA sequence comprises one of SEQ ID NOs: 4680-4682, 4692-4694, 4704-4706, 4716-4718, 4728-4730, 4740-4742, 4752- 4754, 4764-4766, 4776-4778, 4788-4790, 4800-4802, 4812-4814, 4824-4826, 4836-4838, 4848-4850, 4860-4862, 4872-4874 ,4884-4886, 4896-4898, 4908-4910, or 4920-4922.

26. A genome-editing system comprising (i) the LSR variant of any one of claims 1-22 or the isolated nucleic acid of any one of claims 23-25 and (ii) a nucleic acid comprising an AttD site.

27. The genome-editing system of claim 26, wherein the AttD site comprises the nucleic acid sequence of an AttD from any one of SEQ ID NOs: 885-887, 1131-1145, or a nucleic acid sequence having at least 90%, or at least 95% identity to the nucleic acid sequence of an AttD from any one of SEQ ID NOs: 885-887 or 1133-1146.Attorney Docket No: 16290.0011-0304 28. The genome-editing system of claim 26 or claim 27, wherein the genome-editing system is capable of integrating a sequence from the nucleic acid comprising an AttD site at the GS010 integration site.

29. A genome-editing system comprising an LSR variant and a nucleic acid comprising an AttD site comprising any one of SEQ ID NOs: 885-887, 1133-1146, or an AttD site having at least 90%, or at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 885-887 or 1131-1145, wherein the genome-editing system is capable of integrating a sequence from the nucleic acid comprising an AttD site at the GS010 integration site.

30. The genome-editing system of any one of claims 26-29, wherein the nucleic acid encodes a CAR amino acid sequence.

31. The genome-editing system of any one of claims 26-30, wherein the genome-editing system is capable of integrating the CAR sequence at the GS010 integration site in a cell.

32. A method for nucleic acid recombination, comprising contacting one or two nucleic acids comprising a pair of cognate AttA and AttD sites with (i) the LSR variant of any one of claims 1-22, (ii) the isolated nucleic acid of any one of claims 23-25, or (iii) the genome- editing system of any one of claims 26-31.

33. The method of claim 32, wherein the nucleic acid is synthetic DNA.

34. The method of claim 32, wherein the nucleic acid is genomic DNA.

35. The method of claim 32, wherein the nucleic acid is human DNA.

36. The method of claim 32, wherein the nucleic acid is non-human DNA.

37. A method for recombination in a genome, comprising contacting a cell with (i) the LSR variant of any one of claims 1-22, (ii) the isolated nucleic acid of any one of claims 23-25, or (iii) the genome-editing system of any one of claims 26-31.

38. A method for integration of a sequence from a nucleic acid comprising an AttD into a cellular genome, comprising contacting a cell with the genome-editing system of any one of claims 26-31 such that the sequence from the nucleic acid comprising an AttD is integrated into the cellular genome.

39. The method of claim 38, wherein the LSR variant comprises the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233- 235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803-813, 815-854, 856-872, 874-876, 878- 882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, 4593, or an amino acid sequence having at least 90%, at least 95%, or at least 99%Attorney Docket No: 16290.0011-0304 identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, wherein the LSR variant exhibits genome integration activity in a mammalian cell.

40. The method of claim 38 or claim 39, wherein the LSR variant comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593.

41. A method of editing a genome at the GS010 integration site in a human cell, comprising contacting the human cell with the genome-editing system of any one of claims 26-31 such that a sequence from the nucleic acid comprising an AttD is integrated into the cellular genome, wherein the LSR variant comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 3-6, 9, 10-21, 23-25, 27-53, 56-59, 62, 63, 65-68, 70-229, 231, 233-235, 242, 250, 291, 293, 301-320, 322-326, 328, 329, 331-343, 346-349, 351-364, 368-392, 394-404,407-421, 423, 424, 426, 428-441, 443-446, 449, 450, 452-526, 528-544,546, 550, 553, 556, 557, 560, 564, 566, 567, 569-573, 575-582, 584-741, 744-746, 748-756, 760, 761, 763-767, 774-778, 780-789, 792-796, 798-801, 803- 813, 815-854, 856-872, 874-876, 878-882, 891-911, 913, 914, 917-921, 923-1026, 1028, 1031, 1034-1043, 1047-1052, 4114, 4118, 4122, 4126, 4130, 4135, 4139, 4144, 4148, 4152, 4156, 4160, 4164, 4172, 4176, 4180, 4184, 4188, 4192, 4196, 4200, 4204, 4208, 4213, 4217, 4222, 4226, 4230, 4234, 4238, 4242, 4250, 4254, 4258, 4262, 4266, 4270, 4274, 4278, 4282, 4286, 4290, 4294, 4298, 4302, 4306, 4310, 4314, 4320, 4326, 4330, 4334, 4338, 4342, 4346, 4350, 4354, 4358, 4362, 4366, 4370, 4374, 4378, 4382, 4386, 4390, 4394, 4398, 4402, 4406, 4410, 4414, 4418, 4422, 4426, 4523, 4577, 4588, or 4593, and the AttD site comprises a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NOs: 885-887 or 1133-1146.

42. The method of claim 41, wherein the LSR variant comprises the amino acid sequence of any one of SEQ ID NOs: 304, 660, 661, 664, 677, 741, 775, 822, 825, 830, 1010, 1022, 1024, 4278, 4326, 4390, 4394, 4523, 4577, 4588, or 4593.

43. The method of any one of claims 32-42, wherein the method is an in vitro or ex vivo method.

44. The method of any one of claims 32-42, wherein the method is an in vivo method.

45. The method of any one of claims 32-44, wherein the cell is a T cell.

46. The method of any one of claims 32-45, wherein the sequence from the nucleic acid comprising an AttD further encodes a CAR amino acid sequence.Attorney Docket No: 16290.0011-0304 47. An engineered cell made by a process comprising: (i) isolating a cell and (ii) contacting the isolated cell with an effective amount of the genome-editing system of any one of claims 26-31, wherein the genome-editing system integrates a sequence from the nucleic acid comprising an AttD of the genome-editing system into the isolated cell’s genome. 48, The cell of claim 47, wherein the cell is a T cell.

49. A method of treating a disease in a subject, the method comprising: administering to the subject an effective amount of the genome-editing system of any one of claims 26-31, wherein the method comprises integrating a sequence from the nucleic acid comprising an AttD of the genome-editing system into the subject’s genome, thereby treating the disease.

50. A method of treating a disease in a subject, the method comprising: (i) contacting isolated cells with an effective amount of the genome-editing system of any one of claims 26-31, wherein the method comprises integrating a sequence from the nucleic acid comprising an AttD of the genome-editing system into the cells to yield engineered cells and (ii) administering the engineered cells to the subject, thereby treating the disease.

51. The method of claim 49 or claim 50, wherein the subject is human.

52. The method of any one of claims 49-51, wherein the disease is caused by a genetic mutation.

53. The method of any one of claims 49-52, wherein the sequence from the nucleic acid comprising an AttD further encodes a CAR amino acid sequence.

54. The method of any one of claims 49-53, wherein the cells are T cells.

55. The method of any one of claims 49-54, wherein the disease comprises cancer.

56. A method of integrating a nucleic acid sequence at the GS010 integration site in an immune cell such that the protein encoded by the nucleic acid is expressed by the cell, wherein the integration comprises contacting the immune cell with a genome-editing system.

57. The method of claim 56, wherein the genome-editing system comprises the genome- editing system of any one of claims 26-31.

58. The genome-editing system of any one of claims 26-31 for use in treating a disease in a subject, wherein the use comprises administering an effective amount of the genome-editing system to the subject or contacting isolated cells with an effective amount of the genome- editing system to yield engineered cells and administering the engineered cells to the subject.

59. Use of the genome-editing system of any one of claims 26-31 in the manufacture of a medicament for treating a disease.

60. Use of the genome-editing system of any one of claims 26-31 in the manufacture of a host cell to produce a biologic.