Cell line development for protein production

Abstract

The present disclosure provides, in part, methods for generating cell lines, e.g., for the production of recombinant protein, using integrases for genomic insertion into host cells.

Description

Atorney Docket No.: KOM-003PC / 138774-5003CELL LINE DEVELOPMENT FOR PROTEIN PRODUCTIONFIELD

[0001] The present disclosure relates to, inter alia, molecular biology approaches to cell line construction using mutant integrases, positive / negative selection procedures, and methods of using the same, e.g., for cell line development and recombinant protein expression.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 735,972, filed December 19, 2024, and U.S. Provisional Application No. 63 / 843,553, filed July 14, 2025, the entire contents of each of which are incorporated herein by reference.SEQUENCE LISTING

[0003] The instant application contains a sequence listing, which has been submitted in XML format via Patentcenter. The XML file is named “138774-5003_Sequence_Listing,” which was created on December 19, 2025 and is approximately 155,648 bytes in size, the entire contents of which are incorporated herein by reference in their entirety.BACKGROUND

[0004] Recombination-based methods for generating cells lines for stable protein expression have been adapted using a variety of recombinase systems. For example, Cre and Flp recombination techniques have been developed which include the insertion of a Lox or FRT sequence, respectively, into a genomic target site, which is then targeted by the Cre or Flp recombinase to initiate site-specific recombination. Ultimately, the target location becomes the site of a genomic amendment, which enables the cell to stably express a gene of interest over successive cell divisions.

[0005] However, recombination-based techniques for cell line development have caveats, including 1) how to identify “safe harbor” genomic locations, 2) how to accurately place recombination sequences into these safe harbor locations, and 3) how to improve fidelity in targeting each recombination sequence with minimal off-target effects. In addition, these techniques have several limitations including multi-insertion (typically only allow a single insertion per cell), DNA payload size for genomic integration (limited in bp length), efficiency (typically limited to ~1 -3% of clonal cell population), and inherent risks for introduction of unintended mutation or double-strand DNA breaks. Traditional methods also rely on antibiotic selection (e.g., insertionDBl / 164937876.2 1Atorney Docket No.: KOM-003PC / 138774-5003 of one or more resistance cassettes), or auxotrophic cells (e.g., glutamine synthetase (GS) selective pressure), further extending and complicating the selection process as clonal population with random insertion, among other unwanted populations, may survive several rounds of selection.

[0006] Serine integrases are among a class of enzymes that may address the shortcomings of other traditional recombination-based methods. Serine integrases are capable of inserting large DNA cargos at defined sequences. For example, with respect to mammalian cell culture, serine integrases can facilitate recombination between attachment (att) sites, e.g., a phage attachment site, atP (donor sequence), and bacterial attachment site, atB (target sequence). The DNA is protected during cleavage, strand exchange, and re-ligation. No synthesis or degradation of DNA occurs and no sequence homology is required for the donor DNA. This process does not rely on rate-limiting host factors and does not provoke error-prone DNA repair processes caused by nuclease cleavage that can induce a p53-mediated DNA damage response and lead to unwanted selection for cells with an impaired p53 pathway. A limitation of serine integrase technology is that the att site must first be inserted at the target sequence (e.g., in a host cell genome). Fortunately, the small size of at sites has permitted their insertion using HDR into the genomes of mice and human cells to function for site-specific integration.

[0007] There remains a need to develop cell lines with genomic amendments capable of stable expression of target proteins, e.g., for biotherapeutic production in clinically relevant systems. In addition, there remains a need to improve the efficiency of the enzymatic machinery involved in integrase-mediated integration to diversify the repertoire of integration sites, as well as improved methods for cell line development.SUMMARY

[0008] Accordingly, the present disclosure provides, in part, the ability to deliver large transgenes to one or more genomic locus with high efficiency using integrases. In embodiments, the integrases comprise mutant integrases which catalyze the integration of large DNA cargos (e.g., one or more insertion sequences) at attachment (aft) sites. In embodiments, targeting to att sites in the genome utilize one or more genome insertion techniques in addition to integrase-mediated genomic insertion, such as Cas9-directed reverse transcription, zinc finger nucleases (ZFNs), TALENS, etc., where integration occurs at a target “safe harbor” loci. In embodiments, methods herein avoid double-strand breaks, which would ordinarily trigger DNA damage pathways (e.g., p53).DBl / 164937876.2 2Atorney Docket No.: KOM-003PC / 138774-5003

[0009] In aspects, described herein is a method for generating a cell line comprising introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the single genomic locus, and wherein the one or more nucleic acids encodes one or more bacterial attachment (attB) sites and / or one or more phage attachment (attP) sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more integration sequences by one or more integrases, and culturing a clonal cell population from the cell to generate the cell line.

[0010] In embodiments, the one or more nucleic acids encodes one or more one or more attB sites or one or more attP sites, optionally a single attB site or a single attP site. In embodiments, the one or more bacterial attachment (attB) sites comprises a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 1 , 3, and 5; and / or wherein the one or more bacterial attachment (attB) sites is compatible with one or more phage attachment (attP) sites, optionally comprising a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 2, 4, and 6. In embodiments, the one or more bacterial attachment (attP) sites comprises a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 2, 4, and 6; and / or wherein the one or more bacterial attachment (attP) sites is compatible with one or more phage attachment (attB) sites, optionally comprising a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 1 , 3, and 5.

[0011] In embodiments, the one or more nucleic acids comprises one or more homology directed repair (HDR) insulator sequence and one or more attB site and / or attP site flanking one or more kill switch gene, one or one or more promoter, one or more 2A peptide sequence, one or more insertion sequence, and / or one or more polyA tail.

[0012] In embodiments, the promoter comprises one or more of a mammalian gene promoter, viral promoter, inducible promoter, tissue-specific promoter, cell type-specific promoter, and bidirectional promoter. In embodiments, the promoter comprises one or more native promoter. In embodiments, the promoter comprises one or more of a B29 promoter, CAG promoter, CD 14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, CMV promoter, desmin promoter, Ef1 a promoter, EGR1 promoter, elastase-1 promoter, elF2A1 promoter, endoglin promoter, FerH promoter, FerL promoter, fibronectin promoter, Flt-1 promoter, GAPDH promoter, GFAP promoter, GPU b promoter, GRP78 promoter, GRP94 promoter, HSP70 promoter, Hspa5p promoter, ICAM-2 promoter, INF-0 promoter, Nphsl promoter, OG-2 promoter, PGK-1 promoter, ROSA promoter, SP-B promoter, SV40 promoter, SYN1 promoter, ubiquitin B promoter, WASP promoter, -actin promoter, and -kin promoter.DBl / 164937876.2 3Atorney Docket No.: KOM-003PC / 138774-5003

[0013] In embodiments, the donor plasmid encodes one or more 2A peptide, optionally one or more of a T2A, P2A, E2A, and F2A peptide sequence. In embodiments, the one or more 2A peptide sequence comprises one or more amino acid sequence of SEQ ID NOs: 7-10 (e.g., based on consensus sequence of SEQ ID NO: 11). In embodiments, the one or more 2A peptide sequences is located between two or more insertion sequences.

[0014] In embodiments, the kill switch gene comprises one or more of an inducible suicide gene or molecular switch.

[0015] In embodiments, the introducing of the one or more nucleic acids comprises Cas9-directed reverse transcription, optionally by introducing one or more Cas9-directed reverse transcription plasmids. In embodiments, the Cas9-directed reverse transcription uses one or more of a nickase, reverse transcriptase, and Cas9-directed reverse transcription guide RNA (pegRNA) to introduce the one or more nucleic acids into the single genomic locus, optionally wherein the one or more Cas9-directed reverse transcription plasmids encodes one or more of the nickase, reverse transcriptase, and pegRNA. In embodiments, the nickase comprises a Cas9 nickase, optionally a Cas9 fusion protein. In embodiments, the reverse transcriptase comprises PE5. In embodiments, the pegRNA comprises a structured RNA motif to prevent degradation and / or an optimized cr772 guide scaffold. In embodiments, the Cas9-directed reverse transcription uses one or more dominant negative mutant of human mutL homolog 1 (MLH1), optionally wherein the one or more Cas9-directed reverse transcription plasmids encode the one or more dominant negative mutant of human MLH1.

[0016] In embodiments, the one or more Cas9-directed reverse transcription plasmids is configured to introduce the one or more atiB sites and / or attP sites into the single genomic locus without a double-strand DNA break.

[0017] In embodiments, the introducing of the one or more nucleic acids comprises using one or more zinc-finger nucleases (ZFNs), CRISPR / Cas endonucleases, transcription activator-like effector nucleases (TALENs), TALE-derived transcription factors, TALE repeat domain proteins, meganucleases, restriction enzymes, site-specific nucleases, and / or gene-editing systems, optionally selected from one or more of TALENs, ZFNs, RNase P RNA, RNase H, CRISPR / Cas, C2c1 , C2c2, C2c3, Cas9, Cpf1 , TevCas9, Archaea Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and any ortholog or homolog thereof, and further optionally wherein the one or more nucleic acids encodes the one or more zinc-finger nucleases (ZFNs), CRISPR / Cas endonucleases, transcription activator-like effector nucleasesDBl / 164937876.2 4Atorney Docket No.: KOM-003PC / 138774-5003(TALENs), TALE-derived transcription factors, TALE repeat domain proteins, meganucleases, restriction enzymes, site-specific nucleases, and / or gene-editing systems. In embodiments, one or more of the gene editing systems are configured to introduce the one or more attB sites and / or attP sites into the single genomic locus via homology directed repair.

[0018] In embodiments, the one or more attB sites and / or attP sites is introduced into the single genomic locus at a wild-type Bxb1 central dinucleotide sequence comprising guanine-thymine (GT). In embodiments, the one or more attB sites and / or attP sites is introduced into the single genomic locus at a non-canonical Bxb1 central dinucleotide sequence comprising one of the other 15 di nucleotide pairs (e.g., TA, TT, TG, TC, CC, CA, CT, CG, GG, GA, GC, AT, AA, AG, AC).

[0019] In embodiments, the single genomic locus comprises a AAVS1 locus, ACTB locus, ACTB locus, ALB locus, albumin locus, B2M locus, CCR5 locus, CD38 locus, CFTR locus, COL7A1 locus, Factor IX locus, FANCA locus, GBA1 locus, GYS1 locus, Hippl 1 locus, Keppel-19 locus, MACO1 locus, OI6nne-18 locus, Pansio-1 locus, Rosa26 locus, S100A locus, SHS253 locus, Smn1 locus, S100A locus, TRAC locus, Xq22.1 locus, or a homologous or species-equivalent thereof.

[0020] In embodiments, the cell comprises a mammalian cell, plant cell, insect cell, yeast cell, or bacterial cell, optionally a cell suitable for recombinant protein production. In embodiments, the mammalian cell comprises a Chinese hamster ovary (CHO) cell (CHO-K1 , CHO-DHB11, CHO-DXB1 , CHO-S, CHO- DG44, CHO-M), human embryonic kidney (HEK293, HEK293T) cell, K562 human lymphoblast cell, U2OS human osteosarcoma cell, primary human fibroblasts (human dermal fibroblast (HDFa)) cell, baby hamster kidney (BHK) cell, Vero cell (Vero, Vero 76, Vero E6), human cervical carcinoma cell (HELA, 3T3), PERc6 cell, CAP cell, iPSCs, human embryonic stem cells (ESCs), or monkey kidney CV1 cell.

[0021] In embodiments, the cell is an auxotrophic cell, optionally a glutamine synthetase (GS) knockout cell. In embodiments, the cell is not an auxotrophic variant relative to a cognate wild-type cell.

[0022] In embodiments, the introducing of one or more nucleic acids into the cell further comprises using one or more of a lipid-based transfection reagent (e.g., cationic lipid-based reagent), diethylaminoethyl (DEAE)-dextran, liposome, electroporation, sonoporation, chemical reagent (e.g., calcium phosphate), microinjection, or via a non-integrating viral vector (e.g., AAV, lentivirus).

[0023] In embodiments, the clonal cell population is compatible with the one or more integrases to integrate at least two insertion sequences into the single genomic locus, optionally wherein the at least twoDBl / 164937876.2 5Atorney Docket No.: KOM-003PC / 138774-5003 insertion sequences are adjacent and / or operably linked within the genome of the cell. In embodiments, the clonal cell population is compatible with introducing two insertion sequences, three insertion sequences, four insertion sequences, or five or more insertion sequences.

[0024] In embodiments, the one or more integrases comprise one or more Bxb1 integrases and / or one or more PhiC31 integrases.

[0025] In embodiments, the one or more integrases comprises one or more Bxb1 integrases comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12.

[0026] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 154, 155, 156, 157, 158, 159, 231 , 232, 233, 234, 235, 236, 237, 257, 314, 316, 318, 321, 322, 323, and 325, relative to SEQ ID NO: 12, or a position corresponding thereto. In embodiments, the one or more mutations are selected from Y154W, Y154A, R155W, R155H, G156P, S157G, L158T, L158R, L158A, P159S, P159T, A234N, S231A, S231 L, S231G, S231 R, S231Y, S231T, S231 H, A232G,A232S, A232R, A232T, A232V, A232Q, A232P, T233G, T233S, T233W, T233R, T233Y, T233D, T233N,T233H, T233Q, T233A, T233C, A234N, A234G, A234S, A234T, A234H, A234F, K236R, K236S, R237K,R237T, R237Q, R237N, R237C, R237V, D257K, F314M, F314L, F314N, F314R, F314K, G316R, G316W,G316S, G318H, G318P. G318S, G318N, G318P, G318K, G318R, H321 Q, H321 K, H321 L, H321 R, H321Y, H321T, H321 S, H321W, P322A, P322R, P322G, R323L, R323G, R323Y, R323I, R325K, R325Q, and R325Y, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0027] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 5, 14, 20, 24, 29, 35, 40, 45, 49, 50, 51 , 60, 68, 69, 70, 73, 74, 78, 84, 86, 87, 100, 105, 116, 124, 157, 183, 197, 207, 208, 209, 229, 261 , 267, 273, 287, 291 , 333, 342, 343, 347, 361 , 368, 375, 435, 449, 453, 462, 483, and 494, relative to SEQ ID NO: 12, or a position corresponding thereto. In embodiments, the one or more mutations are selected from V5I, D14N, E20K, E20Q, E24K, L29F, W35L, V40I, D45G, A49T, V50I, D51 N, D51 E, D51Y, N60S, E68K, E69D, Q70P, D73G, V74A, V74M, Y78H, Y78N, T84S, S86T, I87V, H100Y, V105I, T116P, A124S, S157G, E183L, P197T, R207Q, R208S, G209V, E229K, A261V, E267D, E273D, E273K, R287P, A291T, K333N, M342V, A343T, A347V, E361 D, V368A, V375I, T435A, A449V, T453A, L462M, E483K, and R494Q, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0028] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 4, 5, 18, 24, 34, 36, 40, 42, 46, 51 , 61, 62, 63, 67, 69, 79, 85, 87, 88, 89, 90, 92,DBl / 164937876.2 6Atorney Docket No.: KOM-003PC / 138774-500395, 99, 100, 105, 106, 110, 111 , 119, 122, 130, 133, 137, 140, 145, 153, 156, 160, 164, 166, 174, 175, 178, 179, 181, 187, 189, 191, 203, 209, 218, 223, 229, 231 , 239, 248, 251 , 254, 261, 264, 268, 272, 278, 280,281, 282, 283, 285, 287, 288, 292, 295, 302, 306, 307, 311 , 313, 319, 321 , 328, 331 , 332, 333, 334, 347,353, 355, 359, 360, 361, 362, 369, 370, 375, 380, 388, 397, 398, 405, 409, 411 , 414, 415, 416, 419, 425,428, 434, 435, 444, 449, 453, 461, 462, 463, 466, 468, 476, 479, 480, 483, 484, 487, 488, 489, 494, 496, and 499, relative to SEQ ID NO: 12, or a position corresponding thereto. In embodiments, the one or more mutations are selected from L4I, G34D, D36A, V40I, V40A, E42K, D51 N, L61 F, R63K, F67S, E68K, E69A, I87V, I87L, H89G, Q92H, H95Y, D99N, H100N, V105A, H111 P, A119S, V122M, E133E, A145T, V179A, V179I, R181 K, V187I, V187A, H189N, H203Y, E229K, M239I, A248T, N251 K, T254S, D257K, A261T, V264A, R272Q, A280T, T285A, A288V, A288T, A311V, R319K, R319G, H321 P, H321 N, F331S, P332H, H334R, A347E, V353I, D355N, D359N, A360T, R362K, A369P, A369E, A369T, A369S, V380I, T388M, A398S, R409H, A411V, A414V, A415S, R416K, A425T, E434G, R444L, T453I, T463I, V466M, G468D, L479I, Q480STQP, Q484K, delL488, R494S, H496N, and M499T, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0029] In embodiments, the one or more Bxb1 integrases comprise a C-terminal deletion of one or more amino acids in the range of Q480-S500 of SEQ ID NO: 12. In embodiments, the C-terminal deletion comprises a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or all 21 amino acids of Q480-S500 relative to SEQ ID NO: 12.

[0030] In embodiments, the one or more amino acid mutations comprises mutation of one or more of residues of Y154-P159; and / or wherein the one or more mutations alters specificity at -7 and / or -6 positions of a loop target DNA site compared to a wild-type Bxb1 of SEQ ID NO: 12. In embodiments, the mutation comprises S157G. In embodiments, S157G is notmutated. In embodiments, the one or more Bxb1 integrases comprise a sequence from Y154-P159 selected from YRGSLS (SEQ ID NO: 13), WWGGTP (SEQ ID NO: 14), YRGGLP (SEQ ID NO:15), AHPGRT (SEQ ID NO: 16), and WRGGAS (SEQ ID NO: 17) relative to SEQ ID NO: 12, or a position corresponding thereto.

[0031] In embodiments, the one or more amino acid mutations alters the dinucleotide preference at position -7 and / or position -6, optionally altering the dinucleotide preference to one of adenine (A) and cytosine (C), adenine (A) and thymine (T), thymine (T) and cytosine (C), or thymine (T) and thymine (T).DBl / 164937876.2 7Atorney Docket No.: KOM-003PC / 138774-5003

[0032] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations of residues S231-R237; and / or wherein the one or more mutations alters specificity at -11 , -10, and / or -9 positions of a 3 bp helix target site compared to a wild-type Bxb1 of SEQ ID NO: 12. In embodiments, L235 is not mutated.

[0033] In embodiments, the one or more amino acid mutations comprises mutation of one or more residues of 231-234 and / or 236-237. In embodiments, A234 is mutated to an asparagine (A234N); and / or wherein this mutation alters specificity at the -10 position to thymine (T) compared to a wild-type Bxb1 of SEQ ID NO: 12. In embodiments, the one or more amino acid mutations comprises mutation of one or more residues of 231 , 233, and 237; and / or wherein the mutation alters specificity at -10 and / or -9 positions of a 3 bp helix target site compared to a wild-type Bxb1 of SEQ ID NO: 12. In embodiments, the Bxb1 integrase comprises 1 , 2, 3, 4, or 5 amino acids mutated between S231-R237 (inclusive) relative to SEQ ID NO: 12. In embodiments, the Bxb1 integrase comprises a sequence from S231-R237 selected from AGGNLKR (SEQ ID NO: 18), LGTNLKR (SEQ ID NO: 19), SGTGLKK (SEQ ID NO: 20), SGSALKT (SEQ ID NO: 21), AAWALRR (SEQ ID NO: 22), GGRSLKR (SEQ ID NO: 23), SGYNLRR (SEQ ID NO: 24), SGWGLKK (SEQ ID NO: 25), SGWALRQ (SEQ ID NO: 26), SARALSR (SEQ ID NO: 27), RADTLRR (SEQ ID NO: 28), YSRNLKR (SEQ ID NO: 29), SRNGLRK (SEQ ID NO: 30), RGHALKN (SEQ ID NO: 31), GGSHLKR (SEQ ID NO: 32), TTRTLKR (SEQ ID NO: 33), AVQNLKR (SEQ ID NO: 34), RAAFLKK (SEQ ID NO: 35), RAWTLKC (SEQ ID NO: 36), RAWSLKR (SEQ ID NO: 37), HGWSLKV (SEQ ID NO: 38), HGCTLKR (SEQ ID NO: 39), YGSALKQ (SEQ ID NO: 40), SQWALKC (SEQ ID NO: 41), YPWSLRR (SEQ ID NO: 42), relative to SEQ ID NO: 12 or a position corresponding thereto.

[0034] In embodiments, the one or more Bxb1 integrases comprise a mutation of D257K compared to a wild-type Bxb1 of SEQ ID NO: 12.

[0035] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations of residues F314-R325; and / or wherein the mutation alters specificity at -19 through -12 positions of a DNA binding site compared to a wild-type Bxb1 of SEQ ID NO: 12. In embodiments, the Bxb1 residue P322 is maintained as a proline. In embodiments, the one or more amino acid mutations comprises mutation of one or more residues of F314, G316, G318, H321 , P322, R323, and R325. In embodiments, the one or more Bxb1 integrases comprise 1 , 2, 3, 4, 5, 6, or 7 amino acids mutated between F314-R325 (inclusive) relative to SEQ ID NO: 12. In embodiments, the one or more Bxb1 integrases comprise a sequence from F314-R325 selected from MAGGHRKQALYR (SEQ ID NO: 43), MAGGPRKKRRYR (SEQ ID NO: 44), LARGSRKLALYRDBl / 164937876.2 8Atorney Docket No.: KOM-003PC / 138774-5003(SEQ ID NO: 45), NARGNRKRGRYR (SEQ ID NO: 46), LARGPRKRAGYK (SEQ ID NO: 47), RAWGKRKYAYYQ (SEQ ID NO: 48), KAWGSRKTRLYR (SEQ ID NO: 49), MARGGRKSAIYY (SEQ ID NO: 50), MASGSRKTAIYY (SEQ ID NO: 51), LARGRRKWARYR (SEQ ID NO: 52), and LARGSRKLALYR (SEQ ID NO: 53), relative to SEQ ID NO: 12, or a position corresponding thereto.

[0036] In embodiments, the one or more Bxb1 integrases comprise one or more mutations and / or deletions, and / or one or more combinations of mutations and / or deletions as described in Table 3 and / or Table 4.

[0037] In embodiments, the one or more Bxb1 integrases comprise at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations.

[0038] In embodiments, the one or more Bxb1 integrases comprise at least 90% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations.

[0039] In embodiments, the one or more Bxb1 integrases comprise at least 95% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or at least 20 mutations.

[0040] In embodiments, the one or more Bxb1 integrases comprise at least 96% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations,DBl / 164937876.2 9Atorney Docket No.: KOM-003PC / 138774-5003 at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or 20 mutations.

[0041] In embodiments, the one or more Bxb1 integrases comprise at least 97% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, or 15 mutations.

[0042] In embodiments, the one or more Bxb1 integrases comprise at least 98% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, or 10 mutations.

[0043] In embodiments, the one or more Bxb1 integrases comprise at least 99% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, or 5 mutations.

[0044] In embodiments, the Bxb1 integrase comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and one or more amino acid mutations at positions selected from L4, E42, R57, R63, V76, H111 , V122, V187, K313, R319, A341 , D359, A369, A398, R416, A425, L479, and M499, relative to SEQ ID NO: 12.

[0045] In embodiments, the Bxb1 integrase comprises one or more amino acid mutations are selected from L4I, E42K, R57K, R63K, V76I, H111 P, V122M, V187, K313R, R319K, A341D, D359A, A369P, A398S, R416K, A425T, L479I, and M499T relative to SEQ ID NO: 12.

[0046] In embodiments, the Bxb1 integrase further comprises one or more amino acid mutations selected from D36N, V40A, A49S, V74I, I87L or I87A or I87S, H89G, V175A, V179A, R287H, A288V, T453I, and H496N relative to SEQ ID NO: 12.

[0047] In embodiments, the Bxb1 integrase further comprises one or more amino acid mutations selected from V40I, D45G, I75V, H95Y, A119S, A280T, A311 V, E434G, and V466M relative to SEQ ID NO: 12.DBl / 164937876.2 10Atorney Docket No.: KOM-003PC / 138774-5003

[0048] In embodiments, one or more of the following amino acids relative to SEQ ID NO: 1 are not mutated: A62, H100, Q191, P195, G209, P295, L302, C307, L387, E419, S428, E483, and R487 relative to SEQ ID NO: 12.

[0049] In embodiments, the one or more amino acid mutations are selected from mutations at position V122 and / or position A369 relative to SEQ ID NO: 12. In embodiments, the mutation at position V122 is V122M. In embodiments, the mutation at position A369 relative to SEQ ID NO: 12 is A369P. In embodiments, the one or more amino acid mutations are selected from mutations at position V122 and position A369 relative to SEQ ID NO: 12. In embodiments, the one or more amino acid mutations are selected from V122M and A369P relative to SEQ ID NO: 12.

[0050] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation position combinations selected from: V76 and V122; V76 and A369; I87 and V122; I87 and A369; H95 and V122; H95 and A369; V122 and E434; A369 and E434; V76, V122, and A369; 187, H95 and A369; 187, V122 and E434; I87, A369 and E434; H95, V122 and A369; H95, V122 and E434; H95, A369 and E434; V122, A369 and E434; I87, H95, V122 and E434; I87, H95, A369 and E434; and I87, V122, A369 and E434 relative to SEQ ID NO: 12.

[0051] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation combinations selected from: V76I and V122M; V76I and A369P; I87L and V122M; I87L and A369P; H95Y and V122; H95Y and A369P; V122M and E434G; A369P and E434G; V76I, V122M, and A369P; I87L, H95Y and A369P; I87L, V122M and A369P; I87L, V122M and E434G; I87L, A369P and E434G; H95, V122M and A369P; H95Y, V122M and E434G; H95Y, A369P and E434G; V122, A369P and E434G; I87L, H95Y, V122M and E434G; I87L, H95Y, A369P and E434G; or I87L, V122, A369P and E434G relative to SEQ ID NO: 12.

[0052] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation position combinations selected from: I87, A369, and E434, V122, A369, and E434, H95, A369, and E434, V122 and E434, V122, A369, E434, and V175, V76 and A119, A119, A369, and E434, 187, V175, and D359, 187, R57, and R63, 187 and A369, 187 and A425, R63 and V122, R63, V122, R319, and V40I, R63, A119, and M499, V122 and A369, H95 and A369, A49, I87, A396, and E434, R57, R63, I87, A396, and E434, D36, V122, A396, and E434, R57, R63 and H95, D36, I87, A396, and E434, I87, A341 , A396, and E434, R57, R63, V122, A396, and E434, V122, R287, A396, and E434, 187, R287, A396, and E434, 187 and A341, 187, R287, A396, E434, and A341 , V122, A396, E434, and A341, H95, and A341 , A49, V122, A396, and E434, D45, A396, and E434, V122, R287, A341, A396, and E434, A49, A396, and E434, I87, A341 , and R287, D36,DBl / 164937876.2 11Atorney Docket No.: KOM-003PC / 138774-5003A396, and E434, V122, D359, A369, and E434, R63 and I87, 187, D359, A369, and E434, V122, A369, A425, and E434, 187, A369, A425, and E434, V40, H95, A369, and E434, L4, H95, A369, and E434, H95, A369, E434, and V466, R63, V122, A369, and E434, V76, 187, and K313, R63, 187, and N194, 187 and D359, L4 and I87, V74 and V76, V122, A369, E434, and R319, 187, A369, E434, and R319, L4, V122, A369, and E434, I87 and A425, E42, V76, and I87, 187 and L479, H95, A369, A425, and E434, R63, 187, A369, and E434, I87 and R319, V76, A369, and E434, H95, A369, E434, and L479, V74, 175, and V76, H95, R319, A369, and E434, L4, 187, A369, and E434, V122, V175, D359, A369, and E434, R57, 187, A369, and E434, E42, H95, A369, and E434, H95, V179, A369, and E434, 175 and V76, L4 and V76, H95 and K313, V40, R63, and H89, A288, A311, A398, R416, T453, H496, and E42, and H111 and E434 relative to SEQ ID NO: 12.

[0053] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation combinations selected from: I87L, A369P, and E434G, V122M, A369P, and E434G, H95Y, A369P, and E434G, I87L and E434G, V122M and E434G, V122M, A369P, E434G, and V175A, V76I and A119S, D36N and A119S, A119S, A369P, and E434G, I87L, V175A, and D359A, I87L, R57K, and R63K, I87L and D36N, I87L and A369P, I87L and A425T, R63K and V122M, R63K, V122M, R319K, and V40I, R63K, A119S, and M499T, H95Y and E434G, V122M and A369P, H95Y and V466M, H95Y and A369P, A49S, I87L, A396P, and E434G, A49S and I87L, R57K, R63K, I87L, A396P, and E434G, D36N, V122M, A396P, and E434G, R57K, R63K and H95Y, D36N, I87L, A396P, and E434G, D36N and H95Y, I87L, A341D, A396P, and E434G, A49S and H95Y, R57K, R63K, V122M, A396P, and E434G, V122M, R287H, A396P, and E434G, I87L and R287H, I87L, R287H, A396P, and E434G, I87L and A341D, I87L, R287H, A396P, E434G, and A341D, V122M, A396P, E434G, and A341D, H95Y and A341D, A49S, V122M, A396P, and E434G, D45G, A396P, and E434G, V122M, R287H, A341D, A396P, and E434G, A49S, A396P, and E434G, I87L, A341D, and R287H, H95Y and R287H, D36N, A396P, and E434G, V122M, D359A, A369P, and E434G, R63K and I87L, I87L and V466M, I87L, D359A, A369P, and E434G, V122M, A369P, A425T, and E434G, I87L, A369P, A425T, and E434G, V40I, H95Y, A369P, and E434G, L4I, H95Y, A369P, and E434G, H95Y, A369P, E434G, and V466M, R63K, V122M, A369P, and E434G, V76I, I87L, and K313R, R63K, I87V, and N194D, I87L and D359A, I87L and L4I V74I and V76I, V122M, A369P, E434G, and R319K, I87L, A369P, E434G, and R319K, L4I, V122M, A369P, and E434G, I87L and A425T, E42K, V76I, and I87L, I87L and L479I, H95Y, A369P, A425T, and E434G, V40I and I87L, R63K, I87L, A369P, and E434G, I87L and R319K, V76I, A369P, and E434G, H95Y, A369P, E434G, and L479I, V74I, I75V, and V76I, H95Y, R319K, A369P, and E434G, L4I, I87L, A369P, and E434G, V122M, V175A, D359A, A369P, and E434G, R57K, I87L, A369P, and E434G, E42K, H95Y, A369P, and E434G, H95Y, V179A, A369P, and E434G, I75V and V76I, I87L and V179A, L4IDBl / 164937876.2 12Atorney Docket No.: KOM-003PC / 138774-5003 and V76I, H95Y and K313R, H95Y and A280T, V40A, R63K, and H89G, A288V, A311V, A398S, R416K, T453I, H496N, and E42K, and H111 P and E434G relative to SEQ ID NO: 12.

[0054] In embodiments, the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12 and having the amino acid mutations I87L, A369P, and E434G relative to SEQ ID NO: 12. In embodiments, the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12 and having the amino acid mutations V122M, A369P, and E434G relative to SEQ ID NO: 12. In embodiments, the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and having the amino acid mutations H95Y, A369P, and E434G relative to SEQ ID NO: 12.

[0055] In embodiments, the one or more PhiC31 integrases comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 54, and one or more amino acid mutations at positions selected from 1 , 2, 12, 14, 18, 24, 32, 36, 41 , 43, 44, 45, 51 , 55, 74, 77, 96, 103, 107, 117, 153, 176, 188, 199, 200, 228, 230, 231 , 235, 238, 240, 252, 255, 259, 262, 264, 266, 269, 274, 278, 302, 320,322, 331, 333, 340, 344, 346, 347, 351 , 355, 359, 362, 364, 378, 382, 393, 396, 397, 399, 406, 410, 424,429, 431, 436, 438, 445, 448, 449, 450, 452, 457, 468, 475, 498, 501 , 505, 512, 516, 517, 520, 535, 536,549, 551 , 552, 563, 580, 585, 586, 587, 590, 592, 600, 603, 604, 609, 616, and 621 , relative to SEQ ID NO54, or a position corresponding thereto.

[0056] In embodiments, the one or more amino acid mutations is selected from M1 E, M1 V, D2V, D2M, S12N, E14G, S18N, D32A, D36A, V41 I, D44A, V51 M, 1153V, E176D, A199T, H228Y, P230S, F231 L, H240R, D252G, A255G, S269N, P274S, M278L, T302A, A333T, A333S, A333D, A340V, G344V, G344D, G346S, R347K, L351V, L351Q, D362N, D362G, L364M, E378K, S396N, S396R, A397T, N406S, A410T, G429S, E431V, W438R, W448R, E449D, A450D, E452K, E475D, G505V, G505S, A516T, P535L, T536A, A580V, K586Q, D590G, D592G, T600S, V603I, V603A, A604S, P609S, A616V, and R621 L relative to SEQ ID NO 54, or a position corresponding thereto.

[0057] In embodiments, the PhiC31 integrase comprises a N-terminal addition of one or more amino acids. In embodiments, the N-terminal addition comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids,DBl / 164937876.2 13Atorney Docket No.: KOM-003PC / 138774-5003 or about 50 amino acids relative to SEQ ID NO: 54. In embodiments, the N-terminal addition comprises a sequence selected from SEQ ID NOs: 55-67.

[0058] In embodiments, the one or more PhiC31 integrases comprise one or more mutations, N- terminal additions, and / or one or more combinations of mutations and / or N-terminal additions as described in Table 3, Table 6, and / or Table 7 relative to SEQ ID NO: 54.

[0059] In embodiments, the one or more PhiC31 integrases have at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations, e.g, as selected from Table 3, Table 6, and / or Table 7 relative to SEQ ID NO: 54.

[0060] In embodiments, the one or more PhiC31 integrases comprise at least 90% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations.

[0061] In embodiments, the one or more PhiC31 integrases comprise at least 95% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, or at least 30 mutations.

[0062] In embodiments, the one or more PhiC31 integrases comprise at least 96% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations.DBl / 164937876.2 14Atorney Docket No.: KOM-003PC / 138774-5003

[0063] In embodiments, the one or more PhiC31 integrases comprise at least 97% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, or at least 15 mutations.

[0064] In embodiments, the one or more PhiC31 integrases comprise at least 98% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, or 10 mutations.

[0065] In embodiments, the one or more PhiC31 integrases comprise at least 99% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, or at least 5 mutations.

[0066] In embodiments, the Bxb1 integrases and / or PhiC31 integrases herein comprise one or more nuclear localization sequences. In embodiments, the one or more nuclear localization sequences is fused at the N-terminus, at the C-terminus, at both the N-terminus and C-terminus, or within the integrase sequence. In embodiments, the one or more nuclear localization sequences comprises a sequence selected from PKKKRKV (SEQ ID NO: 68), NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 69); PAAKRVKLD (SEQ ID NO: 70), RQRRNELKRSF (SEQ ID NO: 71); NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 72), RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 73), VSRKRPRP (SEQ ID NO: 74), PPKKARED (SEQ ID NO: 75), PQPKKKPL (SEQ ID NO: 76), SAUKKKKKMAP (SEQ ID NO: 77), DRLRR (SEQ ID NO: 78), PKQKKRK (SEQ ID NO: 79), RKLKKKIKKL (SEQ ID NO: 80), REKKKFLKRR (SEQ ID NO: 81), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 82), RKCLQAGMNLEARKTKK (SEQ ID NO: 83), DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 84), KRTADGSEFESPKKKRKV (SEQ ID NO: 85),KRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86), GSHHHHHHGSGPKKKRKV (SEQ ID NO: 87), and GSGSGSHHHHHHGSGPKKKRKV (SEQ ID NO: 88). In embodiments, a NLS of the present disclosure comprises one or more mutations (e.g., amino acid substitutions, deletions, and / or insertions) with respect to any sequences herein.

[0067] In aspects, described herein are methods for generating a cell line comprising introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the singleDBl / 164937876.2 15Atorney Docket No.: KOM-003PC / 138774-5003 genomic locus, and wherein the one or more nucleic acids encodes one or more bacterial attachment (attB) sites and / or one or more phage attachment (attP) sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more integration sequences by one or more integrases, one or more inducible kill switch genes wherein the inducible kill switch gene functions to encode a protein which induces apoptosis in cells that do not receive an insertion sequence at the one or more attB sites and / or attP sites, one or more homology directed repair (HDR) insulator sequence compatible with the single genomic locus, wherein the one or more HDR insulator sequence flanks the one or more attB sites and / or attP sites, wherein the one or more nucleic acids does not encode an antibiotic resistance gene or auxotrophic selection gene, wherein the cell is not an auxotrophic variant relative to a cognate wild-type cell, and culturing a clonal cell population from the cell to generate the cell line.

[0068] In aspects, described herein are methods for generating a cell line for expressing one or more recombinant proteins comprising providing a clonal cell population produced by the methods herein, where the clonal cell population comprises one or more one or more bacterial attachment (attB} sites and / or one or more phage attachment (attP) sites, one or more inducible kill switch genes integrated into a single genomic locus at a single allele, one or more homology directed repair (HDR) insulator sequences compatible with the single genomic locus, wherein the one or more HDR insulator sequences flank the one or more attB sites and / or attP sites, the clonal cell population does not encode an antibiotic resistance gene or auxotrophic selection gene, and is not an auxotrophic variant relative to a cognate wild-type cell, introducing into the clonal cell population a first nucleic acid encoding one or more integrases suitable for site-specific integration at the one or more attB sites and / or one or more attP sites, wherein the cell is suitable to express the one or more integrases, and a second nucleic acid encoding one or more insertion sequences encoding the one or more recombinant proteins, wherein the one or more insertion sequences comprise one or more attB sites and / or one or more cognate attP sites that are suitable for site-specific integration at the one or more attB sites and / or one or more attP sites at the single genomic locus of the clonal cell population, integrating the one or more integration sequences into the single genomic locus using the one or more integrases, and inducing apoptosis via the one or more inducible kill switch genes in cells that do not integrate the one or more insertion sequences.

[0069] In embodiments, the one or more inducible kill switch genes (suicide gene) or molecular switch comprise one or more genes which can be induced or triggered for protein expression which results in cell death and / or apoptosis. In embodiments, the one or more inducible kill switch genes (suicide gene) or molecular switch functions as a selectable marker.DBl / 164937876.2 16Atorney Docket No.: KOM-003PC / 138774-5003

[0070] In embodiments, the one of more insertion sequences comprises a double-stranded DNA (dsDNA) molecule ranging in size from about 0.1 kb to about 50 kb or more. In embodiments, the dsDNA molecule is about or at least about 0.5 kb in length, about or at least about 1 kb in length, about or at least about 2 kb in length, about or at least about 3 kb in length, about or at least about 4 kb in length, about or at least about 5 kb in length, about or at least about 6 kb in length, about or at least about 7 kb in length, about or at least about 8 kb in length, about or at least about 9 kb in length, about or at least about 10 kb in length, about or at least about 11 kb in length, about or at least about 12 kb in length, about or at least about 13 kb in length, about or at least about 14 kb in length, about or at least about 15 kb in length, about or at least about 16 kb in length, about or at least about 17 kb in length, about or at least about 18 kb in length, about or at least about 19 kb in length, about or at least about 20 kb in length, about or at least about 30 kb, or about or at least about 50 kb.

[0071] In embodiments, the one or more insertion sequences encodes one or more of an expression cassette encoding a protein, optionally comprising one or more promoter sequence, enhancer sequence, internal ribosome entry site (IRES), and 3’ polyadenine (polyA) sequence.

[0072] In embodiments, the protein comprises a therapeutic protein, optionally an antibody or antibodyformat protein, therapeutic enzyme, fusion protein, secretory protein, protein hormone, and / or protein toxin or antitoxin.

[0073] In embodiments, methods herein comprise using one or more integrases to introduce at least two insertion sequences into a single genomic locus, optionally wherein the at least two insertion sequences are adjacent and / or operably linked within the genome of the cell. In embodiments, genomic insertion is monoallelic at a single loci. In embodiments, this comprises introducing two insertion sequences, three insertion sequences, four insertion sequences, or five or more insertion sequences. In embodiments, this comprises using two integrases, three integrases, four integrases, or five or more integrases, optionally wherein integration of each integration sequence comprises using a distinct integrase.

[0074] In aspects, described herein are cell lines produced by the methods herein.

[0075] In aspects, described herein are methods for generating a cell for expressing one or more recombinant proteins comprising providing a clonal cell population produced by the methods herein, and culturing the clonal cell population under conditions suitable for expression of the one or more recombinant proteins. In embodiments, the methods further comprise recovering the one or more recombinant proteins from the cell and / or the cell culture media.DBl / 164937876.2 17Atorney Docket No.: KOM-003PC / 138774-5003

[0076] In aspects, the present disclosure provides, in part, inducible hamster-specific caspase-9 fusion proteins and nucleic acid constructs thereof, as well as positive and negative cell selection methods using integrases (e.g., wild-type and / or mutant Bxb1).

[0077] In embodiments, the inducible hamster-specific caspase-9 fusion protein comprises an amino acid sequence having about or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 99. In embodiments, the inducible hamster-specific caspase-9 fusion protein comprises about or at least about 92% sequence identity, about or at least about 94% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 99. In embodiments, the inducible hamster-specific caspase-9 fusion protein comprises the amino acid sequence of SEQ ID NO: 99. In embodiments, the inducible hamsterspecific caspase-9 fusion protein consists of the amino acid sequence of SEQ ID NO: 99.

[0078] In embodiments, the inducible hamster-specific caspase-9 fusion protein comprises a hamster caspase-9 domain, a FKBP12 drug-binding domain, and an additional protein sequence or domain. In embodiments, the FKBP12 drug-binding domain is inducible by a small molecule to form a dimer(e.g., chemical inducer of dimerization (CID) or B / B Homodimerizer). In embodiments, the small molecule is or comprises AP20187, AP1903, rimiducid, or rapamycin. In embodiments, the dimerization results in the inducible hamster-specific caspase-9 becoming active and inducing apoptosis in a cell that expresses the inducible hamster-specific caspase-9 amino acid sequence and is exposed to the small molecule.

[0079] In aspects, described herein is a nucleic acid molecule comprising a polynucleotide sequence encoding the inducible hamster-specific caspase-9 fusion protein, as described herein.

[0080] In embodiments, the nucleic acid molecule comprises DNA or RNA. In embodiments, the DNA is circular or linear, or single stranded or double stranded. In embodiments, the DNA is a plasmid, viral vector, or non-viral vector.

[0081] In embodiments, the nucleic acid molecule further comprises a promoter sequence operably linked to the polynucleotide sequence. In embodiments, the promoter sequence is or comprises one or more of an inducible promoter, mammalian gene promoter, viral promoter, tissue-specific promoter, cell typespecific promoter, and bidirectional promoter. In embodiments, the inducible promoter comprises one or more of a Tet-On / Tet-Off, Cumate, Ecdysone, Mifepristone (GeneSwitch), Rapamycin (FKBP / FRB), RU486, Heat Shock, Doxycycline, Copper-inducible, and small mineral-based systems such as Zinc-inducible promoters.DBl / 164937876.2 18Atorney Docket No.: KOM-003PC / 138774-5003In embodiments, the promoter sequence comprises one or more synthetic promoter or composite promoter (e.g., combining elements of two or more different promoters). In embodiments, the promoter sequence is a native promoter (e.g., native mammalian gene promoter, viral promoter, tissue-specific promoter, or cell typespecific promoter). In embodiments, the promoter is or comprises one or more, as described herein.

[0082] In embodiments, the promoter is a CMV promoter that comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 100. In embodiments, the CMV promoter is or comprises the nucleic acid sequence of SEQ ID NO: 100.

[0083] In embodiments, the nucleic acid comprises two or more att sequences. In embodiments, the two or more att sequences comprises bacterial attachment (attB) sequences, or phage attachment (attP) sequences. In embodiments, the two or more att sequences flank the polynucleotide sequence encoding the inducible hamster-specific caspase-9 fusion protein.

[0084] In embodiments, the one or more bacterial attachment (attB) sequences comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95. In embodiments, the one or more bacterial attachment (attB) sequences is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95. In embodiments, the one or more bacterial attachment (attB) sequences is compatible with one or more phage attachment (attP) sequences comprising about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97.DBl / 164937876.2 19Atorney Docket No.: KOM-003PC / 138774-5003

[0085] In embodiments, the one or more phage attachment (attP) sequences comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97. In embodiments, the one or more phage attachment (attP) sequences is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97. In embodiments, the one or more phage attachment (attP) sequences is compatible with one or more bacterial attachment (attB) sequences comprising about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95.

[0086] In embodiments, the nucleic acid further comprises one or more homology directed repair (HDR) insulator sequences, one or more splice acceptor sequences, one or more splice donor sequences, one or more nucleic acid sequences encoding a 2A peptide, one or more insertion sequences, and / or one or more transcription terminator sequences.

[0087] In embodiments, the one or more 2A peptide comprises one or more of a T2A, P2A, E2A, and F2A peptide sequence. In embodiments, the one or more 2A peptide sequences is or comprises one or more amino acid sequence of SEQ ID NOs: 7-10. In embodiments, the one or more 2A peptide sequences is or comprises one or more amino acid sequence having the consensus sequence of SEQ ID NO: 11. In embodiments, the one or more 2A peptide sequences is located between two or more protein-coding sequences. In embodiments, one or more 2A peptide sequences is located between the inducible hamsterspecific caspase-9 amino acid sequence and one or more protein-coding sequences (e.g., for a protein of interest (POI)).

[0088] In embodiments, the one or more transcription terminator sequences is or comprises one or more polyA sequences / signals (e.g., polyA tail). In embodiments, the one or more transcription terminator sequences is or comprises the consensus sequence aauaaa.DBl / 164937876.2 20Atorney Docket No.: KOM-003PC / 138774-5003

[0089] In embodiments, the one or more transcription terminator sequences is or comprises a transcription terminator sequence derived from a nucleic acid sequence of rabbit beta globin (rBG), simian vacuolating virus 40 (SV40), human growth hormone (hGH), or bovine growth hormone (BGH).

[0090] In embodiments, the one or more transcription terminator sequences is or comprises a rabbit beta globin sequence (rBG). In embodiments, the rabbit beta globin sequence (rBG) comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101. In embodiments, the rabbit beta globin sequence (rBG) is or comprises the nucleic acid sequence of SEQ ID NO: 101.

[0091] In embodiments, the one or more splice acceptor sequences comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 102. In embodiments, the one or more splice acceptor sequences is or comprises the nucleic acid sequence of SEQ ID NO: 102.

[0092] In aspects, described herein is a “donor DNA-specific” nucleic acid molecule comprising a first nucleic acid sequence encoding a promoter, and a second nucleic acid sequence encoding at least one start codon, at least one splice donor or splice acceptor sequence, and at least one att sequence, where the promoter is operably linked to drive expression from the start codon and the start codon is not linked to a protein-coding sequence.

[0093] In embodiments, the at least one aft sequence is compatible with a wild-type or mutant Bxb1.

[0094] In embodiments, the promoter sequence is or comprises one or more of an inducible promoter, mammalian gene promoter, viral promoter, tissue-specific promoter, cell type-specific promoter, and bidirectional promoter. In embodiments, the inducible promoter comprises one or more of a Tet-On / Tet-Off, Cumate, Ecdysone, Mifepristone (GeneSwitch), Rapamycin (FKBP / FRB), RU486, Heat Shock, Doxycycline, Copper-inducible, and small mineral-based systems such as Zinc-inducible promoters. In embodiments, the promoter sequence comprises one or more synthetic promoter or composite promoter (e.g., combiningDBl / 164937876.2 21Atorney Docket No.: KOM-003PC / 138774-5003 elements of two or more different promoters). In embodiments, the promoter sequence is a native promoter (e.g., native mammalian gene promoter, viral promoter, tissue-specific promoter, or cell type-specific promoter). In embodiments, the promoter is or comprises one or more, as described herein.

[0095] In embodiments, the CMV promoter comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 100. In embodiments, the CMV promoter is or comprises the nucleic acid sequence of SEQ ID NO: 100.

[0096] In embodiments, the first nucleic acid sequence is upstream (5’) of the second nucleic acid sequence and located substantially adjacent such that the CMV promoter sequence is suitable to drive expression from the start codon in the second nucleic acid sequence. In embodiments, the end of the promoter sequence is within about 1000 bp of the start codon.

[0097] In embodiments, the at least one att sequence is a bacterial attachment (attB) sequence. In embodiments, the attB sequence comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95, or wherein the attB sequence is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, and 89-95; and / or wherein the attB sequence is compatible with one or more phage attachment (attP) sequences having about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence one of any one of SEQ ID NOs: 2, 4, 6, and 96-97.

[0098] In embodiments, the at least one att sequence is a phage attachment (attP) sequence. In embodiments, the attP sequence comprises about or at least about 70% sequence identity, about or at leastDBl / 164937876.2 22Atorney Docket No.: KOM-003PC / 138774-5003 about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97, or wherein the attP sequence is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97; and / or wherein the attP sequence is compatible with one or more bacterial attachment (attB) sequences having about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, and 89-95.

[0099] In embodiments, the attB sequence or the attP sequence has an ac core dinucleotide sequence.

[0100] In embodiments, the donor DNA sequence comprises at least one attB sequence and at least one splice donor sequence. In embodiments, the at least one splice donor or splice acceptor sequence comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 102. In embodiments, the at least one splice donor or splice acceptor sequence is or comprises the nucleic acid sequence of SEQ ID NO: 102.

[0101] In embodiments, the second nucleic acid sequence comprises about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, about or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 105. In embodiments, the second nucleic acid sequence is or comprises the nucleic acid sequence of SEQ ID NO: 105.DBl / 164937876.2 23Atorney Docket No.: KOM-003PC / 138774-5003

[0102] In aspects, described herein is a “landing pad” nucleic acid molecule comprising an aft sequence, at least one splice acceptor or splice donor sequence, a protein-coding nucleic acid sequence, wherein the protein-coding sequence lacks a start codon, encodes a selectable marker protein, and is located downstream of at least one splice acceptor or splice donor sequence and the att sequence, and a transcriptional terminator sequence downstream of the protein-coding nucleic acid sequence.

[0103] In embodiments, the selectable marker protein is an antibiotic resistance enzyme. In embodiments, the antibiotic resistance enzyme is for detoxifying an antibiotic that is toxic to eukaryotic cells. In embodiments, the antibiotic resistance enzyme is or comprises an enzyme for detoxifying hygromycin B, geneticin (G418), gentamicin, puromycin, blasticidin, neomycin, amphotericin B, actinomycin D, zeocin (bleomycin), mycophenolic acid.

[0104] In embodiments, the antibiotic resistance enzyme is hygromycin B phosphotransferase. In embodiments, the hygromycin B phosphotransferase comprises about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 104, or wherein the Hygromycin B phosphotransferase is or comprises the amino acid sequence of SEQ ID NO: 104.

[0105] In embodiments, the protein-coding nucleic acid sequence encodes Hygromycin B phosphotransferase and comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 103, or is or comprises the nucleic acid sequence of SEQ ID NO: 103.

[0106] In embodiments, the transcriptional terminator sequence terminates transcription of the proteincoding nucleic acid sequence encoding a selectable marker protein. In embodiments, the one or more transcription terminator sequences is or comprises one or more polyA sequences / signals (e.g., polyA tail), optionally wherein the one or more transcription terminator sequences is or comprises the consensus sequence aauaaa.DBl / 164937876.2 24Atorney Docket No.: KOM-003PC / 138774-5003

[0107] In embodiments, the one or more transcription terminator sequences is or comprises a transcription terminator sequence derived from a nucleic acid sequence of rabbit beta globin (rBG), simian vacuolating virus 40 (SV40), human growth hormone (hGH), or bovine growth hormone (BGH).

[0108] In embodiments, the one or more transcription terminator sequences is or comprises a rabbit beta globin sequence (rBG). In embodiments, the rabbit beta globin sequence (rBG) comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101. In embodiments, the rabbit beta globin sequence (rBG) is or comprises the nucleic acid sequence of SEQ ID NO: 101.

[0109] In embodiments, the aft sequence is compatible with a wild-type or mutant Bxb1.

[0110] In embodiments, the at least one att sequence is a bacterial attachment (attB) sequence. In embodiments, the attB sequence comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, and 89-95, or wherein the attB sequence is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, and 89-95; and / or wherein the attB sequence is compatible with one or more phage attachment (affP) sequences comprising about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97.

[0111] In embodiments, the at least one att sequence is a phage attachment (affP) sequence. In embodiments, the attP sequence comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequenceDBl / 164937876.2 25Atorney Docket No.: KOM-003PC / 138774-5003 identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97, or wherein the attP sequence is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97; and / or wherein the attP sequence is compatible with one or more bacterial attachment (attB) sequences comprising about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, and 89-95.

[0112] In embodiments, the attB sequence or the attP sequence has an accore dinucleotide sequence.

[0113] In embodiments, the landing pad sequence comprises at least one splice acceptor sequence. In embodiments, the at least one splice donor or splice acceptor sequence comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 102. In embodiments, the at least one splice donor or splice acceptor sequence is or comprises the nucleic acid sequence of SEQ ID NO: 102.

[0114] In aspects, described herein isacell comprising one or more genomically-integrated nucleic acid molecules encoding the inducible hamster-specific caspase-9 fusion protein.

[0115] In aspects, described herein is a cell comprising one or more genomically-integrated landing pad nucleic acid molecules.

[0116] In aspects, described herein is a method for negative cell selection comprising providing a population of cells comprising one or more genomically-integrated nucleic acid molecules encoding the inducible hamster-specific caspase-9 fusion protein, introducing into the population of cells an exogenous nucleic acid molecule comprising two or more att sequences compatible with a Bxb1 recombinase flanking an intervening DNA sequence, culturing the population of cells under conditions suitable for Bxb1 recombinase-mediated cassette exchange between a genomically-integrated inducible hamster-specific caspase-9 fusion protein coding sequence and the two or more att sequences compatible with a Bxb1DBl / 164937876.2 26Atorney Docket No.: KOM-003PC / 138774-5003 recombinase flanking an intervening DNA sequence, such that the inducible hamster-specific caspase-9 fusion protein coding sequence is replaced by the intervening DNA sequence, and inducing apoptosis in cells that did not undergo the Bxb1 recombinase-mediated cassette exchange based on expression of the inducible hamster-specific caspase-9 fusion protein.

[0117] In embodiments, inducing apoptosis comprises contacting the population of cells with a small molecule. In embodiments, the FKBP12 drug-binding domain is inducible by a small molecule to form a dimer, such as a chemical inducer of dimerization (CID) or B / B Homodimerizer, as known in the art. For example, in embodiments, the small molecule is selected from AP20187, AP1903, rimiducid, or rapamycin.

[0118] In embodiments, the method comprises culturing only the cells that underwent Bxb1 recombinase-mediated cassette exchange.

[0119] In embodiments, the population of cells comprises eukaryotic cells or prokaryotic cells. In embodiments, the cell is suitable for recombinant protein production. In embodiments, the eukaryotic cells comprise mammalian cells, plant cells, yeast cells, or insect cells. In embodiments, the mammalian cells comprise Chinese hamster ovary (CHO) cells (CHO-K1 , CHO-DHB11, CHO-DXB1 , CHO-S, CHO-DG44, CHO-M), human embryonic kidney cells (HEK293, HEK293T), K562 human lymphoblast cells, U2OS human osteosarcoma cells, primary human fibroblasts (human dermal fibroblast (HDFa)), baby hamster kidney (BHK) cells, Vero cells (Vero, Vero 76, Vero E6), human cervical carcinoma cells (HELA, 3T3), PERc6 cells, CAP cells, iPSCs, human embryonic stem cells (ESCs), or monkey kidney CV1 cells.

[0120] In embodiments, the introducing step further comprises using one or more of a lipid-based transfection reagent (cationic lipid-based reagent), diethylaminoethyl (DEAE)-dextran, liposome, electroporation, sonoporation, chemical reagent (calcium phosphate), microinjection, or via a non-integrating episome or viral vector (e.g., AAV, lentivirus).

[0121] In aspects, described herein is a method for positive cell selection comprising providing a population of cells comprising one or more genomically-integrated landing pad nucleic acid molecules, introducing into the population of cells a donor DNA-specific nucleic acid molecule, as described herein, culturing the population of cells under conditions suitable for Bxb1 recombinase-mediated cassette exchange, such that a promoter and start codon from the donor DNA-specific nucleic acid molecule is now operatively linked to a genomically-integrated protein-coding sequence to drive expression of the proteincoding sequence, and selecting for the cells that underwent Bxb1 recombinase-mediated cassette exchange based on expression of the protein-coding sequence.DBl / 164937876.2 27Atorney Docket No.: KOM-003PC / 138774-5003

[0122] In embodiments, upon insertion of the donor DNA-specific nucleic acid molecule an intronic sequence is formed comprising at least one splice acceptor or donor of a landing pad nucleic acid molecule and at least one splice acceptor or donor of the donor DNA-specific nucleic acid molecule, and an intervening recombined att sequence (e.g., attR). In embodiments, the start codon from the donor DNA-specific nucleic acid molecule and the genomically-integrated protein-coding sequence are configured to be expressed from the promoter.

[0123] In embodiments, the selecting further comprises contacting the cells with an antibiotic, in embodiments, the antibiotic is selected from hygromycin B, geneticin (G418), gentamicin, puromycin, blasticidin, neomycin, amphotericin B, actinomycin D, zeocin (bleomycin), and mycophenolic acid.

[0124] In embodiments, the method comprises culturing only the cells that underwent Bxb1 recombinase-mediated cassette exchange.

[0125] In embodiments, the population of cells comprises eukaryotic cells or prokaryotic cells. In embodiments, the cell is suitable for recombinant protein production. In embodiments, the eukaryotic cells comprise mammalian cells, plant cells, yeast cells, or insect cells. In embodiments, the mammalian cells comprise Chinese hamster ovary (CHO) cells (CHO-K1, CHO-DHB11 , CHO-DXB1 , CHO-S, CHO-DG44, CHO-M), human embryonic kidney cells (HEK293, HEK293T), K562 human lymphoblast cells, U2OS human osteosarcoma cells, primary human fibroblasts (human dermal fibroblast (HDFa)), baby hamster kidney (BHK) cells, Vero cells (Vero, Vero 76, Vero E6), human cervical carcinoma cells (HELA, 3T3), PERc6 cells, CAP cells, iPSCs, human embryonic stem cells (ESCs), or monkey kidney CV1 cells.

[0126] In embodiments, the introducing step further comprises using one or more of a lipid-based transfection reagent (cationic lipid-based reagent), diethylaminoethyl (DEAE)-dextran, liposome, electroporation, sonoporation, chemical reagent (calcium phosphate), microinjection, or via a non-integrating episome or viral vector (e.g., AAV, lentivirus).

[0127] In embodiments, the introducing step of methods herein further comprises introducing one or more nucleic acid sequence that encodes a wild-type or one or more mutant Bxb1 integrases for Bxb1 recombinase-mediated cassette exchange. In embodiments, the Bxb1 used in the methods described herein comprises one or more Bxb1 integrases as described herein (e.g., wild-type and / or mutant). In embodiments, mutant Bxb1 integrases herein comprise one or more non-naturally-occurring mutants or combination of mutants. In embodiments, mutant Bxb1 integrases herein comprise one or more nuclear localizationDBl / 164937876.2 28Atorney Docket No.: KOM-003PC / 138774-5003 sequence that improve nuclear retention and targeting to the nucleus. In embodiments, mutant Bxb1 integrases herein comprise one or more stabilization sequences.

[0128] In aspects, described herein are cell lines or clonal cell populations produced by the methods herein. In embodiments, the methods are performed as substantially depicted in Fig. 9. In embodiments, the methods are performed as substantially depicted in Fig. 10.DESCRIPTION OF THE DRAWINGS

[0129] Fig. 1 depicts a non-limiting illustrative graphical representation of Bxb1-mediated genomic integration. One or more attB site is introduced into the genome of a host cell. Then, Bxb1 -mediated integration occurs between the attB and one or more attP in an insertion plasmid carrying one or more insertion sequence. The resultant reaction results in an integrated insertion sequence, flanked by attL and attR sites.

[0130] Fig. 2 depicts a non-limiting illustrative graphical representation of multiplexed gene stacking or the ability to introduce one or more insertion sequences adjacent to one or more previously integrated insertion sequences within the same genomic location by one or more integrases.

[0131] Fig. 3 depicts a graphical representation of site-specific genomic integration of a 6.6 kbp donor DNA sequence using wild-type Bxb 1 compared against mutant Bxb1 at the TRAC locus. Percent integration was measured using ddPCR.

[0132] Fig. 4 depicts a graphical representation of site-specific genomic integration of a 6.6 kbp donor DNA sequence at the TRAC locus using wild-type Bxb1 and select Bxb1 mutants compared against cognate recombinases with nuclear localization sequence (NLS). Percent integration was measured using ddPCR.

[0133] Fig. 5 depicts a graphical representation of Cas9-directed reverse transcription and site-specific genomic integration of a 6.6 kbp donor DNA sequence at the ROSA26 locus in HEK293T cells using wildtype Bxb1 and select Bxb1 mutants compared against cognate recombinases with nuclear localization sequence (NLS). Percent integration was measured using ddPCR.

[0134] Fig. 6 depicts a graphical representation of dual Cas9-directed reverse transcription and sitespecific genomic integration of a 6.6 kbp donor DNA sequence using C-terminal NLSs with wild-type Bxb1 and select Bxb1 mutants. Percent integration was measured using ddPCR.DBl / 164937876.2 29Atorney Docket No.: KOM-003PC / 138774-5003

[0135] Fig. 7 depicts a graphical representation of Cas9-directed reverse transcription and site-specific genomic integration of a 6.6 kbp donor DNA sequence using with wild-type Bxb1 and select Bxb1 mutants compared against cognate recombinases with NLSs. Percent integration was measured using ddPCR.

[0136] Fig. 8 depicts a graphical representation of Cas9-directed reverse transcription and site-specific genomic integration of a 6.6 kbp donor DNA sequence using with wild-type Bxb1 and select Bxb1 mutants compared against cognate recombinases with NLSs. Percent integration was measured using ddPCR.

[0137] Fig. 9 depicts a non-limiting illustrative graphical representation of a negative selection process using a genomically-integrated inducible hamster-specific caspase-9 fusion protein gene (2) flanked by Bxb1 att sites (1) and Bxb1-mediated recombination to replace the hamster-specific caspase-9 fusion protein gene with a gene of interest (GOI) (4) flanked by Bxb1 att sites (3) that results in a genome-inserted GOI flanked by new att sites (5), resulting in inducible apoptosis in cells that did not undergo Bx b 1 -mediated recombination (e.g., selecting against cells having the gene).

[0138] Fig. 10 depicts a non-limiting illustrative graphical representation of a positive selection process using a genomically-integrated selectable marker lacking a functional promoter and start codon and Bxb1- mediated recombination to replace an upstream portion with a functional promoter and start codon, resulting in operably linked promoter and start codon such that expression of the selectable marker occurs (e.g., selecting for cells that have the functional gene).

[0139] Fig. 11 depicts a non-limiting diagrammatic representation of plasmid design for site-specific genomic insertion using mutant integrases herein for manufacturing cell lines. The Donorl plasmid cell line incorporates two attB sites recognized by a Bxb1 integrase. Between the two attB sites, a cassette coexpresses green fluorescence protein (GFP) and the herpes simplex virus (HSV) thymidine kinase (TK) suicide gene, separated by a T2A peptide sequence. GFP is used to enrich cells with on-target genomic insertion and to verify that expression levels from the insertion site remain consistent over time. TK enables the selection of on-target knock-in of the gene-of-interest (GOI). An on-target knock-in replaces this cassette with the GOI (e.g., an antibody with a heavy and light chain portion is shown as a non-limiting example). Through ganciclovir treatment, parental cells and those with random insertion, both containing TK, are eliminated leaving only the cells with the correct insertion.

[0140] Fig. 12 depicts a non-limiting diagrammatic representation of a knock-in strategy for making cell lines herein. The suicide gene is first inserted to the desired locus in the genome. Next, the integrase replaces the suicide gene with the gene-of-interest (GOI). There are three possible cell populations that can result:DBl / 164937876.2 30Atorney Docket No.: KOM-003PC / 138774-5003 those with successful, site-specific knock-in, those with failed knock-in, and those with random insertion. Selection kills the cells containing suicide gene (the latter two groups), leaving only cells with site-specific genomic knock-in of the GOI.

[0141] Figs. 13A-13B depict a representative gel electrophoresis (Fig. 13A) and flow cytometry (Fig. 13B) showing CRISPR HDR was successfully used to insert GFP and a thymidine kinase (TK) suicide gene flanked by att sites at the S100A locus in CHO cells. Clones were generated that contained the intended insert which was verified by PCR (Fig. 13A) showing products that contained the junction of the insert and genome. The clonally expanded cells were >99% GFP positive as measured by flow cytometry (Fig. 13B). Control cells without the HDR insertion did not produce PCR products (middle two lanes of Fig. 13A) or express GFP (“neg control” 0% in Fig. 13B).

[0142] Figs. 14A-14C depict representative flow cytometry data (Figs. 14A and 14C) and an illustrative diagrammatic schematic (Fig. 14B) which show site-specific integrase-mediated insertion of a GOI with concomitant removal of GFP and suicide gene expression cassette. Fig. 14A shows GFP expression by flow cytometry which confirmed stable site-specific genomic integration of GFP and the suicide gene in CHO cells. Fig. 14B depicts that at GOI (-14 kb) was used in combination with the mutant Bxb1 described herein to knock-in the GOI where the GFP / suicide gene is present. This results in a negative selection procedure that removes all cells that failed to knock-in the GOI at the GFP / suicide gene location, as shown in Fig. 14C.

[0143] Fig. 15 depicts a graphical representation of stable production of an antibody (trastuzymab) in a Bxb1 -engineered cell line. The cell line was able to achieve approximately 0.7 g / L yield at about 1 week growth, as measured by enzyme-linked immunosorbent assay (ELISA). Data are mean ± standard deviation (SD).

[0144] Figs. 16A-16D depict representative flow cytometry data (Figs. 16A and 16C) and quantitative analyses of the same (Figs. 16B and 16D) which show that an inducible hamster-specific caspase-9 fusion protein works well for cell selection procedures.DETAILED DESCRIPTION

[0145] The present disclosure provides, in part, methods of cell line development for stable, genomic integration of sequences, e.g., for therapeutic protein expression. In embodiments, methods herein enable the ability to integrate large transgenes to a single genomic locus with high efficiency. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks and / or antibiotic selectionDBl / 164937876.2 31Atorney Docket No.: KOM-003PC / 138774-5003 or cellular auxotrophy. In embodiments, methods described herein facilitate the integration of large insertion sequence cargos at attachment (att) sites. In embodiments, targeting to aft sites in the genome utilize Cas9- directed reverse transcription or zinc finger nucleases (ZFNs), which are integrated at “safe harbor” loci. In embodiments, methods of insertion herein avoids double-strand breaks, which would ordinarily trigger DNA damage pathways (e.g., p53).

[0146] In embodiments, the insertion sequences are single or multi-gene cargos. In embodiments, the integration rates are as high as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of target chromosomes in host cells. In embodiments, present methods permit insertion of one or more insertion sequences ranging in size from about 0.1 kb to about 20 kb or more (e.g., about 0.5 kb, about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, and larger).

[0147] In embodiments, the methods and compositions described accelerate therapeutic protein production.

[0148] In embodiments, integrases described herein find use various methods including, without limitation, (i) site-specific integration of insertion sequences into the genomes of cell lines for industrial biotechnology, the production of pharmaceuticals, and other expression of proteins; (ii) introduction of desirable traits into host cells, such as disease resistance, enhanced nutritional content, or to confer increased functionality; (iii) insertion of disease-related genes into genomes of animal models to study the mechanisms of various disorders and / or test therapeutic interventions; (iv) integration of genetic modules into synthetic genomes, enabling the creation of biological memory devices or genetic logic gates; (v) attachment of epitope tags or fusion proteins to study gene regulation and localization in situ and / or in vivo, and / or (vi) delivering therapeutic genes into the genomes of patients' cells.

[0149] In embodiments, methods herein include two stages, where the first stage includes generating a clonal cell population which comprises at a single genomic locus (monoallelic insertion) one or more attB and / or attP sequences flanked by HDR insulator sequences. In embodiments, this clonal cell population does not require or use antibiotic or auxotrophic (e.g., glutamine synthetase (GS)) selection pressure. In embodiments, this clonal cell population includes at the single genomic locus one or more inducible “kill switch” gene, or “suicide” gene, which refers to a coding sequence that encodes a protein that is configured to induce cell death in any cells of the clonal cell population which do not receive an insertion sequence inDBl / 164937876.2 32Atorney Docket No.: KOM-003PC / 138774-5003 the second stage, thereby eliminating any cells that lack the genetic amendment and eliminating any cells which underwent random integration (e.g., integration outside the intended single genomic locus). In embodiments, the terms “kill switch” or “suicide” in reference to a gene or protein is interchangeable to which refers to a coding sequence that encodes a protein that is configured to induce apoptosis.

[0150] In embodiments, methods herein include a second stage which includes using the clonal cell population generated by the first stage and integrating one or more insertion sequences within the att site using one or more integrases. In embodiments, the integration of the one or more insertion sequences is performed using a new promoter to prevent any potential silencing during biologies production. In embodiments, this new promoter functions as a “molecular switch,” which is used to control expression of one or more proteins (e.g., confine expression to only during production when the cells are at high density), prevent the burden and toxicity of the high protein production during the early cell growth, and prevent potential promoter silencing due to short-term periods of protein expression.

[0151] In embodiments, methods herein enable the use of “gene stacking,” or the ability to introduce a new insertion sequence next to a previous insertion sequence within the same genomic loci by different integrases (e.g., 1, 2, 3, 4, 5, or 6 or more integrases). In embodiments, this enables increased insertion size within the same locus to generate multi-specific products and new complex genomic engineering that is not possible by the current technologies.

[0152] In embodiments, described herein is an adaptation of a method of Cas9-directed reverse transcription established as a tool for precise targeting of short sequences to desired loci with advantages over HDR including high efficiency in both dividing and non-dividing cells as well as a reduction in DSBs. In embodiments, Cas9-directed reverse transcription uses a Cas9 nickase that targets the DNA using a Cas9- directed reverse transcription guide RNA (pegRNA) that encodes a reverse transcriptase template (RTT) which is “written” into the target site by a reverse transcriptase (RT). In embodiments, Cas9-directed reverse transcription is used for DNA substitutions, short insertions, and deletions. In embodiments, methods herein utilize several recent improvements to Cas9-directed reverse transcription, including pegRNA engineering, optimized Cas9-directed reverse transcription architecture, and twin Cas9-directed reverse transcription have enabled the insertion of sequences >50 bp at high efficiencies.Methods of Generating Cell Lines Enabled for Genomic Integration and / or Protein Expression

[0153] In embodiments, described herein are methods of generating cell lines comprising introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the singleDBl / 164937876.2 33Atorney Docket No.: KOM-003PC / 138774-5003 genomic locus, and wherein the one or more nucleic acids encodes one or more bacterial attachment (attB sites and / or one or more phage attachment (attP) sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more integration sequences by one or more integrases, and culturing a clonal cell population from the cell to generate the cell line.

[0154] In embodiments, described herein are methods of generating cell lines comprising introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the single genomic locus, and wherein the one or more nucleic acids encodes one or more bacterial attachment (attB) sites and / or one or more phage attachment (attP) sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more integration sequences by one or more integrases, encodes one or more inducible kill switch genes wherein the inducible kill switch gene functions to encode a protein which induces apoptosis in cells that do not receive an insertion sequence at the one or more attB sites and / or attP sites, and encodes one or more homology directed repair (HDR) insulator sequence compatible with the single genomic locus, wherein the one or more HDR insulator sequence flanks the one or more attB sites and / or attP sites, where the one or more nucleic acids does not encode an antibiotic resistance gene or auxotrophic selection gene, and where the cell is not an auxotrophic variant relative to a cognate wild-type cell, and culturing a clonal cell population from the cell to generate the cell line.

[0155] In embodiments, described herein are methods of generating a cell line comprising introducing one or more nucleic acids at a single genomic locus in a cell, where the insertion is monoallelic at the single genomic locus, and where the one or more nucleic acids encodes: (i) one or more attB sites and / or (ii) one or more attP sites, and where the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more insertion sequences by one or more integrases, where the one or more integrases comprises one or more mutant Bxb1 integrases having one or more mutations relative to SEQ ID NO: 12. In embodiments, the method further comprises and culturing a cell population from the cell to generate the cell line.

[0156] In embodiments, described herein are methods of generating a cell line comprising (a) introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the single genomic locus, and wherein the one or more nucleic acids encodes: (i) one or more attB sites and / or one or more attP sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more insertion sequences by one or more integrases, (ii) oneDBl / 164937876.2 34Atorney Docket No.: KOM-003PC / 138774-5003 or more inducible kill switch genes wherein the one or more inducible kill switch genes encodes a protein which induces apoptosis in cells that do not receive an insertion sequence at the one or more attB sites and / or attP sites, (iii) one or more homology directed repair (HDR) insulator sequences compatible with the single genomic locus, wherein the one or more HDR insulator sequences flanks the one or more attB sites and / or attP sites, where the one or more nucleic acids does not encode an antibiotic resistance gene or auxotrophic selection gene, and where the cell is not an auxotrophic variant relative to a cognate wild-type cell. In embodiments, the method further comprises culturing a cell population from the cell to generate the cell line. In embodiments, the one or more integrases comprises one or more mutant Bxb1 integrases having one or more mutations relative to SEQ ID NO: 12.

[0157] In embodiments, described herein are methods of generating a cell line comprising (a) introducing one or more nucleic acids into a population of cells at a single genomic locus, where the one or more nucleic acids comprises two or more attB sites and / or attP sites flanking an intervening DNA sequence, where the two or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration with one or more integrases, and where the population of cells comprises a genomically-integrated nucleic acid molecule encoding one or more inducible kill switch gene, wherein the one or more inducible kill switch comprises an inducible hamster-specific caspase-9 fusion protein having about or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 99, (b) culturing the population of cells under conditions suitable for recombinase-mediated cassette exchange by the one or more integrases between the genomically-integrated nucleic acid molecule encoding the inducible hamster-specific caspase-9 fusion protein and the two or more attB sites and / or attP sites of the one or more nucleic acids, such that the inducible hamster-specific caspase-9 fusion protein coding sequence is replaced by the intervening DNA sequence, and (c) inducing apoptosis in cells that did not undergo the recombinase-mediated cassette exchange based on expression of the inducible hamster-specific caspase-9 fusion protein to generate the cell line.

[0158] In embodiments, described herein are methods of positive cell selection to generate a cell line comprising: (a) introducing a donor DNA-specific nucleic acid molecule into a population of cells, each comprising a genomically-integrated landing pad nucleic acid molecule at a single genomic locus, (i) where the donor DNA-specific nucleic acid molecule comprises: a first nucleic acid sequence encoding a promoter, and a second nucleic acid sequence encoding at least one start codon, at least one splice donor or splice acceptor, and one or more attB sites and / or attP sites, where the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration with one or more integrases, and where theDBl / 164937876.2 35Atorney Docket No.: KOM-003PC / 138774-5003 promoter is operably linked to drive expression from the start codon and the start codon is not linked to a protein-coding sequence, and (ii) wherein the genomically-integrated landing pad nucleic acid molecule comprises: one or more attB sites and / or attP sites, wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration with the one or more integrases, at least one splice donor or splice acceptor sequence, a protein-coding nucleic acid sequence, where the protein-coding sequence lacks a start codon, encodes a selectable marker protein, and is located downstream (3’) of the at least one splice donor or splice acceptor sequence and the att sequence, and a transcriptional terminator sequence downstream of the protein-coding nucleic acid sequence, (c) culturing the population of cells under conditions suitable for recombinase-mediated cassette exchange by the one or more integrases, such that the promoter and the at least one start codon from the donor DNA-specific nucleic acid molecule becomes operatively linked to the protein-coding sequence of the genomically-integrated landing pad nucleic acid molecule to drive expression of the protein-coding sequence, and (d) selecting for the cells that underwent recombinase-mediated cassette exchange based on expression of the protein-coding sequence to generate the cell line.

[0159] In embodiments, methods herein utilize mutant integrases which have altered functionality relative to cognate wild-type integrases, e.g., as described herein. In embodiments, the integrase (e.g., sometimes referred to as a recombinase, or phage integrase) is bacterial, e.g., such as Pa01 (Pseudomonas). In embodiments, the integrase is a serine integrase. In embodiments, the serine integrase is Bxb1 (Mycobacterium). In embodiments, the serine integrase is PhiC31 (Streptomyces). In embodiments, the serine integrase is R4 (Streptomyces). In embodiments, the serine integrase is TP901 (Lactococcus). In embodiments, the serine integrase is yb (E. coli). In embodiments, the serine integrase is gin (phage Mu) (E. coli). In embodiments, the serine integrase is Tn3 (Klebsiella).

[0160] In embodiments, nucleic acids and methods herein comprise one or more integrase that is a large serine recombinase selected from Bxb1 , PhiC31 , R4, phiBTI , MJ1 , MR11, TP901-1 , A118, V153, phiRVI, phi370.1 , TG1 , WB, BL3, SprA, phiJoe, phiK38, Int2, Int3, Int4, Int7, Int8, Int9, IntIO, Inti 1 , Int12, Inti 3, L1, peaches, Bxz2, and SV1. Persons skilled in the art, with the benefit of this disclosure in its entirety, will understand how to utilize structure-function analysis to take mutations / modifications from Bxb1 to make equivalent mutations in other large serine recombinases and test their efficiency against wild-type enzymes e.g., using the methods described herein and in the art.DBl / 164937876.2 36Atorney Docket No.: KOM-003PC / 138774-5003

[0161] In embodiments, the one or more nucleic acids encodes one or more one or more attB sites or one or more attP sites. In embodiments, methods herein utilize a single attB site and a single attP site.

[0162] In embodiments, the attB sites used herein comprise about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89- 95. In embodiments, the attB site used herein comprises the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95. In embodiments, the attB sites used herein are compatible with one or more attP sites comprising about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97.

[0163] In embodiments, the attP sites used herein comprise about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96- 97. In embodiments, the attP site used herein is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97. In embodiments, the attP sites used herein are compatible with one or more attB sites comprising about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95.

[0164] In embodiments, the att sequence used in nucleic acids herein is or comprises an att nucleic acid sequence selected from Table 1 , or a sequence having about or at least about 70% sequence identity thereto. In embodiments, att sequences used herein are selected from Table 1.

[0165] In embodiments, the terms “sites,” or “sequences,” when referring to an att (attB or attP) are interchangeable and referring to a nucleic acid sequence and / or location that can function as an att for recombinase-mediated exchange. In embodiments, the terms “integrase,” or “recombinase,” are interchangeable, for example, when referring to Bxb1. In embodiments, “compatibility,” when referring to att sites includes the ability for the two sequences to be suitable for recombinase-mediated exchange, forDBl / 164937876.2 37Atorney Docket No.: KOM-003PC / 138774-5003 example, as catalyzed by an integrase, such as Bxb1 , PhiC31, or any other. In embodiments, compatibility between att sites is measured by ddPCR, gel electrophoresis, western blotting, flow cytometry, etc.Table 1 : Illustrative attB and attP sites for integrases Bxb1 , Phi3C1 , and Pa01 , with illustrative, wild-type minimal aft sequences and / or core dinucleotide sequences underlined and bolded.DBl / 164937876.2 38Atorney Docket No.: KOM-003PC / 138774-5003

[0166] In embodiments, the one or more bacterial attachment (attB) sequences comprises about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95. In embodiments, the one or more bacterial attachment (attB) sequences is compatible with one or more phage attachment (attP) sequences comprising about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97. In embodiments, the one or more bacterial attachment (attB) sequences comprises a nucleic acid sequence selected from Table 1.

[0167] In embodiments, the one or more phage attachment (attP) sequences comprises about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to one of SEQ ID NOs: SEQ ID NOs: 2, 4, 6, and 96-97. In embodiments, the one or more phage attachment (attP) sequences is compatible with one or more bacterial attachment (attB) sequences comprising about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: SEQ ID NOs: 1, 3, 5, and 89-95. In embodiments, the one or more phage attachment (attP) sequences comprises a nucleic acid sequence selected from Table 1.

[0168] In embodiments, methods herein include introducing a nucleic acid having one or more att sequences into a cell. In embodiments, methods herein include Bxb1 recombinase-mediated catalysis between one or more attB and one or more attP.DBl / 164937876.2 39Atorney Docket No.: KOM-003PC / 138774-5003

[0169] In embodiments, the one or more atB sites and / or atP, or two or more attB sites and / or atP sites is suitable for recombination at a central dinucleotide sequence comprising guanine-thymine (GT).

[0170] In embodiments, the one or more atB sites and / or attP, or two or more attB sites and / or atP sites is suitable for recombination at a central dinucleotide sequence comprising adenine-cytosine (AC).

[0171] In embodiments, methods herein include introducing the one or more bacterial attachment (attB) sites includes introducing one or more nucleic acids into the cell.

[0172] In embodiments, one or more nucleic acids herein is introduced into a host cell simultaneously, e.g., in a single transfection and / or transduction, or in series (one nucleic acid at a time). In embodiments, the one or more nucleic acid includes one or more Cas9-directed reverse transcription plasmid, one or more integrase expression plasmid, and / or one or more integration sequence. In embodiments, introduction of nucleic acids into host cells herein is achieved using one or more of a lipid-based transfection reagent (e.g., LIPOFECTAMINE (INVITROGEN), TRANSIT-2020 (MIRUS BIO), HELA-MONSTER (MIRUS BIO), NEON TRANSFECTION SYSTEM (INVITROGEN), diethylaminoethyl (DEAE)-dextran, liposomes, cationic lipid- based reagents, electroporation, sonoporation, chemical reagent (e.g., calcium phosphate, etc.), microinjection, or via a non-integrating episome or viral vector system (e.g., AAV, lentiviral, etc.). In embodiments, nucleic acids are delivered as DNA (e.g., plasmid, vector, or minicircle) or RNA (e.g., mRNA).

[0173] In embodiments, the one or more nucleic acids comprise one or more homology directed repair (HDR) insulator sequence and one or more attB and / or atP site flanking one or more kill switch gene, one or one or more promoter, one or more 2A peptide sequence, one or more insertion sequence, and / or one or more polyA tail.

[0174] In embodiments, HDR insulators herein function as barrier insulators to prevent euchromatin silencing by the spread of neighboring heterochromatin in the cellular genome. In embodiments, this is done to support expression of one or more elements within the flanking HDR insulator sequences. Potent endogenous insulators are known to exist at several mammalian loci. Without wishing to be bound by theory, loci described herein are chosen (in part) due to their endogenous insulator capabilities to allow expression of genes placed therein. In embodiments, insulator sequences can be selectable by using preferred integration locations (e.g., AAV integration “host-spots”), for example with the AAVS1 locus.

[0175] In embodiments, “insertion sequence” herein refers to a transcribable / translatable element, including protein-coding and non-protein-coding sequences of interest (e.g., including sequences that areDBl / 164937876.2 40Atorney Docket No.: KOM-003PC / 138774-5003 translated from an mRNA or pre-mRNA transcript transcribed from the insertion sequence). In embodiments, the terms, “insertion sequence,” “integration sequence,” “intervening DNA sequence,” and “protein-coding sequence” can be used interchangeably to refer to a transcribable / translatable element, including proteincoding and non-protein-coding sequences of interest that is to be recombined into a host cell genome.

[0176] In embodiments, the promoter comprises one or more of a mammalian gene promoter, viral promoter, inducible promoter, tissue-specific promoter, cell type-specific promoter, and bidirectional promoter. In embodiments, the promoter comprises one or more native promoter. In embodiments, the promoter comprises one or more synthetic promoter or composite promoter (e.g., combining elements of two or more different promoters). In embodiments, the promoter comprises one or more of a B29 promoter, CAG promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, CMV promoter, desmin promoter, Ef1 a promoter, EGR1 promoter, elastase-1 promoter, elF2A1 promoter, endoglin promoter, FerH promoter, FerL promoter, fibronectin promoter, Flt-1 promoter, GAPDH promoter, GFAP promoter, GPIIb promoter, GRP78 promoter, GRP94 promoter, HSP70 promoter, Hspa5p promoter, ICAM-2 promoter, INF- P promoter, Nphsl promoter, OG-2 promoter, PGK-1 promoter, ROSA promoter, SP-B promoter, SV40 promoter, SYN1 promoter, ubiquitin B promoter, WASP promoter, -actin promoter, and -kin promoter.

[0177] In embodiments, the nucleic acid encodes one or more 2A peptide. In embodiments, the 2A peptides comprises one or more of a T2A, P2A, E2A, and F2A peptide sequence. In embodiments, the 2A peptide is located between two or more protein-coding sequences. In embodiments, the 2A peptide is located between two or more insertion sequences (e.g., between protein-coding regions). In embodiments, 2A peptides refer to a class of 18-22 amino acid-long peptides, which induce ribosomal skipping during translation. In embodiments, these peptides share a core sequence motif of DXEXNPGP (SEQ ID NO: 11 , where “X” denotes any amino acid) and are found in a wide range of viral families. In embodiments, 2A peptides are introduced to help generate polyproteins from a single open reading frame (ORF) in the genomic loci by causing the ribosome to fail at making a peptide bond during translation of the sequence, and then resume translation. In embodiments, 2A and 2A-like peptides for ribosomal skipping can be found for example in de Lima JGS and Lanza DCF, “2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology,” Viruses, (2021), Vol, 13, No. 11 :2160, the entire contents of which are incorporated by reference.DBl / 164937876.2 41Atorney Docket No.: KOM-003PC / 138774-5003

[0178] In embodiments, the 2A peptide comprises one or more sequence from Table 2. In embodiments, the donor plasmid encodes one or more 2A peptide of SEQ ID NOs: 7-10, and / or one or more 2A peptide having the consensus sequence of SEQ ID NO: 8.Table 2: Illustrative 2A peptide sequences and linker / spacer sequences.

[0179] In embodiments, the donor plasmid includes one or more kill switch genes, which function to induce apoptosis and cell death under certain conditions based on the genomic insertion status of the cell.

[0180] In embodiments, the nucleic acid does not need to encode one or more metabolic enzymes, for example, for cell selection based on genomic integration status. In embodiments, methods herein do not rely on or require metabolic enzymes, such as glutamine synthetase (GS) (e.g., for use in a GS-knockout or GS- impaired cell). In embodiments, methods herein do not rely on or require metabolic enzymes, such as phosphoglycerate kinase (PGK) (e.g., for use in a PGK-knockout or PGK-impaired cell). In embodiments, the nucleic acids herein for integration into the host cell do not encode one or more antibiotic resistance cassettes for antibiotic selection. In embodiments, methods herein represent advantages over traditional methods in that they circumvent the need for antibiotic use in developing and maintaining the cell lines and clonal cell populations, the improvements include for example, avoiding the stresses induced by typical antibiotic usage or metabolic stress due to auxotrophy. In embodiments, methods herein improve upon traditional cell line development by circumventing the need for antibiotic-based selection and / or metabolic enzyme expressionbased selection.

[0181] In embodiments, methods herein utilize a process referred to as Cas9-directed reverse transcription for site-specific genomic integration of one or more att site. In embodiments, methods herein utilize one or more Cas9-directed reverse transcription plasmids. In embodiments, a Cas9-directed reverse transcription plasmid encodes one or more of a nickase, reverse transcriptase, and Cas9-directed reverse transcription guide RNA (pegRNA). In embodiments, a Cas9-directed reverse transcription plasmid encodesDBl / 164937876.2 42Atorney Docket No.: KOM-003PC / 138774-5003 each of the nickase, reverse transcriptase, and pegRNA. In embodiments, the epegRNA is targeted to a single loci in the host cell genome.

[0182] In embodiments, the nickase comprises any endonuclease which “nicks” orcleaves only a single strand of dsDNA target, as opposed to cleaving (ds-break). In embodiments, nickases include bacterial, viral, and mammalian endonucleases, for example, a Cas-based fusion protein.

[0183] In embodiments, Cas9-directed reverse transcription (and the Cas9-directed reverse transcription plasmid thereof) includes a fusion protein comprising a Cas9 nickase and a reverse transcriptase PE5, as well as a pegRNA. In embodiments, the pegRNA is a modified single guide RNA (sgRNA) which both direct the PE5 to the target genomic sequence and encodes the desired edit. In embodiments, both the fusion protein and / or pegRNA are encoded in a Cas9-directed reverse transcription plasmid or vector. In embodiments, the pegRNA contains a 5’ spacer sequence that directs PE5 to nick a target sequence. In embodiments, the primer binding site (PBS) found on the 3’ end of the pegRNA anneals to the non-target DNA strand following the nick in the target strand. In embodiments, the pegRNA includes a reverse transcriptase template (RTT) that is reverse transcribed to generate a 3’ single-strand flap of DNA that encodes the inserted DNA, such as the att site. During twin Cas9-directed reverse transcription, in embodiments, a second pegRNA directs the insertion of an adjacent flap that includes overlapping homologous sequence so that the two flaps form a double-stranded intermediate. Upon resolution of the intermediate, in embodiments, the new sequence becomes permanently inserted into the host genome.

[0184] For example, in embodiments, Cas9-directed reverse transcription efficiently inserts sequences <100 bp. In embodiments, Cas9-directed reverse transcription is first used to insert a first att site (e.g., ~50 bp) at a desired genomic locus. In embodiments, the att site acts as a genomic integration site pad for one or more integrases, which can efficiently insert multi-kb sequences. In embodiments, during a second step, the integrase recombines the att site found in the genome with a cognate att site found on one or more insertion sequences (e.g., a dsDNA plasmid encoding one or more recombinant protein). Following recombination, in embodiments, the entire donor plasmid becomes inserted into the genome and the attB and attP sites are rearranged to form attL and attR sites flanking the insertion (e.g., as shown in Fig. 1). Because the integrase cannot recombine the newly formed attL and attR sites, in embodiments, the insertion sequence plasmid contains one or more sequences, e.g., as described herein, that is permanently inserted into the host cell genome.DBl / 164937876.2 43Atorney Docket No.: KOM-003PC / 138774-5003

[0185] Cas9-directed reverse transcription efficiency is generally higher for shorter inserts, thus in embodiments, the pegRNA is designed for inserting a minimal atB site and / or minimal atP site (e.g., as described in Table 1).

[0186] In embodiments, the minimal atB site is about or at least about 25 nucleotides in length, about or at least about 30 nucleotides in length, about or at least about 35 nucleotides in length, about or at least about 40 nucleotides in length, about or at least about 41 nucleotides in length, about or at least about 42 nucleotides in length, about or at least about 43 nucleotides in length, about or at least about 44 nucleotides in length, about or at least about 45 nucleotides in length, about or at least about 46 nucleotides in length, about or at least about 47 nucleotides in length, about or at least about 48 nucleotides in length, about or at least about 49 nucleotides in length, about or at least about 50 nucleotides in length, about or at least about 51 nucleotides in length, about or at least about 52 nucleotides in length, about or at least about 53 nucleotides in length, about or at least about 54 nucleotides in length, about or at least about 55 nucleotides in length, or about or at least about 60 nucleotides in length.

[0187] In embodiments, the minimal atP site is about or at least about 25 nucleotides in length, about or at least about 30 nucleotides in length, about or at least about 35 nucleotides in length, about or at least about 40 nucleotides in length, about or at least about 41 nucleotides in length, about or at least about 42 nucleotides in length, about or at least about 43 nucleotides in length, about or at least about 44 nucleotides in length, about or at least about 45 nucleotides in length, about or at least about 46 nucleotides in length, about or at least about 47 nucleotides in length, about or at least about 48 nucleotides in length, about or at least about 49 nucleotides in length, about or at least about 50 nucleotides in length, about or at least about 51 nucleotides in length, about or at least about 52 nucleotides in length, about or at least about 53 nucleotides in length, about or at least about 54 nucleotides in length, about or at least about 55 nucleotides in length, or about or at least about 60 nucleotides in length.

[0188] In embodiments, the one or more att site is inserted into the single genomic locus at a wild-type Bxb1 central dinucleotide sequence comprising guanine-thymine (GT). In embodiments, the atB and / or atP site targets to a central dinucleotide sequence comprising guanine-thymine (GT). There are 16 possible dinucleotide combinations (e.g., of A, T, C, and G). In embodiments, the one or more atB sites and / or atP sites is introduced into the single genomic locus at a non-canonical Bxb1 central dinucleotide sequence comprising one of the other 15 di nucleotide pairs (e.g., TA, TT, TG, TC, CC, CA, CT, CG, GG, GA, GC, AT, AA, AG, AC). In embodiments, one or more integrases used in methods herein are mutable to alter theDBl / 164937876.2 44Atorney Docket No.: KOM-003PC / 138774-5003 dinucleotide sites for genomic integration, altering the specificity and repertoire of locations for subsequent rounds of integration.

[0189] To improve Cas9-directed reverse transcription efficiency, in embodiments, methods herein use an engineered pegRNA containing a structured RNA motif to prevent degradation and / or an optimized cr772 guide scaffold. In embodiments, methods herein use Cas9-directed reverse transcription where PE5 is expressed from a PE5max plasmid with an improved editor architecture that includes a dominant negative mutant of human mutL homolog 1 (MLH1) involved in DNA mismatch repair which has been shown to enhance Cas9-directed reverse transcription. In embodiments, PE5max and twin epegRNA expression plasmids for each target site are introduced into the host cell.

[0190] In embodiments, the att site found on the pegRNA expression plasmid may recombine with the att site on the donor plasmid, rendering both plasmids nonfunctional, which reduces the amount of pegRNA available for Cas9-directed reverse transcription, as well as reduce the number of donor plasmids available for integrase insertion. To reduce this effect, in embodiments, the epegRNA expression plasmids are engineered to contain a partial att site. In embodiments, during “twin Cas9-directed reverse transcription,” each inserted 3’ single strand flap contains only part of the att site with an overlapping sequence to form a double-stranded intermediate. In embodiments, the remaining single-stranded sequence of the att sites are subsequently filled in to promote insertion of the full att site. In embodiments, this strategy is sufficient for reducing or preventing unwanted integrase recombination.

[0191] In embodiments, methods herein, combine the Cas9-directed reverse transcription and integrase expression plasmids into a single nucleic acid introduction event (e.g., in a single transfection). In embodiments, this strategy allows for the insertion of one or more att site using Cas9-directed reverse transcription followed by the insertion of one or more integration sequence by one or more integrases at the target sequence.

[0192] In embodiments, methods herein are compatible with a variety of methods for knock-in of att sequence within the host cell genome. In embodiments, such methods include, without limitation, introducing single strand DNA breaks (e.g., nick-mediated insertion or tandem paired nicking), double strand DNA breaks (e.g., with non-homologous end joining (NHEJ) or homology-directed repair (HDR)), transposon-based insertion, and the like. Persons skilled in the art, with the benefit of this disclosure in its entirety, will be aware of the various techniques that can be used to insert att sites within host cell genomes for use with methods of integrases herein.DBl / 164937876.2 45Atorney Docket No.: KOM-003PC / 138774-5003

[0193] In embodiments, methods herein include the use of one or more zinc-fi nger nucleases (ZFNs) to introduce the one or more att sites into the host cell genome. For example, in embodiments, the one or more nucleic acids encoding the one or more att site can be integrated into the host cell genome using ZFN mRNA and one or more ULTRAMER nucleic acid construct from INTEGRATED DNA TECHNOLOGIES. In embodiments, the ZFN generates a double-strand break that facilitates integration via homology directed repair (HDR) (e.g., double-stand DNA break). In embodiments, ZFNs include engineered ZFNs (e.g., Fokl nuclease engineered domains).

[0194] In embodiments, methods herein include the use of one or more CRISPR / Cas endonucleases, transcription activator-like effector nucleases (TALENs), TALE-derived transcription factors, TALE repeat domain proteins, meganucleases, restriction enzymes, site-specific nucleases, and / or gene-editing systems to introduce the one or more att sites into the host cell genome. In embodiments, such systems include one or more proteins and / or nucleic acids working in concert, including without limitation, TALENs, ZFNs, RNase P RNA, RNase H, CRISPR / Cas, C2c1, C2c2, C2c3, Cas9, Cpf1 , TevCas9, Archaea Cas9, CasY.1 , CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and / or any ortholog or homolog thereof. In embodiments, the gene editors can also include an RNA molecule (e.g., gRNA, which, as used herein, refers to guide RNA). In embodiments, the gRNA is sequence complimentary to a coding or a non-coding sequence and can be tailored to the particular sequence to be targeted (e.g., targeting to a particular genomic locus). In embodiments, the gRNA sequence can be a sense or anti-sense nucleic acid sequence. In embodiments, when a gene editor composition is administered herein, preferably without limitation, including two or more gRNAs; however, a single gRNA can also be used.

[0195] In embodiments, the methods herein target to a single loci of the host cell genome. In embodiments, the loci includes a AAVS1 locus, ACTB locus, ACTB locus, ALB locus, albumin locus, B2M locus, CCR5 locus, CD38 locus, CFTR locus, COL7A1 locus, Factor IX locus, FANCA locus, GBA1 locus, GYS1 locus, Hippl 1 locus, Keppel-19 locus, MACO1 locus, Olonne-18 locus, Pansio-1 locus, Rosa26 locus, S100A locus, SHS253 locus, Smn1 locus, S100A locus, TRAC locus, Xq22.1 locus, or a homologous or species-equivalent thereof, or a homologous or species-equivalent thereof, e.g., in a non-human cell line. In embodiments, these sites are amenable to becoming safe harbor sites for site-specific targeted genomic insertion without substantive deleterious effects on the cell within the context of recombinant protein production, e.g., on growth rate, number of cell splitting cycles, cell cycle control, protein production, etc. In embodiments, the methods herein target to a chromosomal location of one or more of chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 6, chromosome 7, chromosome 8,DBl / 164937876.2 46Atorney Docket No.: KOM-003PC / 138774-5003 chromosome 10, chromosome 11 , chromosome 12, chromosome 13, chromosome 17, chromosome 19, chromosome 20, or chromosome 22. In embodiments, the loci is located at a region of increased gene expression (RIDGE). Persons skilled in the art, with the benefit of this disclosure in its entirety, will be aware of the various methods and techniques used for selecting host cell loci locations for genomic insertion.

[0196] In embodiments, the efficiency of insertion of aft sites into host cell genomes ranges from 0.1% to 90% or more of a population of host cells subjected to the fusion protein and pegRNA. In embodiments, the efficiency of insertion of aft sites into host cell genomes is about or at least about 0.5%, about or at least about 1 .0%, about or at least about 5.0%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 30%, about or at least about 40%, about or at least about 50%, about or at least about 60%, about or at least about 70%, or about or at least about 90%. In embodiments, efficiency of insertion of att sites is measurable using amplicon sequencing with genomic primers flanking the insert followed by confirmation by droplet digital PCR (ddPCR), which is a form of quantitative PCR that reports highly accurate copy number of genomic inserts over a wide range of template concentrations. In embodiments, methods herein utilize any form of PCR-based next-gen sequencing (NGS) to evaluate insertion efficiency and / or fidelity.

[0197] In embodiments, methods herein are amenable to a variety of host cell types, including cells for expression of therapeutic proteins and / or for producing clonal cell populations maintaining stable genetic amendments. In embodiments, the host cell is a mammalian cell, plant cell, insect cell, yeast cell, or bacterial cell. In embodiments, the host cell is a plant cell. In embodiments, the host cell is an insect cell. In embodiments, the host cell is a mammalian cell.

[0198] In embodiments, the host cell is suitable for recombinant protein production. In embodiments, the mammalian host cell the mammalian cell comprises a Chinese hamster ovary (CHO) cell (e.g., CHO-K1 , CHO-DHB11 , CHO-DXB1 , CHO-S, CHO-DG44, CHO-M), human embryonic kidney (e.g., HEK293, HEK293T) cell, K562 human lymphoblast cell, U2OS human osteosarcoma cell, primary human fibroblasts (e.g., human dermal fibroblast (HDFa)) cell, baby hamster kidney (BHK) cell, Vero cell (e.g., Vero, Vero 76, Vero E6), human cervical carcinoma cell (e.g., HELA, 3T3), PERc6 cell, CAP cell, iPSCs, human embryonic stem cells (ESCs), or monkey kidney CV1 cell.

[0199] In embodiments, methods herein are adaptable to use with cells which are auxotrophic, lacking the ability to produce a particular nutrient necessary for survival. For example, in embodiments, such methods include introducing one or more sequences encoding one or more enzymes to enable the cell to surviveDBl / 164937876.2 47Atorney Docket No.: KOM-003PC / 138774-5003 under specific cell conditions, despite its auxotrophy, for example, encoding glutamine synthetase (GS) for a GS-knock-out cell line (e.g., CHO-DG44). Alternatively, in embodiments, the cell is not an auxotrophic variant relative to a cognate wild-type cell (e.g., a cell with unaltered metabolic capacities).

[0200] In embodiments, methods herein are adaptable for multiplex gene stacking, or the ability to introduce one or more insertion sequences adjacent to one or more previously integrated insertion sequences within the same genomic location by one or more integrases (e.g., as shown in Fig. 2). In embodiments, the integration of one or more insertion sequences is operably linked to one or more additional insertion sequences, e.g., in a single ORF using a 2A peptide, or transcribed from one or more shared genetic elements, such as a promoter or enhancer sequence (or transcribed from separate, new promoters). In embodiments, methods herein include (or are adaptable for) introducing two insertion sequences, three insertion sequences, four insertion sequences, or five or more insertion sequences at a single locus. In embodiments, methods herein include (or are adaptable for) utilizing one integrase mutant, two integrase mutants, three integrase mutants, four integrase mutants, or five or more integrase mutants. In embodiments, methods herein include substantially simultaneous host cell expression of the one or more integrase variants; alternatively, in embodiments, expression is cascaded, where integrase mutants are expressed sequentially after each previous insertion sequence is integrated. Without wishing to be bound by theory, methods herein allow for increased insertion sizes within a single genomic locus to generate multi-specific gene products not possible by the current technologies.

[0201] In embodiments, methods herein demonstrate specificity to substantially only target a single chromosomal site (e.g., single allele at a locus), resulting in monoallelic insertion.

[0202] In embodiments, the one or more insertion sequences are single or multi-gene cargos. In embodiments, the integration rates are as high as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of target chromosomes (e.g., single loci, monoallelic) in host cells.

[0203] In aspects, described herein are methods of generating a cell line for expressing one or more recombinant proteins comprising providing a clonal cell population produced by the methods described herein, wherein the clonal cell population comprises one or more one or more bacterial attachment (attB) sites and / or one or more phage attachment attP) sites and one or more inducible kill switch genes integrated into a single genomic locus at a single allele, does not encode an antibiotic resistance gene or auxotrophic selection gene, and is not an auxotrophic variant relative to a cognate wild-type cell, and introducing into the clonal cell population a first nucleic acid encoding one or more integrases (e.g., as described herein) suitableDBl / 164937876.2 48Atorney Docket No.: KOM-003PC / 138774-5003 for site-specific integration at the one or more attB sites and / or one or more attP sites, wherein the cell is suitable to express the one or more integrases, and a second nucleic acid encoding one or more insertion sequences encoding the one or more recombinant proteins, wherein the one or more insertion sequences comprise one or more attB sites and / or one or more cognate attP sites that are suitable for site-specific integration at the one or more attB sites and / or one or more attP sites at the single genomic locus of the clonal cell population, integrating the one or more integration sequences into the single genomic locus using the one or more integrases, and inducing apoptosis via the one or more inducible kill switch genes in cells that do not integrate the one or more insertion sequences.

[0204] In embodiments, clonal cell selection is performed by triggering apoptosis using one or more gene products of the one or more inducible kill switch genes. In embodiments, cells are treated with a small molecule, where teh small molecule triggers apoptosis in cells that lack the site-specific integration of the one or more insertion sequences which disrupt the one or more kill switch genes (suicide genes) or molecular switches. In embodiments, the treatment will not affect cells with site-specific as the kill switch gene will have been disrupted from the site-specific integration of the one or more insertion sequences.

[0205] In embodiments, the genomic insertion comprises insertion of one of more insertion sequences comprising double-stranded DNA (dsDNA) ranging in size from about 0.1 kb to about 50 kb or more. In embodiments, the genomic integration includes knock-in of one or more nucleic acids of about or at least about 0.5 kb in length, about or at least about 1 kb in length, about or at least about 2 kb in length, about or at least about 3 kb in length, about or at least about 4 kb in length, about or at least about 5 kb in length, about or at least about 6 kb in length, about or at least about 7 kb in length, about or at least about 8 kb in length, about or at least about 9 kb in length, about or at least about 10 kb in length, about or at least about 11 kb in length, about or at least about 12 kb in length, about or at least about 13 kb in length, about or at least about 14 kb in length, about or at least about 15 kb in length, about or at least about 16 kb in length, about or at least about 17 kb in length, about or at least about 18 kb in length, about or at least about 19 kb in length, about or at least about 20 kb in length, about or at least about 30 kb, or about or at least about 50 kb.

[0206] In embodiments, the one of more insertion sequences encodes one or more of an expression cassette encoding a protein, optionally comprising one or more promoter sequence, enhancer sequence, internal ribosome entry site (IRES), and 3’ polyadenine (polyA) sequence.DBl / 164937876.2 49Atorney Docket No.: KOM-003PC / 138774-5003

[0207] In embodiment, the protein comprises a therapeutic protein. In non-limiting embodiments, the recombinant protein comprises one or more of an antibody or antibody-format protein, therapeutic enzyme, fusion protein, secretory protein, protein hormone, and / or protein toxin or antitoxin.

[0208] For example, in non-limiting embodiments, methods herein are suitable for production of antibodies or antibody-format proteins, such as one or more of an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), diabody, nanobody (VHH), linear antibody, monoclonal antibody, polyclonal antibody, bispecific antibody, multi-specific antibody, chimeric antibody, humanized antibody, human antibody, non-human antibody, and fusion protein comprising an antigen-binding portion of an antibody.

[0209] For example, in non-limiting embodiments, methods herein are suitable for production of therapeutic proteins / enzymes, such as asparaginase, glucarpidase, p-glucocerebrosidase, phenylalanine hydroxylase (PAH), ocriplasmin, tissue-type plasminogen activator (tPA), fibrinogen, anti-clotting factor or clotting factor (factor VIII, factor IX, von Willebrand factor,), tissue-nonspecific alkaline phosphatase (TNAP), lysosomal acid lipase, and fusions thereof.

[0210] For example, in non-limiting embodiments, methods herein are suitable for production of proteins with one or more amino-acid linker and / or fusion domain, such as antibody Fc fusions (e.g., IgG, IgGi, lgG2, IgGs, I gG4), extended recombinant polypeptide (XTENylation), albumin fusion (e.g., human serum albumin), PAS (e.g., proline-alanine-serine repeat) fusion, elastin-like peptide (ELP), glycine-rich homo-amino-acid polymer (HAP), proline / alanine / serine (PAS) repeats, artificial gelatin-like protein (GLK), C-terminal peptide (CTP) of human chorionic gonadotropin -subunit, maltose binding protein (MBP), FLAG tag or hexahistidine tag, e.g., for half-life improvement, reduction in organ sinking and / or renal clearance, for manufacturing and purification purposes, etc.

[0211] For example, in non-limiting embodiments, methods herein are suitable for production of cellular factors or secretable proteins, either endogenously secreted or modified for secretable expression, e.g., by incorporation of one or more secretion signal peptides or tags, for example for production of cytokines (e.g., interferons, interleukins, and fusions thereof), granulocyte colony-stimulating factor (G-CSF), coagulation factors, growth factors, and other signaling molecules.

[0212] For example, in non-limiting embodiments, methods herein are suitable for production of peptide / protein hormones, which have long-standing success as biotherapeutics, such as glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), insulin, leptin (metreleptin), fibroblast growth protein,DBl / 164937876.2 50Atorney Docket No.: KOM-003PC / 138774-5003 human growth hormone (HGH), growth hormone (GH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), parathyroid hormone or parathyroid hormone-related protein (PTH or PTHrP), human chorionic gonadotropin (HCG), and erythropoietin.

[0213] For example, in non-limiting embodiments, methods herein are suitable for production of toxins and / or antitoxins, e.g., for the production of therapeutic proteins, antitoxins, or for vaccine development (e.g., as is done with acellular Pertussis toxin), such as botulinum toxin (BoNT), Shiga toxin, cholera toxin, among other bacterial, fungal, and viral toxins.

[0214] In embodiments, cell line host cell type is selected based on desired PTM, for example CHO cells are attractive for production of proteins that require glycosylation for bioefficacy.

[0215] In embodiments, the insertion sequence encodes a chimeric receptor (e.g., anti-CD19 chimeric antigen receptor (CAR)), a promoter or enhancer sequence, and / or a genetic cassette encoding a therapeutic protein corrective protein (e.g., a non-mutant form of a protein that is mutant or otherwise defective in the host cell, for example, for protein replacement therapy).

[0216] In aspects, described herein are methods for producing one or more recombinant proteins using one or more clonal cell populations generated by the methods described herein. In embodiments, methods include culturing the cell under conditions suitable for expression of the recombinant protein.

[0217] In embodiments, methods of culturing cells include incubating the clonal cell populations with one or more excipients, e.g., such as cell media (e.g., MEM, DMEM, RPM1 1640, Ham’s F-12K, Ham’s F-10, GMEM, BME, McCoy's 5A, M200, Leibovitz's L-15, or IMDM), fetal bovine serum (FBS), amino acids (L- glutamate, non-essential amino acids (NEAA)), sodiumpyruvate, pH buffer (HEPES, etc.), NaCI, pH indicator (e.g., phenol red), hydrocortisone, dexamethasone, cell factors, (e.g., epidermal growth factor, insulin, etc.).

[0218] In embodiments, methods further comprise recovering the one or more recombinant proteins from the cell and / or the cell culture media (e.g., for secreted products). Persons skilled in the art, upon the benefit of this disclosure in its entirety will be aware of the various techniques and methods for recombinant protein purification and isolation from host cell lysates and / or cell media.

[0219] In embodiments, methods herein include suspending clonal cell populations in one or more cryoprotectant to protect cells for freeze-thaw cycles, e.g., for cell line development. In embodiments, cryoprotectants generally regarded appropriate for cells include DMSO, glycerol, sucrose, ethanol, ethylene glycol (EG), and propylene glycol (PG).DBl / 164937876.2 51Atorney Docket No.: KOM-003PC / 138774-5003Integrases for Use in Methods of Cell Line Development and Recombinant Protein Expression

[0220] The present disclosure provides, in embodiments, methods utilizing recombinant “mutant” integrases with altered functionality relative to their cognate wild-type (e.g, non-mutant variant) counterparts. As used herein, in embodiments, “wild-type” refers to a naturally-occurring nucleic acid and / or amino acid sequence, including natural polymorphisms and variants, which has not been modified. In embodiments, the altered functionality includes an improved functionality with respect to one or more functions of the integrase, including increased affinity and / or binding to nucleic acid target sites, altered recognition (e.g., different specificity at DNA target sites, including for example, at attB / attP sites -19 through -12, -11 through -9, -7, - 6, and / or +4, or at attP sites -24 through -17, -11 through -9, -7, and -6) increased integration (e.g., as measured as integration at one or both loci in a chromosome, and / or calculated as a percent, ratio, or amount of host cells with integration compared to the total population of cells), increased probability of integration, decreased off-target integration, etc. In embodiments, “hyperactive,” “increased activity,” or “improved functionality,” with respect to evovled integrases refers to an increase in activity and / or functionality, relative to a cognate wild-type integrase, of about or at least about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (e.g., 1-fold), 150%, 200% (e.g., 2-fold), 250%, 300% (e.g., 3-fold), 350%, 400% (e.g., 4-fold), 450%, 500% (e.g., 5-fold), 1000% (e.g., 10-fold), 1500% (e.g., 15-fold), 2000% (e.g., 20-fold) or more, in one or more respects.

[0221] In embodiments, the integrase is an enzyme which catalyzes DNA integration using one or more attachment (att) sites. In embodiments, the integrase described herein interacts between attB and / or attP sites. In embodiments, att sites are double-stranded DNA (dsDNA). For example with respect to Bxb1 integrases, in embodiments, the integrases herein make contacts with dsDNA at attB sites -19 through -12 (e.g, hairpin target), -11 through -9 (e.g, helix target), -7 and -6 (e.g, loop target); and / or makes contacts with dsDNA at attP sites -24 through -17 (e.g, hairpin target), -11 through -9 (e.g, helix target), -7 and -6 (e.g, loop target). In embodiments, integrases herein adopt higher order tertiary and quaternary structures (e.g, homodimers, homotetramers, etc.) which make symmetrical contacts on either side of a central dipeptide (e.g, often denoted -1 / +1), where negative (-) and positive (+) denotes a left and right half-site, respectively. In embodiments, integrases (and mutations, addition, and / or deletions made thereto) alter binding interaction within a half-site (e.g, left or right) which may symmetrically effect the binding interaction in the other half-site.

[0222] Illustrative sequence of Bxb1 and PhiC31 integrases are shown in Table 3.DBl / 164937876.2 52Atorney Docket No.: KOM-003PC / 138774-5003Table 3: Illustrative wild-type amino acid sequence of serine integrases Bxb1 and PhiC31. Residues in bold and underlined represent illustrative mutable amino acids.

[0223] In embodiments, the Bxb1 integrase includes one or more mutants at one or more amino acid positions of 4, 5, 14, 18, 20, 24, 29, 34, 35, 36, 40, 42, 45, 46, 49, 50, 51 , 57, 60, 61 , 62, 63, 67, 68, 69, 70, 73, 74, 75, 76, 78, 79, 84, 85, 86, 87, 88, 89, 90, 92, 95, 99, 100, 104, 105, 106, 110, 111, 116, 119, 122, 124, 130, 133, 137, 140, 145, 153, 156, 157, 158, 160, 164, 166, 174, 175, 178, 179, 181 , 183, 187, 189,191, 194, 197, 203, 207, 208, 209, 218, 223, 229, 231 , 232, 233, 234, 236, 237, 239, 248, 251 , 254, 257,261, 264, 267, 268, 272, 273, 278, 280, 281, 282, 283, 285, 287, 288, 291 , 292, 295, 302, 306, 307, 311 ,313, 314, 316, 318, 319, 321 , 322, 323, 325, 328, 331 , 332, 333, 334, 341 , 342, 343, 347, 353, 355, 359,360, 361, 362, 368, 369, 370, 375, 380, 388, 396, 397, 398, 405, 409, 411 , 414, 415, 416, 419, 425, 428,434, 435, 444, 449, 453, 461 , 462, 463, 466, 468, 476, 479, 480, 483, 484, 487, 488, 489, 494, 496, and / or 499 relative to SEQ ID NO: 12, or equivalent positions thereof in related integrases.

[0224] In embodiments, the Bxb1 integrase includes one or more mutants selected from one or more of L4I, V5V, V5I, DUN, S18S, E20K, E20Q, E24E, E24K, L29F, G34D, W35L, D36A, V40I, V40A, V40V, E42K, D45G, V46V, A49T, V50I, D51D, , D51N, D51 E, D51Y, N60S, L61 F, A62A, R63K, F67S, E68K, E69A,DBl / 164937876.2 53Atorney Docket No.: KOM-003PC / 138774-5003E69D, E69E, Q70P, D73G, V74A, V74I, V74M, 175V, V76I, Y78H, Y78N, R79R, T84S, R85R, S86T, 187V, I87L, R88R, H89G, L90L, Q92H, Q92Q, H95Y, D99N, H100N, H100Y, V105A, V105I, S106S, A110A, H111P, T116P, A119S, V122M, A124S, A130A, E133E, 11371, R140R, A145T, K153K, S157G, G156G, P160P, L164L, T166I, L174L, V175V, P178P, V179A, V179I, R181 K, E183L, V187I, V187V, H189N, Q191Q, P197T, H203Y, R207Q, R208S, G209G, G209V, A218A, R223R, E229K, S231S, A232G, A232S, A232R, A232T, A232V, A232P, A232Q, T233G, T233W, T233R, T233Y, T233D, T233G, T233N, T233H, T233S, T233Q, T233A, T233C, A234N, A234G, A234S, A234T, A234H, A234F, A234S, K236R, K236S, R237K, R237Q, R237N, R237V, R237C, M239I, A248T, N251K, N251N, T254S, D257K, A261T, A261V, V264A, E267D, P268P, R272Q, E273D, E273K, L278L, A280T, A280A, E281E, L282L, V283V, T285A, R287R, R287P, A288V, A288T, A291T, P292P, P295P, L302L, V306V, C307C, A311V, K313R, K313K, F314M, F314L, F314N, F314R, F314K, G316R, G316W, G318H, G318HP, G318S, G318N, G318K, R319K, R319G, H321P, H321Q, H321K, H321L, H321R, H321Y, H321T, H321S, P322A, P322R, P322G, P322L, R323L, R323G, R323Y, R323I, R325K, R325Q, R325Y, S328S, F331S, P332H, K333K, K333N, H334R, M342T, M342V, A343T, A347E, A347V, V353I, D355N, D359N, D359D, A360T, E361E, E361D, R362K, V368A, A369P, A369E, A369T, A369S, G370G, V375V, V375I, V380I, T388M, R397R, A396P, A398S, A405A, R409H, A411V, A414A, A414V, A415S, R416K, E419E, A425T, A425A, S428S, E434G, T435T, T435A, R444L, A449V, T453I, T453A, R461R, L462M, T463I, V466M, V466V, G468D, G468G, F476F, L479I, Q480STOP, E483E, E483K, Q484K, R487R, del L488 (del L488 frameshift), G489G, R494S, R494R, R494Q, H496N, and / or M499T relative to SEQ ID NO: 12, or equivalent positions thereof in related integrases. In embodiments, these mutants relate to the combinations of the lists of mutants herein.

[0225] In embodiments, methods herein utilize one or more Bxb1 integrase comprising an amino acid sequence that is at least 90% identical to amino acids 1-488 of SEQ ID NO: 12.

[0226] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 154, 155, 156, 157, 158, 159, 231, 232, 233, 234, 235, 236, 237, 257, 314, 316, 318, 321, 322, 323, and 325, or a position corresponding thereto. In embodiments, the one or more mutations are selected from Y154W, Y154A, R155W, R155H, G156P, S157G, L158T, L158R, L158A, P159S, P159T, A234N, S231A, S231L, S231G, S231R, S231Y, S231T, S231H, A232G, A232S, A232R, A232T, A232V, A232Q, A232P, T233G, T233S, T233W, T233R, T233Y, T233D, T233N, T233H, T233Q, T233A, T233C, A234N, A234G, A234S, A234T, A234H, A234F, K236R, K236S, R237K, R237T, R237Q, R237N, R237C, R237V, D257K, F314M, F314L, F314N, F314R, F314K, G316R, G316W, G316S, G318H, G318P. G318S, G318N, G318P, G318K, G318R, H321Q, H321 K, H321L, H321R, H321Y, H321T, H321S, H321W, P322A,DBl / 164937876.2 54Atorney Docket No.: KOM-003PC / 138774-5003P322R, P322G, R323L, R323G, R323Y, R323I, R325K, R325Q, and R325Y, or a position corresponding thereto.

[0227] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 5, 14, 20, 24, 29, 35, 40, 45, 49, 50, 51, 60, 68, 69, 70, 73, 74, 78, 84, 86, 87, 100, 105, 116, 124, 157, 183, 197, 207, 208, 209, 229, 261, 267, 273, 287, 291, 333, 342, 343, 347, 361, 368, 375, 435, 449, 453, 462, 483, and 494, or a position corresponding thereto. In embodiments, the one or more mutations are selected from V5I, D14N, E20K, E20Q, E24K, L29F, W35L, V40I, D45G, A49T, V50I, D51N, D51E, D51Y, N60S, E68K, E69D, Q70P, D73G, V74A, V74M, Y78H, Y78N, T84S, S86T, I87V, H100Y, V105I, T116P, A124S, S157G, E183L, P197T, R207Q, R208S, G209V, E229K, A261V, E267D, E273D, E273K, R287P, A291T, K333N, M342V, A343T, A347V, E361D, V368A, V375I, T435A, A449V, T453A, L462M, E483K, and R494Q, or a position corresponding thereto.

[0228] In embodiments, the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 4, 5, 18, 24, 34, 36, 40, 42, 46, 51, 61, 62, 63, 67, 69, 79, 85, 87, 88, 89, 90, 92, 95, 99, 100, 105, 106, 110, 111, 119, 122, 130, 133, 137, 140, 145, 153, 156, 160, 164, 166, 174, 175, 178, 179, 181, 187, 189, 191, 203, 209, 218, 223, 229, 231, 239, 248, 251, 254, 261, 264, 268, 272, 278, 280,281, 282, 283, 285, 287, 288, 292, 295, 302, 306, 307, 311, 313, 319, 321, 328, 331, 332, 333, 334, 347,353, 355, 359, 360, 361, 362, 369, 370, 375, 380, 388, 397, 398, 405, 409, 411, 414, 415, 416, 419, 425,428, 434, 435, 444, 449, 453, 461, 462, 463, 466, 468, 476, 479, 480, 483, 484, 487, 488, 489, 494, 496, and 499, or a position corresponding thereto. In embodiments, the one or more mutations are selected from L4I, G34D, D36A, V40I, V40A, E42K, D51N, L61F, R63K, F67S, E68K, E69A, I87V, I87L, H89G, Q92H, H95Y, D99N, H100N, V105A, H111 P, A119S, V122M, E133E, A145T, V179A, V179I, R181 K, V187I, V187A, H189N, H203Y, E229K, M239I, A248T, N251K, T254S, D257K, A261T, V264A, R272Q, A280T, T285A, A288V, A288T, A311V, R319K, R319G, H321P, H321N, F331S, P332H, H334R, A347E, V353I, D355N, D359N, A360T, R362K, A369P, A369E, A369T, A369S, V380I, T388M, A398S, R409H, A411V, A414V, A415S, R416K, A425T, E434G, R444L, T453I, T463I, V466M, G468D, L479I, Q480STOP, Q484K, delL488, R494S, H496N, and M499T, or a position corresponding thereto.

[0229] In embodiments, the Bxb1 integrase comprises a C-terminal deletion of one or more amino acids in the range of L488-S500 relative to SEQ ID NO: 12 (e.g., a deletion of 1-13 amino acids from the C-terminal end). In embodiments, the C-terminal deletion comprises a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 aminoDBl / 164937876.2 55Atorney Docket No.: KOM-003PC / 138774-5003 acids, 11 amino acids, 12 amino acids, or all 13 amino acids of L488-S500 relative to SEQ ID NO: 12. In embodiments, the C-terminal deletion includes mutating a residue selected from the range of L488-S500, relative to SEQ ID NO: 12, to a stop codon (e.g., L488STOP). In embodiments, a deletion of one or more amino acids of L488-S500 relative to SEQ ID NO: 12 results in no change in functionality of Bxb1 integrase relative to wild-type. In embodiments, a deletion of one or more amino acids of L488-S500 relative to SEQ ID NO: 12 results in improved functionality of Bxb1 integrase relative to wild-type.

[0230] In embodiments, the Bxb1 integrase comprises a C-terminal deletion of one or more amino acids in the range of Q480-S500 relative to SEQ ID NO: 12 (e.g., a deletion of 1 -21 amino acids from the C-terminal end). In embodiments, the C-terminal deletion comprises a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or all 21 amino acids of Q480-S500 relative to SEQ ID NO: 12. In embodiments, the C-terminal deletion includes mutating a residue selected from the range of Q480-S500, relative to SEQ ID NO: 12, to a stop codon (e.g., Q488STOP). In embodiments, a deletion of one or more amino acids of Q480-S500 relative to SEQ ID NO: 12 results in no change in functionality of Bxb1 integrase relative to wild-type. In embodiments, a deletion of one or more amino acids of Q480-S500 relative to SEQ ID NO: 12 results in improved functionality of Bxb1 integrase relative to wildtype.

[0231] In embodiments, Bxb1 residues Y154-P159 are mutable to alter specificity at -7 and -6 positions of the loop target DNA site. In embodiments, the mutation of Bxb1 is selected from residues S157, L158, and P160. In embodiments, mutation of Bxb1 L158 alters specificity at -7 and -6 positions of the loop target DNA site. In embodiments, the mutation is S157G in the Bxb1 att DNA loop recognition site. In embodiments, mutation at this position alters the dinucleotide preference to adenine (A) or thymine (T) at positions -7 and cytosine (C) or guanine (G) at position -6.

[0232] In embodiments, the one or more Bxb1 integrases comprise a sequence from Y154-P159 selected from YRGSLS (SEQ ID NO: 13), WWGGTP (SEQ ID NO: 14), YRGGLP (SEQ ID NO: 15), AHPGRT (SEQ ID NO: 16), and WRGGAS (SEQ ID NO: 17), where the residues mutated from the wild-type sequence (e.g., SEQ ID NO: 12) are bolded and underlined.

[0233] In embodiments, the Bxb1 S231-R237 region recognizes the 3 bp helix target site flanking the DNA cleavage site (e.g., -11 , -10, -9) in the target DNA site. In embodiments, this stretch of amino acidsDBl / 164937876.2 56Atorney Docket No.: KOM-003PC / 138774-5003 forms an alpha helix that docks with the target DNA site, reminiscent of the interaction between a zinc finger (ZF) and its target trinucleotide sequence. In embodiments, this site is mutated to a non-native sequence to alter target specificity, e.g., non-native genomic sites. Because this section recognizes 3 DNA bps, in embodiments, mutations are made that target all 64 possible DNA triplets at positions -11 , -10, and -9. In embodiments, L235 is not mutated because it is analogous to the residue often found at +4 of a zinc finger (ZF) recognition helix, .e.g., leaving residues 231-234 and 236-237 to be mutated to alter specificity at positions -11 , -10, and -9. In embodiments, A234 is mutated to an asparagine (e.g., A234N) to alter the specificity at the -10 position to thymine (T). In embodiments, the 3 bp helix target site flanking the DNA cleavage site (e.g., -11 , -10, -9) is altered as a function of mutation of residues S231-R237.

[0234] In embodiments, residues 231 , 233, and / or 237 are mutable to alter specificity at -10 and -9 DNA target sites. In embodiments, the Bxb1 integrase comprises a sequence from S231-R237 relative to SEQ ID NO: 1 selected from AGGNLKR (SEQ ID NO: 18), LGTNLKR (SEQ ID NO: 19), SGTGLKK_(SEQ ID NO: 20), SGSALKT (SEQ ID NO: 21), AAWALRR (SEQ ID NO: 22), GGRSLKR (SEQ ID NO: 23), SGYNLRR (SEQ ID NO: 24), SGWGLKK (SEQ ID NO: 25), SGWALRQ (SEQ ID NO: 26), SARALSR (SEQ ID NO: 27), RADILRR (SEQ ID NO: 28), YSRNLKR (SEQ ID NO: 29), SRNGLRK (SEQ ID NO: 30), RGHALKN (SEQ ID NO: 31), GGSHLKR (SEQ ID NO: 32), TTRTLKR (SEQ ID NO: 33), AVQNLKR (SEQ ID NO: 34), RAAFLKK_(SEQ ID NO: 35), RAWTLKC (SEQ ID NO: 36), RAWSLKR (SEQ ID NO: 37), HGWSLKV (SEQ ID NO: 38), HGCTLKR (SEQ ID NO: 39), YGSALKQ (SEQ ID NO: 40), SQWALKC (SEQ ID NO: 41), YPWSLRR (SEQ ID NO: 42), where the residues mutated from the wild-type sequence (e.g., SEQ ID NO: 12) are bolded and underlined. In embodiments, 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues are mutated between S231-R237 (inclusive) relative to SEQ ID NO: 12.

[0235] In embodiments, mutation of Bxb1 residues F314-R325 alter specificity at DNA interaction sites -19 through -12 (e.g., DNA hairpin target sequence). In embodiments, these residues form the interfacial region with the DNA major grove and / or influence DNA hairpin structure. In embodiments, Bxb1 residue P322 is maintained as a proline. In embodiments, one or more of Bxb1 residues F314, G316, G318, H321 , P322, R323, and / or R325 are mutated. In embodiments, residues K314-R325 are mutated to one of the following sequences: MAGGHRKQALYR (SEQ ID NO: 43), MAGGPRKKRRYR (SEQ ID NO: 44), LARGSRKLALYR (SEQ ID NO: 45), NARGNRKRGRYR (SEQ ID NO: 46), LARGPRKRAGYK (SEQ ID NO: 47), RAWGKRKYAYYQ (SEQ ID NO: 48), KAWGSRKTRLYR (SEQ ID NO: 49), MARGGRKSAIYY (SEQ ID NO: 50), MASGSRKIA1YY (SEQ ID NO: 51), LARGRRKWARYR (SEQ ID NO: 52), and LARGSRKLALYR (SEQ ID NO: 53), where the residues mutated from the wild-type sequence (e.g., SEQ ID NO: 12) are bolded andDBl / 164937876.2 57Atorney Docket No.: KOM-003PC / 138774-5003 underlined. In embodiments, 1 residue, 2 residues, 3 residues, 4 residues, 5 residues, 6 residues, or 7 residues are mutated between F314-R325 (inclusive) relative to SEQ ID NO: 12.

[0236] In embodiments, the Bxb1 integrase comprises one or more mutations and / or deletions, and / or one or more combinations of mutations and / or deletions as described in Table 3 and / or Table 4.Table 4: Illustrative Bxb1 integrase mutants and illustrative mutant combinations. “X” refers to any amino acid different from the wild-type residue. Illustrative changes are relative to SEQ ID NO: 1. bpNLS = bipartite NLS KRTADGSEFESPKKKRKV (SEQ ID NO: 85): npNLS = nucleoplasmin NLS KRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86).DBl / 164937876.2 58Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 59Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 60Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 61Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 62Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 63Atorney Docket No.: KOM-003PC / 138774-5003

[0237] In embodiments, the Bxb1 integrase has at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations,DB1 / 164937876.2 64Atorney Docket No.: KOM-003PC / 138774-5003 or at least 50 mutations, e.g., as selected from Table 3 and / or Table 4 relative to SEQ ID NO: 12, or equivalent positions thereof.

[0238] In embodiments, the Bxb1 integrase comprises at least 90% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations, e.g., as selected from Table 3 and / or Table 4 relative to SEQ ID NO: 12, or equivalent positions thereof.

[0239] In embodiments, the Bxb1 integrase comprises at least 95% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or at least 20 mutations, e.g., as selected from Table 3 and / or Table 4 relative to SEQ ID NO: 12, or equivalent positions thereof.

[0240] In embodiments, the Bxb1 integrase comprises at least 96% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or 20 mutations, e.g., as selected from Table 3 and / or Table 4 relative to SEQ ID NO: 12, or equivalent positions thereof.

[0241] In embodiments, the Bxb1 integrase comprises at least 97% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, or 15 mutations, e.g., as selected from Table 3 and / or Table 4 relative to SEQ ID NO: 12, or equivalent positions thereof.DBl / 164937876.2 65Atorney Docket No.: KOM-003PC / 138774-5003

[0242] In embodiments, the Bxb1 integrase comprises at least 98% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, or 10 mutations, e.g., as selected from Table 3 and / or Table 4 relative to SEQ ID NO: 12, or equivalent positions thereof.

[0243] In embodiments, the Bxb1 integrase comprises at least 99% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, or 5 mutations, e.g., as selected from Table 3 and / or Table 4 relative to SEQ ID NO: 12, or equivalent positions thereof.

[0244] In embodiments, the Bxb1 integrase comprises at least 90% sequence identity to residues 1- 480 of SEQ ID NO: 12 and having one or more mutations selected from Table 3 and / or Table 4. In embodiments, the Bxb1 integrase comprises at least 92% sequence identity to residues 1-480 of SEQ ID NO: 12 and having one or more mutations selected from Table 3 and / or Table 4. In embodiments, the Bxb1 integrase comprises at least 95% sequence identity to residues 1-480 of SEQ ID NO: 12 and having one or more mutations selected from Table 3 and / or Table 4. In embodiments, the Bxb1 integrase comprises at least 96% sequence identity to residues 1 -480 of SEQ ID NO: 12 and having one or more mutations selected from Table 3 and / or Table 4. In embodiments, the Bxb1 integrase comprises at least 97% sequence identity to residues 1-480 of SEQ ID NO: 12 and having one or more mutations selected from Table 3 and / or Table 4. In embodiments, the Bxb1 integrase comprises at least 98% sequence identity to residues 1-480 of SEQ ID NO: 1 and having one or more mutations selected from Table 3 and / or Table 4. In embodiments, the Bxb1 integrase comprises at least 99% sequence identity to residues 1-480 of SEQ ID NO: 12 and having one or more mutations selected from Table 3 and / or Table 4.

[0245] In embodiments, the Bxb1 integrase the an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 and one or more amino acid mutations at positions selected from L4, E42, R57, R63, V76, H111 , V122, V187, K313, R319, A341 , D359, A369, A398, R416, A425, L479, and M499, relative to SEQ ID NO: 1.

[0246] In embodiments, the Bxb1 amino acid is at least 97% identical to the amino acid sequence of SEQ ID NO: 1 and has one or more amino acid mutations at positions selected from L4, E42, R57, R63, V76, H111 , V122, V187, K313, R319, A341, D359, A369, A398, R416, A425, L479, and M499, relative to SEQ ID NO: 1.DBl / 164937876.2 66Atorney Docket No.: KOM-003PC / 138774-5003

[0247] In embodiments, the Bxb1 amino acid is at least 98% identical to the amino acid sequence of SEQ ID NO: 1 and has one or more amino acid mutations at positions selected from L4, E42, R57, R63, V76, H111 , V122, V187, K313, R319, A341, D359, A369, A398, R416, A425, L479, and M499, relative to SEQ ID NO: 1.

[0248] In embodiments, the Bxb1 amino acid is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and has one or more amino acid mutations at positions selected from L4, E42, R57, R63, V76, H111 , V122, V187, K313, R319, A341, D359, A369, A398, R416, A425, L479, and M499, relative to SEQ ID NO: 1.

[0249] In embodiments, the Bxb1 integrase comprises one or more amino acid mutations selected from L4I, E42K, R57K, R63K, V76I, H111 P, V122M, V187, K313R, R319K, A341 D, D359A, A369P, A398S, R416K, A425T, L479I, and M499T relative to SEQ ID NO: 1. In embodiments, the Bxb1 integrase comprises one or more amino acid mutations selected from D36N, V40A, A49S, V74I, I87L or I87A or I87S, H89G, V175A, V179A, R287H, A288V, T453I, and H496N relative to SEQ ID NO: 1 (e.g., in combination with the one or more amino acid mutations selected from L4I, E42K, R57K, R63K, V76I, H111 P, V122M, V187, K313R, R319K, A341 D, D359A, A369P, A398S, R416K, A425T, L479I, and M499T relative to SEQ ID NO: 1). In embodiments, the Bxb1 integrase comprises one or more amino acid mutations selected from V40I, D45G, I75V, H95Y, A119S, A280T, A311 V, E434G, and V466M relative to SEQ ID NO: 1 (e.g., in combination with the one or more amino acid mutations selected from L4I, E42K, R57K, R63K, V76I, H111 P, V122M, V187, K313R, R319K, A341 D, D359A, A369P, A398S, R416K, A425T, L479I, and M499T relative to SEQ ID NO: 1).

[0250] In embodiments, the Bxb1 integrase comprises one or more of the following amino acids, e.g., relative to SEQ ID NO: 1 , which are not mutated: A62, H100, Q191 , P195, G209, P295, L302, C307, L387, E419, S428, E483, and R487 relative to SEQ ID NO: 1.

[0251] In embodiments, the Bxb1 integrase comprises one or more amino acid mutations selected from mutations at position V122 and / or position A369 relative to SEQ ID NO: 1 . In embodiments, the mutation at position V122 relative to SEQ ID NO: 1 is V122M. In embodiments, the mutation at position A369 relative to SEQ ID NO: 1 is A369P. In embodiments, the Bxb1 integrase comprises mutations at position V122 and position A369 relative to SEQ ID NO: 1. In embodiments, the mutations are V122M and A369P relative to SEQ ID NO: 1.DBl / 164937876.2 67Atorney Docket No.: KOM-003PC / 138774-5003

[0252] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation position combinations selected from: V76 and V122; V76 and A369; I87 and V122; I87 and A369; H95 and V122; H95 and A369; V122 and E434; A369 and E434; V76, V122, and A369; I87, H95 and A369; I87, V122 and E434; I87, A369 and E434; H95, V122 and A369; H95, V122 and E434; H95, A369 and E434; V122, A369 and E434; I87, H95, V122 and E434; I87, H95, A369 and E434; and I87, V122, A369 and E434 relative to SEQ ID NO: 1.

[0253] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation combinations selected from: V76I and V122M; V76I and A369P; I87L and V122M; I87L and A369P; H95Y and V122; H95Y and A369P; V122M and E434G; A369P and E434G; V76I, V122M, and A369P; I87L, H95Y and A369P; I87L, V122M and A369P; I87L, V122M and E434G; I87L, A369P and E434G; H95, V122M and A369P; H95Y, V122M and E434G; H95Y, A369P and E434G; V122, A369P and E434G; I87L, H95Y, V122M and E434G; I87L, H95Y, A369P and E434G; or I87L, V122, A369P and E434G relative to SEQ ID NO: 1.

[0254] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation position combinations selected from: I87, A369, and E434, V122, A369, and E434, H95, A369, and E434, V122 and E434, V122, A369, E434, and V175, V76 and A119, A119, A369, and E434, 187, V175, and D359, 187, R57, and R63, 187 and A369, 187 and A425, R63 and V122, R63, V122, R319, and V40I, R63, A119, and M499, V122 and A369, H95 and A369, A49, I87, A396, and E434, R57, R63, I87, A396, and E434, D36, V122, A396, and E434, R57, R63 and H95, D36, I87, A396, and E434, I87, A341, A396, and E434, R57, R63, V122, A396, and E434, V122, R287, A396, and E434, 187, R287, A396, and E434, 187 and A341, 187, R287, A396, E434, and A341, V122, A396, E434, and A341, H95, and A341, A49, V122, A396, and E434, D45, A396, and E434, V122, R287, A341, A396, and E434, A49, A396, and E434, I87, A341, and R287, D36, A396, and E434, V122, D359, A369, and E434, R63 and I87, 187, D359, A369, and E434, V122, A369, A425, and E434, 187, A369, A425, and E434, V40, H95, A369, and E434, L4, H95, A369, and E434, H95, A369, E434, and V466, R63, V122, A369, and E434, V76, 187, and K313, R63, 187, and N194, 187 and D359, L4 and I87, V74 and V76, V122, A369, E434, and R319, 187, A369, E434, and R319, L4, V122, A369, and E434, I87 and A425, E42, V76, and I87, 187 and L479, H95, A369, A425, and E434, R63, 187, A369, and E434, I87 and R319, V76, A369, and E434, H95, A369, E434, and L479, V74, 175, and V76, H95, R319, A369, and E434, L4, 187, A369, and E434, V122, V175, D359, A369, and E434, R57, 187, A369, and E434, E42, H95, A369, and E434, H95, V179, A369, and E434, 175 and V76, L4 and V76, H95 and K313, V40, R63, and H89, A288, A311, A398, R416, T453, H496, and E42, and H111 and E434 relative to SEQ ID NO: 1.DBl / 164937876.2 68Atorney Docket No.: KOM-003PC / 138774-5003

[0255] In embodiments, the Bxb1 integrase comprises one or more amino acid mutation combinations selected from: I87L, A369P, and E434G, V122M, A369P, and E434G, H95Y, A369P, and E434G, I87L and E434G, V122M and E434G, V122M, A369P, E434G, and V175A, V76I and A119S, D36N and A119S, A119S, A369P, and E434G, I87L, V175A, and D359A, I87L, R57K, and R63K, I87L and D36N, I87L and A369P, I87L and A425T, R63K and V122M, R63K, V122M, R319K, and V40I, R63K, A119S, and M499T, H95Y and E434G, V122M and A369P, H95Y and V466M, H95Y and A369P, A49S, I87L, A396P, and E434G, A49S and I87L, R57K, R63K, I87L, A396P, and E434G, D36N, V122M, A396P, and E434G, R57K, R63K and H95Y, D36N, I87L, A396P, and E434G, D36N and H95Y, I87L, A341 D, A396P, and E434G, A49S and H95Y, R57K, R63K, V122M, A396P, and E434G, V122M, R287H, A396P, and E434G, I87L and R287H, I87L, R287H, A396P, and E434G, I87L and A341 D, I87L, R287H, A396P, E434G, and A341 D, V122M, A396P, E434G, and A341 D, H95Y and A341 D, A49S, V122M, A396P, and E434G, D45G, A396P, and E434G, V122M, R287H, A341 D, A396P, and E434G, A49S, A396P, and E434G, I87L, A341 D, and R287H, H95Y and R287H, D36N, A396P, and E434G, V122M, D359A, A369P, and E434G, R63K and I87L, I87L and V466M, I87L, D359A, A369P, and E434G, V122M, A369P, A425T, and E434G, I87L, A369P, A425T, and E434G, V40I, H95Y, A369P, and E434G, L4I, H95Y, A369P, and E434G, H95Y, A369P, E434G, and V466M, R63K, V122M, A369P, and E434G, V76I, I87L, and K313R, R63K, I87V, and N194D, I87L and D359A, I87L and L4I, V74I and V76I, V122M, A369P, E434G, and R319K, I87L, A369P, E434G, and R319K, L4I, V122M, A369P, and E434G, I87L and A425T, E42K, V76I, and I87L, I87L and L479I, H95Y, A369P, A425T, and E434G, V40I and I87L, R63K, I87L, A369P, and E434G, I87L and R319K, V76I, A369P, and E434G, H95Y, A369P, E434G, and L479I, V74I, I75V, and V76I, H95Y, R319K, A369P, and E434G, L4I, I87L, A369P, and E434G, V122M, V175A, D359A, A369P, and E434G, R57K, I87L, A369P, and E434G, E42K, H95Y, A369P, and E434G, H95Y, V179A, A369P, and E434G, I75V and V76I, I87L and V179A, L4I and V76I, H95Y and K313R, H95Y and A280T, V40A, R63K, and H89G, A288V, A311V, A398S, R416K, T453I, H496N, and E42K, and H111 P and E434G relative to SEQ ID NO: 1 .

[0256] In embodiments, the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and has the amino acid mutations I87L, A369P, and E434G relative to SEQ ID NO: 1.

[0257] In embodiments, the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and having the amino acid mutations V122M, A369P, and E434G relative to SEQ ID NO: 1.DBl / 164937876.2 69Atorney Docket No.: KOM-003PC / 138774-5003

[0258] In embodiments, the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and having the amino acid mutations H95Y, A369P, and E434G relative to SEQ ID NO: 1.

[0259] In embodiments, the Bxb1 integrase comprises one or more nuclear localization sequences (NLSs). In embodiments, "nuclear localization signal" or "NLS" refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., International PCT application, PCT / EP2000 / 011690, filed November 23, 2000, published as WQ / 2001 / 038547 on May 31, 2001; and in Kosugi et al., J. Biol. Chem. (2009) 284: 478-485, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. Nuclear localization sequences, in embodiments, include the nuclear localization sequence of the SV40 virus large T-antigen the minimal functional unit of which is the seven amino acid sequence PKKKRKV (SEQ ID NO: 68). Other examples of nuclear localization sequences include the nucleoplasmin bipartite NLS with the sequence NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 69); the c-myc nuclear localization sequence having the amino acid sequence PAAKRVKLD (SEQ ID NO: 70) or RQRRNELKRSF (SEQ ID NO: 71); and the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 72). Further examples of nuclear localization sequences are: the sequenceRMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 73) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 74) and PPKKARED (SEQ ID NO: 75) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 76) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 77) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 78) and PKQKKRK (SEQ ID NO: 79) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 80) of the Hepatitis virus delta antigen; and the sequence REKKKFLKRR (SEQ ID NO: 81) of the mouse Mx1 protein. It is also possible, in embodiments, to use bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 82) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 83) of the steroid hormone receptors (human) glucocorticoid. In embodiments, a NLS of the present disclosure comprises the amino acid sequence SV40 Large T-antigen NLS comprising the amino acid sequence DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 84). In embodiments, the NLS comprises the sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 85). In embodiments, the NLS comprises the sequenceDBl / 164937876.2 70Atorney Docket No.: KOM-003PC / 138774-5003KRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86). In embodiments, the NLS comprises the sequence GSHHHHHHGSGPKKKRKV (SEQ ID NO: 87). In embodiments, the NLS comprises the nucleic acid sequence GSGSGSHHHHHHGSGPKKKRKV (SEQ ID NO: 88). In embodiments, a NLS of the present disclosure comprises one or more mutations (e.g., amino acid substitutions, deletions, and / or insertions) with respect to any one of above sequences.

[0260] In embodiments, the one or more NLSs further improve the efficiency to the Bxb1 integrase by shunting the Bxb1 into the nucleus where it carries out its recombination. In embodiments, the Bxb1 has a single NLS. In embodiments, the NLS is located at the N-terminus. In embodiments, the NLS is located at the C-terminus. In embodiments, a NLS is located at the N-terminus and the C-terminus. In embodiments, the NLS is located within the protein sequence.

[0261] In embodiments, integrases herein comprise one or more stabilization tags. In embodiments, “stabilization sequence,” "stabilization tag," "stability tag, or “stability sequence," refers to an amino acid sequence that influences one or more of stability, solubility, folding, aggregation, degradation, purification, isolation, expression tracking, and / or subcellular localization, and the like, e.g., by promoting improvements of protein functionality relative to a cognate protein lacking the stability tag / sequence. Stabilization sequences for proteins are known in the art and would be apparent to the skilled artisan depending upon the desired functionality. For example, stabilization sequences are described in Khurana et al., “Chapter Seventeen - Elucidating the role of an immunomodulatory protein in cancer: From protein expression to functional characterization,” Methods in Enzymology, Vol. 629 (2019): pp. 307-60, the contents of which are incorporated herein by reference for their disclosure of exemplary stabilization tag sequences.

[0262] In embodiments, the stabilization sequence is or comprises about 5 amino acids to about or at least about 300 amino acids in length (e.g., about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids or more in length, including lengths therebetween). In embodiments, the stabilization tag is or comprises a “C-stab” tag (SEQ ID NO: 106). In embodiments, the stabilization tag is or comprises a “C-term stab-tag np” tag (SEQ ID NO: 107). In embodiments, the stabilization tag is or comprises a “N-term stab-tag np” tag (SEQ ID NO: 108). In embodiments, the stabilization tag is or comprises a “C-term stab-tag bp” tag (SEQ ID NO: 109). In embodiments, the stabilization tag is or comprises a “N -term stab-tag bp” tag (SEQ ID h 0: 110). In embodiments, the one or more stabilization sequences is or comprises the amino acid sequence of any one of SEQ ID NOs: 106-110, or an amino acid sequence having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes thereto. In embodiments,DBl / 164937876.2 71Atorney Docket No.: KOM-003PC / 138774-5003 the stabilization tag is or comprises the amino acid sequence of any one of SEQ ID NOs: 106-110, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes thereto. In embodiments, the stabilization tag is or comprises a sequence selected from Table 5.Table 5: Illustrative Stabilization Sequences for Integrases herein.

[0263] Stabilization sequences, in embodiments, include poly-amino acid sequences, such as poly-Cys, poly-Arg, poly-Asp, poly-Phe, and poly-His (e.g., hexahistidine or His6x) tags. Other exemplary stabilization tag sequences include AU1 tag, AU5 tag, Glu-Glu tag EYMPME, FLAG tag, strep-tag II, HA tag, c-Myc tag, T7 tag, VSV-G tag, KT3 epitope tag, HSV tag, Protein C tag, V5 tag, S-tag, twin-strep tag, SBP tag, CBD tag, protein eXact tag. In embodiments, the stabilization tag is a protein domain, or a portion thereof, for example of ubiquitin, human serum albumin (HSA), SmbP, SUMO, Trx, Skp, Ecotin, SNAP-tag, slyd, DsbA, GST, Halo tag, MBP, NusA, p-galactosidase, Fc-domain fusion (e.g., lgG1, lgG2, lgG3, lgG4, etc.), orCaBP. In embodiments, the stabilization tag is a protein domain, or a portion thereof useful for detection, e.g., such as RFP, GFP, CFP, mCherry, mOrange, SEAP, and luciferase. In embodiments, the stabilization sequence is or comprises an amino acid sequence that is or comprises a recognition site that is cleavable by one or more of thrombin, enterokinase, TEV protease, Factor Xa, SUMO protease, or HRV 3C protease.

[0264] In embodiments, integrases herein are fusion proteins comprising one or more DNA-binding domains. In embodiments, the one or more DNA-binding domains helps to target the integrase to a genomic aft site by increasing the overall level of interaction (e.g., instead of relying purely on loop, helix, hairpin interactions, such as the case with Bxb1). In embodiments, the one or more DNA-binding domain comprisesDBl / 164937876.2 72Atorney Docket No.: KOM-003PC / 138774-5003 one or more TAL domains, one or more Cas proteins (Cas + guide RNA), and / or one or more zinc finger protein domains.

[0265] In embodiments, the one or more DNA-binding domains comprise one or more zinc finger proteins (ZFPs). Zinc finger proteins (ZFPs), in embodiments, are protein that contain zinc finger structural motifs, which are specific DNA-binding domains. In embodiments, these structural motifs are used to target and bind to specific DNA sequences within the genome. In embodiments, the one or more ZFPs comprises a C2H2 (Cys2HiS2) zinc finger (e.g., illustrative PROSITE identifier PS00028), composed of a 0-hairpin and an a-helix stabilized by a zinc ion, and recognizes about or at least about 3-4 base pairs of a DNA sequence. Exemplary native proteins with native ZFPs include MYST family histone acetyltransferases, Myt1 myelin transcription factor, and ST18 tumor suppressor protein. In embodiments, the one or more ZFPs is or comprises a Gag knuckle structural motif (e.g., illustrative PROSITE identifier PS50158), treble clef structural motif , zinc ribbon structural motif (e.g., illustrative PROSITE identifier PS51134), Zn2Cyse structural motif (e.g., illustrative PROSITE identifier cd00067), and / or TAZ2 domain motif. In embodiments, ZFPs have modular designs to target DNA triplets which are fused to the N-terminus, C-terminus, and / or within, the Bxb1 integrase. In embodiments, other DNA-binding domain ZFPs and similar DNA-binding domains comprise one or more of B-box zinc finger, DNA-binding protein, FPG lleRS zinc finger, Kriippel associated box, RING finger domain, TAL effector, transcription activator-like effector nuclease, zinc finger inhibitor, zinc finger nuclease, and zinc finger transcription factor, and / or are useful for structure-based design based on a designed genomic sequence.

[0266] Persons skilled in the art, with the benefit of this disclosure in its entirety, will understand how to design and clone TALE repeat domains, Cas domains, and ZFP domains into integrases (e.g., Bxb1) herein, for example using standard mutagenesis techniques, as well as design tools. For instance, non-limiting examples of zinc finger design tools include ZFtools, as described in Mandell and Barbas, “Zinc Fi nger Tools: custom DNA-binding domains for transcription factors and nucleases,” Nucleic Acids Research, Vol. 34 (2006): pp. W516-23; ZiFiT, as described in Sander et al., “Zinc Finger Targeter (ZiFiT): an engineered zinc finger / target site design tool,” Nucleic Acids Research, Vol. 35, (2007): pp. W599-W605; ZifBASE, as described in Jayakanthan et al., “ZifBASE: a database of zinc finger proteins and associated resources,” BMC Genomics, Vol. 10, No. 421 (2009); and ZF Design, as described in Ichikawa, et al. “A universal deeplearning model for zinc finger design enables transcription factor reprogramming,” Nat Biotechnol, Vol. 41 , (2023): pp. 1117-29, the entire contents of each are incorporated herein by reference in their entireties.DBl / 164937876.2 73Atorney Docket No.: KOM-003PC / 138774-5003

[0267] In embodiments, mutant Bxb1 integrases herein comprise an amino acid linker sequence, e.g., between a NLS and the N-term or C-term of the Bxb1 . Amino acid linker sequences are known in the art, for example, for producing fusion proteins, etc. Exemplary amino acid linker sequences, in embodiments, include glycine and serine (e.g., GG, GS, SS, SG), as well as repeated units of glycine-serine, aspartic acid (D) residues, and the like. Amino acid linkers, in embodiments, includes lengths of a single amino acid, about 2 amino acids in length, about 4 amino acids in length, about 6 amino acids in length, about 10 amino acids in length, or about 15 amino acids in length, including lengths therebetween. In embodiments, amino acid linkers are included between NLS peptides or sequence (e.g., bipartite sequences).

[0268] In embodiments, mutant integrases herein (e.g., Bxb1) comprise an amino acid linker sequence. In embodiments, the amino acid linker is located between a NLS and the N-term or C-term of the integrase, between sections of a NLS, between a stabilization sequence and the N-term or C-term of the integrase, between a NLS and stabilization sequence, and / or a combination thereof. Amino acid linker sequences are known in the art, for example, for producing fusion proteins, etc. Exemplary amino acid linker sequences, in embodiments, include glycine and serine (e.g., GG, GS, SS, SG, GGS, SSG, SGS, GSG, etc.), as well as repeated motifs and units thereof, repeated units / motifs consisting of glycine and serine residues, aspartic acid (D) residues, and the like. Amino acid linkers, in embodiments, are about 1-30 amino acids, such as a single amino acid in length, about 2 amino acids in length, about 3 amino acids in length, about 4 amino acids in length, about 5 amino acids in length, about 6 amino acids in length, about 8 amino acids in length, about 10 amino acids in length, about 12 amino acids in length, about 15 amino acids in length, about 20 amino acids in length, about 25 amino acids in length, or about 30 amino acids in length, including lengths therebetween. In embodiments, amino acid linkers are included between NLS peptides / signals (e.g., bipartite sequences). In embodiments, an amino acid linker is between the NLS and the enzyme. In embodiments, an amino acid linker is between a stabilization sequence (stability tag) and the enzyme. In embodiments, an amino acid linker is between a stabilization sequence (stability tag) and a NLS. In embodiments, an amino acid linker is between one or more DNA-binding domains (e.g., one or more ZFPs) and / or between one or more DNA-binding domains (e.g., one or more ZFPs) and the Bxb1 sequence.

[0269] In embodiments, an N-terminal addition site of Bxb1 is between M1 and R2 residues, e.g., of SEQ ID NO: 12. In embodiments, the N-terminal extension is include N-terminal relative to M1 of SEQ ID NOL 12. In embodiments, an extension of one or more amino acids at the N-terminus relative to SEQ ID NO: 12 results in no adverse change in functionality of Bxb1 integrase relative to wild-type. In embodiments, anDBl / 164937876.2 74Atorney Docket No.: KOM-003PC / 138774-5003 extension of one or more amino acids at the N-terminus relative to SEQ ID NO: 12 results in improved functionality of Bxb1 integrase relative to wild-type.

[0270] In embodiments, the N-terminal addition site of Bxb1 is between M1 and R2 residues, e.g., of SEQ ID NO: 12. In embodiments, the N-terminal extension is include N-terminal relative to M1 of SEQ ID NOL 12. In embodiments, an extension of one or more amino acids at the N-terminus relative to SEQ ID NO: 12 results in no adverse change in functionality of Bxb1 integrase relative to wild-type. In embodiments, an extension of one or more amino acids at the N-terminus relative to SEQ ID NO: 12 results in improved functionality of Bxb1 integrase relative to wild-type.

[0271] In embodiments, the one or more PhiC31 integrases comprises an amino acid sequence that is at least 90% identical to an amino acids sequence of SEQ ID NO: 54, one or more amino acid mutations at positions selected from 1 , 2, 12, 14, 18, 24, 32, 36, 41 , 43, 44, 45, 51 , 55, 74, 77, 96, 103, 107, 117, 153,176, 188, 199, 200, 228, 230, 231, 235, 238, 240, 252, 255, 259, 262, 264, 266, 269, 274, 278, 302, 320,322, 331 , 333, 340, 344, 346, 347, 351 , 355, 359, 362, 364, 378, 382, 393, 396, 397, 399, 406, 410, 424,429, 431, 436, 438, 445, 448, 449, 450, 452, 457, 468, 475, 498, 501 , 505, 512, 516, 517, 520, 535, 536,549, 551, 552, 563, 580, 585, 586, 587, 590, 592, 600, 603, 604, 609, 616, and 621 , relative to SEQ ID NO 54, or a position corresponding thereto.

[0272] In embodiments, the one or more PhiC31 integrases comprises one or more amino acid mutations selected from M1 E, M1V, D2V, D2M, S12N, E14G, S18N, D32A, D36A, V411, D44A, V51 M, 1153V, E176D, A199T, H228Y, P230S, F231 L, H240R, D252G, A255G, S269N, P274S, M278L, T302A, A333T, A333S, A333D, A340V, G344V, G344D, G346S, R347K, L351V, L351 Q, D362N, D362G, L364M, E378K, S396N, S396R, A397T, N406S, A410T, G429S, E431V, W438R, W448R, E449D, A450D, E452K, E475D, G505V, G505S, A516T, P535L, T536A, A580V, K586Q, D590G, D592G, T600S, V603I, V603A, A604S, P609S, A616V, and R621 L.

[0273] In embodiments, the one or more PhiC31 integrases comprises a N-terminal addition of one or more amino acids. In embodiments, the N-terminal extension comprises an addition of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, or about 50 amino acids, relative to SEQ ID NO: 54. In embodiments, the N- terminal addition is selected from Table 6.DBl / 164937876.2 75Atorney Docket No.: KOM-003PC / 138774-5003Table 6: Illustrative N-terminal additions for PhiC31 integrases.

[0274] In embodiments, the PhiC31 integrase comprises one or more nuclear localization sequences (NLSs), as described herein. In embodiments, the NLS comprises one or more sequence selected from PKKKRKV (SEQ ID NO: 68), NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 69), PAAKRVKLD (SEQ ID NO: 70), RQRRNELKRSF (SEQ ID NO: 71), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 72), RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 73), VSRKRPRP (SEQ ID NO: 74), PPKKARED (SEQ ID NO: 75), PQPKKKPL (SEQ ID NO: 76), SALIKKKKKMAP (SEQ ID NO: 77), DRLRR (SEQ ID NO: 78), PKQKKRK (SEQ ID NO: 79), RKLKKKIKKL (SEQ ID NO: 80), REKKKFLKRR (SEQ ID NO: 81), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 82), RKCLQAGMNLEARKTKK (SEQ ID NO: 83), DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 84), KRTADGSEFESPKKKRKV (SEQ ID NO: 85),KRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86), GSHHHHHHGSGPKKKRKV (SEQ ID NO: 87), and GSGSGSHHHHHHGSGPKKKRKV (SEQ ID NO: 88). In embodiments, a NLS of the present disclosure comprises one or more mutations (e.g., amino acid substitutions, deletions, and / or insertions) with respect to any one of above sequences.

[0275] In embodiments, the one or more NLSs further improve the efficiency to the PhiC31 integrase by shunting the PhiC31 into the nucleus where it carries out its recombination. In embodiments, the PhiC31 has a single NLS. In embodiments, the NLS is located at the N-terminus. In embodiments, the NLS is located at the C-terminus. In embodiments, a NLS is located at the N-terminus and the C-terminus. In embodiments, the NLS is located within the protein sequence.DBl / 164937876.2 76Atorney Docket No.: KOM-003PC / 138774-5003

[0276] In embodiments, mutant PhiC31 integrases herein comprise an amino acid linker sequence, e.g., between a NLS and the N-term or C-term of the PhiC31 . Amino acid linker sequences are known in the art, for example, for producing fusion proteins, etc. Exemplary amino acid linker sequences, in embodiments, include glycine and serine (e.g., GG, GS, SS, SG), as well as repeated units of glycine-serine, aspartic acid (D) residues, and the like. Amino acid linkers, in embodiments, includes lengths of a single amino acid, about 2 amino acids in length, about 4 amino acids in length, about 6 amino acids in length, about 10 amino acids in length, or about 15 amino acids in length, including lengths therebetween. In embodiments, amino acid linkers are included between NLS peptides or sequence (e.g., bipartite sequences).

[0277] In embodiments, the NLS and / or N-terminal addition site is between M1 and D2 residues, e.g., of SEQ ID NO: 54. In embodiments, the N-terminal extension is include N-terminal relative to M1 of SEQ ID NO: 54. In embodiments, an extension of one or more amino acids at the N-terminus relative to SEQ ID NO: 54 results in no change in functionality of PhiC31 integrase relative to wild-type. In embodiments, an extension of one or more amino acids at the N-terminus relative to SEQ ID NO: 54 results in improved functionality of PhiC31 integrase relative to wild-type.

[0278] In embodiments, the PhiC31 integrase comprises one or more mutations, N-terminal additions, and / or one or more combinations of mutations and / or N-terminal additions as described in Table 6 and / or Table 7. In embodiments, the PhiC31 integrase comprises one or more mutations and / or N-terminal additions selected from Table 6 and / or Table 7.Table 7: Illustrative PhiC31 integrase mutants and illustrative mutant combinations. “X” refers to any amino acid different from the wild-type residue. Illustrative changes are relative to SEQ ID NO: 54.DBl / 164937876.2 77Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 78Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 79Atorney Docket No.: KOM-003PC / 138774-5003

[0279] In embodiments, the one or more PhiC31 integrases have at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations, e.g, as selected from Table 1 , Table 3, and / or Table 4 relative to SEQ ID NO: 54.

[0280] In embodiments, the one or more PhiC31 integrases comprise at least 90% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations.

[0281] In embodiments, the one or more PhiC31 integrases comprise at least 95% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, or at least 30 mutations.

[0282] In embodiments, the one or more PhiC31 integrases comprise at least 96% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at leastDB1 / 164937876.2 80Atorney Docket No.: KOM-003PC / 138774-500314 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations.

[0283] In embodiments, the one or more PhiC31 integrases comprise at least 97% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, or at least 15 mutations.

[0284] In embodiments, the one or more PhiC31 integrases comprise at least 98% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, or 10 mutations.

[0285] In embodiments, the one or more PhiC31 integrases comprise at least 99% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, or at least 5 mutations.

[0286] In embodiments, “mutation” herein (e.g., “X” in Tables 4 and 6) refers to a mutation that differs from the wild-type amino acid. In embodiments, the mutation (e.g., “X” in Tables 4 and 6) refers to a mutation from an amino acid to a stop codon, which truncates the polypeptide at that position. In embodiments, the mutation (e.g., “X” in Tables 4 and 6) refers to a deletion of an amino acid at that position. In embodiments, the mutation is a conservative substitution (e.g., mutation to an amino acid with similar physicochemical properties). For example, in embodiments, the amino acid is mutated from a first hydrophobic (or non-polar) amino acid to a second hydrophobic (or non-polar) amino acid (e.g., selected from the group consisting of glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and tryptophan (W)); from a first polar amino acid to a second polar amino acid (e.g., selected from the group consisting of serine (S), threonine (T), cysteine (C), asparagine (N), glutamine (Q), and tyrosine (Y)); from a first basic amino acid to a second basic amino acid (e.g., selected from the group consisting of arginine (R), lysine (K), and histidine (H)); from a first acidic amino acid to a second amino acid (e.g., selected from the group consisting of aspartate (D) and glutamate (E)). In embodiments, the mutation is a non-conservative substitution, where an amino acid from a first physicochemical group (e.g., hydrophobic / non-polar, polar, basic, acidic, etc.) is mutated to an amino acid from a physicochemical second group, where the groups differ in physicochemical properties.DBl / 164937876.2 81Atorney Docket No.: KOM-003PC / 138774-5003

[0287] In embodiments, the mutation (e.g., “X” in Tables 4 and 6; or the bolded underline residues in SEQ ID NOs: 12 and 54) refers to a mutation from the indicated amino acid to a deletion, a stop codon, or to an amino acid selected from alanine (A), arginine (R), asparagine (N), aspartate (D), cysteine (C), glutamate (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).Inducible Hamster-Specific Caspase-9 Fusion Protein

[0288] In aspects, described herein is an inducible hamster-specific caspase-9 fusion protein. In embodiments, methods herein include the use the inducible hamster-specific caspase-9 fusion protein, e.g., for positive and / or negative selection methods for cell line development.

[0289] In embodiments, the inducible hamster-specific caspase-9 fusion protein comprises about or at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 99. In embodiments, the inducible hamster-specific caspase-9 fusion protein comprises about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 92% sequence identity, about or at least about 94% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 99. In embodiments, the inducible hamster-specific caspase-9 amino acid sequence comprises the amino acid sequence of SEQ ID NO: 99. In embodiments, the inducible hamster-specific caspase-9 amino acid sequence consists of the amino acid sequence of SEQ ID NO: 99.

[0290] In embodiments, the inducible hamster-specific caspase-9 fusion protein comprises the amino acid sequence and / or is encoded by the nucleic acid sequence listed in Table 8.

[0291] In embodiments, the inducible hamster-specific caspase-9 is a fusion protein comprising a hamster caspase-9 fused to an FKBP12 drug-binding domain with an additional protein sequence or domain (e.g., such as a linker or stabilization sequence, as described herein). In embodiments, the hamster caspase- 9 fused at the N-terminus to the FKBP12 drug-binding domain. In embodiments, the hamster caspase-9 fused at the C-terminus to the FKBP12 drug-binding domain.

[0292] In embodiments, the inducible hamster-specific caspase-9 fusion protein forms a dimer (or other higher order forms) to adopt an active form that induces apoptosis in a host cell. In embodiments, the inducible hamster-specific caspase-9 fusion protein is inducible by a small molecule, e.g., such as AP20187,DBl / 164937876.2 82Atorney Docket No.: KOM-003PC / 138774-5003AP1903, rimiducid, or rapamycin. In embodiments, the induced dimerization results in the inducible hamsterspecific caspase-9 fusion protein becoming active and inducing apoptosis of a cell that expresses the inducible hamster-specific caspase-9 amino acid sequence.

[0293] Without withing to be bound by theory, the hamster-specific caspase-9 functions substantially similarly or better than a human-specific caspase-9 for cell selection purposes in certain cell lines (e.g., hamster cells, CHO cells), for example, as measured by cell toxicity due to fusion protein expression (before induction of dimerization with a small molecule inducer) and / or fidelity of cell apoptosis (efficacy of apoptosis after induction of dimerization with a small molecule inducer).Table 8. Illustrative nucleic acid sequence and amino acid sequence for inducible hamster-specific caspase- 9.DBl / 164937876.2 83Atorney Docket No.: KOM-003PC / 138774-5003Nucleic Acid Constructs

[0294] In aspects, described herein are nucleic acid molecules comprising a polynucleotide sequence encoding one or more elements for producing cell lines.

[0295] In embodiments, the one or more nucleic acids encode one or more cassettes as shown in one or more of Figs. 9-12. In embodiments, the one or more nucleic acids encode one or more integrases. In embodiments, the one or more nucleic acids encode a suicide gene, such as a fusion protein comprising a inducible hamster-specific caspase-9, thymidine kinase (TK), or other apoptosis-inducible protein. In embodiments, the one or more nucleic acids encode one or more protein-coding sequences. In embodiments, the one or more protein-coding sequences are separated by individual promoters, or 2A peptide sequences for ribozyme stalling.

[0296] In embodiments, the nucleic acid molecule comprises DNA (e.g., a genomically-integrated sequence) or RNA (e.g., a transcript or RNA-based intermediate). In embodiments, the DNA is circular or linear, or single stranded or double stranded, for example, as a non-integrating episomal DNA or long halflife plasmid or vector, or as a genomically-integrated sequence. In embodiments, the DNA is a plasmid, episome, viral vector, or non-viral vector.DB1 / 164937876.2 84Atorney Docket No.: KOM-003PC / 138774-5003

[0297] In embodiments, non-limiting schematic diagrams of nucleic acid constructs are illustrated in Figs. 9-12. In embodiments, the nucleic acid is a “donor DNA-specific” nucleic acid molecule. In embodiments, the nucleic acid is a “landing pad” nucleic acid molecule. All elements of nucleic acid molecules and nucleic acid constructs herein are understood to be adaptable across each.

[0298] In embodiments, nucleic acid molecules herein comprise one or more nucleic acid sequences selected from Table 9. In embodiments, nucleic acid molecules herein comprise one or more nucleic acid sequences selected from Table 16a. In embodiments, the nucleic acid molecules (and methods herein) comprise the Donor 1 and / or Donor sequences from Table 16a..Table 9: Illustrative sequences for use in nucleic acids herein.DBl / 164937876.2 85Atorney Docket No.: KOM-003PC / 138774-5003

[0299] In embodiments, the nucleic acid molecule comprises a promoter sequence operably linked to a polynucleotide sequence. In embodiments, the promoter comprises one or more of a mammalian gene promoter, viral promoter, inducible promoter, tissue-specific promoter, cell type-specific promoter, and bidirectional promoter. In embodiments, the inducible promoter comprises one or more of a Tet-On / Tet-Off, Cumate, Ecdysone, Mifepristone (GeneSwitch), Rapamycin (FKBP / FRB), RU486, Heat Shock, Doxycycline, Copper-inducible, and small mineral-based systems such as Zinc-inducible promoters.

[0300] In embodiments, the promoter comprises one or more native promoter (e.g., native mammalian gene promoter, viral promoter, tissue-specific promoter, or cell type-specific promoter). In embodiments, the promoter comprises one or more synthetic promoter or composite promoter (e.g., combining elements of two or more different promoters).

[0301] In embodiments, the promoter comprises one or more of a B29 promoter, CAG promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, CMV promoter, desmin promoter, Ef1a promoter, EGR1 promoter, elastase-1 promoter, elF2A1 promoter, endoglin promoter, FerH promoter, FerLDB1 / 164937876.2 86Atorney Docket No.: KOM-003PC / 138774-5003 promoter, fibronectin promoter, Flt-1 promoter, GAPDH promoter, GFAP promoter, GPIIb promoter, GRP78 promoter, GRP94 promoter, HSP70 promoter, Hspa5p promoter, ICAM-2 promoter, INF-0 promoter, Nphsl promoter, OG-2 promoter, PGK-1 promoter, ROSA promoter, SP-B promoter, SV40 promoter, SYN1 promoter, ubiquitin B promoter, WASP promoter, 0-actin promoter, and 0-kin promoter.

[0302] In non-limiting embodiments, the promoter is or comprises a CMV promoter which comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 100. In embodiments, the CMV promoter is or comprises the nucleic acid sequence of SEQ ID NO: 100.

[0303] In embodiments, the nucleic acid construct comprising the inducible hamster-specific caspase- 9 fusion protein comprises two or more att sequences. In embodiments, the two or more att sequences flank the polynucleotide sequence encoding the inducible hamster-specific caspase-9 fusion protein. In embodiments, the two or more att sequences are bacterial attachment (attB) sequences. In embodiments, the two or more att sequences are phage attachment (attP) sequences.

[0304] In embodiments, the att sequences herein belong to any integrase, or are compatible with any integrase. In embodiments, the att sequences is a stretch of nucleic acids that is suitable to accommodate integrase-mediated recombination between two nucleic acids. In embodiments, the integrase is a serine integrase. In embodiments, the serine integrase is Bxb1 (Mycobacterium). In embodiments, the serine integrase is PhiC31 (Streptomyces). In embodiments, the serine integrase is R4 (Streptomyces). In embodiments, the serine integrase is TP901 (Lactococcus). In embodiments, the serine integrase is y5 (E. coli). In embodiments, the serine integrase is gin (phage Mu) (E. coli). In embodiments, the serine integrase is Tn 3 (Klebsiella).

[0305] In embodiments, nucleic acids and methods herein comprise one or more integrase that is a large serine recombinase selected from Bxb1 , PhiC31 , R4, phiBTI , MJ1 , MR11, TP901-1 , A118, V153, phiRVI, phi370.1 , TG1 , WB, BL3, SprA, phiJoe, phiK38, Int2, Int3, Int4, Int7, Int8, Int9, IntIO, Inti 1 , Int12, Inti 3, L1, peaches, Bxz2, and SV1. Persons skilled in the art, with the benefit of this disclosure in its entirety, will understand how to utilize structure-function analysis to take mutations / modifications from Bxb1 to makeDBl / 164937876.2 87Atorney Docket No.: KOM-003PC / 138774-5003 equivalent mutations in other large serine recombinases and test their efficiency against wild-type enzymes e.g., using the methods described herein and in the art.

[0306] In embodiments, the att sequence used in nucleic acids herein is or comprises an att nucleic acid sequence selected from Table 1 , or a sequence having about or at least about 70% sequence identity thereto. In embodiments, att sequences used herein are selected from Table 1.

[0307] In embodiments, the one or more bacterial attachment (attB) sequences comprises about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95. In embodiments, the one or more bacterial attachment (attB) sequences is compatible with one or more phage attachment (attP) sequences comprising about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97. In embodiments, the one or more bacterial attachment (attB) sequences comprises a nucleic acid sequence selected from Table 1.

[0308] In embodiments, the one or more phage attachment (attP) sequences comprises about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to one of SEQ ID NOs: SEQ ID NOs: 2, 4, 6, and 96-97. In embodiments, the one or more phage attachment (attP) sequences is compatible with one or more bacterial attachment (attB) sequences comprising about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to the nucleic acid sequence of any oneDBl / 164937876.2 88Atorney Docket No.: KOM-003PC / 138774-5003 of SEQ ID NOs: SEQ ID NOs: 1, 3, 5, and 89-95. In embodiments, the one or more phage attachment (attP) sequences comprises a nucleic acid sequence selected from Table 1.

[0309] In embodiments, methods herein include introducing a nucleic acid having one or more at sequences into a cell. In embodiments, methods herein include Bxb1 recombinase-mediated catalysis between one or more attB and one or more attP.

[0310] In embodiments, nucleic acids herein comprise one or more homology directed repair (HDR) insulator sequences, one or more splice acceptor sequences, one or more splice donor sequences, one or more nucleic acid sequences encoding a 2A peptide, one or more insertion sequences, and / or one or more transcription terminator sequences.

[0311] In embodiments, nucleic acids herein comprise one or more homology directed repair (HDR) insulators. In embodiments, HDR insulators herein function as barrier insulators to prevent euchromatin silencing by the spread of neighboring heterochromatin in the cellular genome. In embodiments, this is done to support expression of one or more elements within the flanking HDR insulator sequences. Potent endogenous insulators are known to exist at several mammalian loci. Without wishing to be bound by theory, loci described herein are chosen (in part) due to their endogenous insulator capabilities to allow expression of genes placed therein. In embodiments, insulator sequences can be selectable by using preferred integration locations (e.g., AAV integration “host-spots”), for example with the AAVS1 locus.

[0312] In embodiments, the nucleic acid encodes one or more 2A peptide. In embodiments, the 2A peptides comprises one or more of a T2A, P2A, E2A, and F2A peptide sequence. In embodiments, the 2A peptide is located between two or more insertion sequences (e.g., between protein-coding regions), where the peptide enables ribosome skipping to translation two or more separate polypeptides from a single open reading frame. In embodiments, 2A peptides refer to a class of 18-22 amino acid-long peptides, which induce ribosomal skipping during translation. In embodiments, these peptides share a core sequence motif of DXEXNPGP (SEQ ID NO: 11 , where “X” denotes any amino acid) and are found in a wide range of viral families. In embodiments, 2A peptides are introduced to help generate polyproteins from a single open reading frame (ORF) in the genomic loci by causing the ribosome to fail at making a peptide bond during translation of the sequence, and then resume translation. In embodiments, 2A and 2A-like peptides for ribosomal skipping can be found for example in de Lima JGS and Lanza DCF, “2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology,” Viruses, (2021), Vol, 13, No. 11 :2160, the entire contents of which are incorporated by reference.DBl / 164937876.2 89Atorney Docket No.: KOM-003PC / 138774-5003

[0313] In embodiments, the 2A peptide comprises one or more sequence from Table 2. In embodiments, the donor plasmid encodes one or more 2A peptide of SEQ ID NOs: 7-10, and / or one or more 2A peptide having the consensus sequence of SEQ ID NO: 11 .

[0314] In embodiments, one or more 2A peptide is located between two or more protein-coding sequences. In embodiments, one or more 2A peptide sequences is located between the inducible hamsterspecific caspase-9 fusion protein and one or more protein-coding sequences for a protein of interest (POI).

[0315] In embodiments, nucleic acids comprise one or more transcription terminator sequences. In embodiments, the one or more transcription terminator sequences is or comprises one or more polyA sequences / signals (e.g., polyA tail). In embodiments, one or more transcription terminator sequences is placed at the end of a protein-coding sequence, e.g., for transcription and / or translational stop and / or to increase the half-life of any RNA transcripts derived from a protein-coding gene. In embodiments, the one or more transcription terminator sequences is or comprises a prokaryotic transcription terminator sequence, or a transcription terminator sequence derived from a prokaryotic organism or a virus that infects a prokaryotic organism. In embodiments, the one or more transcription terminator sequences is or comprises a eukaryotic transcription terminator sequence, or a transcription terminator sequence derived from a eukaryotic organism or a virus that infects a eukaryotic organism.

[0316] In embodiments, the one or more transcription terminator sequences is or comprises a sequence commonly used in mammalian expression plasmids to create mRNA (e.g., protein coding sequence transcription from plasmids). In embodiments, commonly used mammalian terminators include rabbit beta globin (rBG), simian vacuolating virus 40 (SV40), human growth hormone (hGH), and bovine growth hormone (BGH). In embodiments, the one or more transcription terminator sequences is or comprises a transcription terminator sequence derived from a nucleic acid sequence of rBG, SV40, hGH, and BGH. In embodiments, the terminator comprises the consensus sequence motif aauaaa, which effectuates both polyadenylation and termination.

[0317] In embodiments, the one or more transcription terminator sequences is or comprises a rabbit beta globin sequence (rBG), or a sequence derived therefrom. In embodiments, the rabbit beta globin sequence (rBG) comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at leastDBl / 164937876.2 90Atorney Docket No.: KOM-003PC / 138774-5003 about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101. In embodiments, the rabbit beta globin sequence (rBG) is or comprises the nucleic acid sequence of SEQ ID NO: 101 .

[0318] In embodiments, the one or more transcription terminator sequences is adjacent to one or more polyA sequences (e.g., is located immediately downstream of a polyA sequence). For example, in non-limiting embodiments, a polyA sequence is located immediate upstream of a rabbit beta globin sequence (rBG), or a sequence derived therefrom.

[0319] In embodiments, the one or more splice acceptor or splice donor sequences comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 102. In embodiments, the one or more splice acceptor sequences is or comprises the nucleic acid sequence of SEQ ID NO: 102.

[0320] In aspects, described herein are nucleic acids referred to as a “donor DNA-specific” nucleic acid molecule or construct which comprises a first nucleic acid sequence encoding a promoter and a second nucleic acid sequence encoding at least one start codon, at least one splice acceptor or splice donor sequence, and an att sequence. In embodiments, the second nucleic acid sequence comprises an attB sequence and at least one splice donor sequence. In embodiments, the promoter is operably linked to drive expression from the start codon and the start codon is not linked to a protein-coding sequence (e.g., is not currently associated with a downstream protein-coding sequence, but will become linked to a protein-coding sequence after Bxb1 -mediated recombination in a cell).

[0321] In embodiments, the “donor DNA-specific” construct comprises the construct formula: Promoter- ATG-SA / SD-aff, where the “Promoter” is any promoter (e.g., as described herein), “ATG” is a start codon, and the “SA / SD” sequence comprises a splice acceptor or splice donor, and “att” is any att sequence described herein. In embodiments, the “donor DNA-specific” construct is a mix-and-match nucleic acid sequence that is suitable for Bxb1 -based recombination and is configured to be inserted upstream of a gene in a host cell genome to drive its expression after Bxb1 -mediated recombination (e.g., as illustrated in Fig. 10). In embodiments, the “mix-and-match” aspect is that the construct is compatible with a variety of promoters, aft sequences, and selectable marker proteins, as needed. In embodiments, the “donor DNA-DBl / 164937876.2 91Atorney Docket No.: KOM-003PC / 138774-5003 specific” construct is knocked-in upstream of a genomically-i ntegrated sequence to drive expression of one or more genomically-integrated sequence (e.g., as illustrated in Fig. 10). In embodiments, this knock-in donor DNA-specific sequence creates a new open reading frame (ORF) where one did not exist previously.

[0322] In embodiments, the first nucleic acid sequence (e.g., promoter or enhancer sequence) is upstream (5’) of the second nucleic acid sequence (e.g., start codon, SA / SD, and / or att) and located substantially adjacent thereto such that the promoter sequence is suitable to drive expression from the start codon in the second nucleic acid sequence. In embodiments, “substantially adjacent” refers to being within close enough proximity that gene expression is possible, including for example, a distance of up to about or at least about 1000 bp in distance. Persons skilled in the art, with the benefit of this disclosure in its entirety, will understand how to detect and measure gene expression to determine the distances and placement of nucleic acid elements herein for the purposes described herein.

[0323] In embodiments, the donor DNA-specific construct comprises one or more att sequence, e.g., such as an att sequence upstream of the promoter, and located within a region downstream of the promoter and start codon. In embodiments, the nucleic acid molecule comprises at least two aft sequences, where the at least two att sequences flank the first and second nucleic acid sequence.

[0324] In embodiments, the donor DNA-specific nucleic acid molecule promoter comprises any promoter described herein, including modifications thereof. For example, in non-limiting embodiments, the first nucleic acid sequence comprises a CMV promoter that has about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, or about or at least 99% sequence identity to the nucleic acid of SEQ ID NO: 100. In embodiments, the CMV promoter is or comprises the nucleic acid sequence of SEQ ID NO: 100.

[0325] In embodiments, the donor DNA-specific nucleic acid molecule att comprises any aft sequences described herein, including modifications thereof. For example, in non-limiting embodiments, the second nucleic acid sequence comprises an attP sequence having about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, orDBl / 164937876.2 92Atorney Docket No.: KOM-003PC / 138774-5003 about or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NOs: 2, 4, 6, 96-97. In embodiments, the attP sequence has an ac core dinucleotide sequence (e.g., SEQ ID NO: 96 or 97).

[0326] In embodiments, the donor DNA-specific nucleic acid molecule comprises any splice acceptor and / or splice donor sequences described herein, including modifications thereof. For example, in non-limiting embodiments, the second nucleic acid sequence comprises at least one splice donor or splice acceptor sequence having about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 102. In embodiments, the at least one splice donor or splice acceptor sequence is or comprises the nucleic acid sequence of SEQ ID NO: 102.

[0327] In embodiments, the donor DNA-specific nucleic acid molecule comprises a second nucleic acid sequence having about or at least 70% sequence identity, about or at least 75% sequence identity, about or at least 80% sequence identity, about or at least 85% sequence identity, about or at least 90% sequence identity, about or at least 95% sequence identity, about or at least 96% sequence identity, about or at least 97% sequence identity, about or at least 98% sequence identity, about or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 105. In embodiments, the second nucleic acid sequence is or comprises the nucleic acid sequence of SEQ ID NO: 105.

[0328] In embodiments, the donor DNA-specific nucleic acid molecule comprises a first nucleic acid sequence encoding a CMV promoter having at least 70% sequence identity to SEQ ID NO: 100, and a second nucleic acid sequence encoding a start codon, slice donor, and attP sequence having at least 70% sequence identity to SEQ ID NO: 105.

[0329] In aspects, the nucleic acid molecule is a “landing pad” nucleic acid molecule or construct that substantially corresponds to the nucleic acid construct denoted by 1, 2, 3 in Fig. 10, where 1 is an att sequence (attB or attP), 2 is comprises at least one splice acceptor or splice donor sequence, and 3 is a protein-coding sequence (e.g., selectable marker protein or resistance marker protein).

[0330] In embodiments, the landing pad nucleic acid molecule comprises one or more att sequence, one or more splice acceptor or splice donor sequence, and one or more protein-coding nucleic acid sequence, where the protein-coding sequence lacks a start codon, but encodes the remainder of a selectableDBl / 164937876.2 93Atorney Docket No.: KOM-003PC / 138774-5003 marker protein, and is located downstream of the splice acceptor or splice donor sequence and the att sequence. In embodiments, a transcriptional terminator sequence is located downstream of the proteincoding nucleic acid sequence.

[0331] In embodiments, the landing pad nucleic acid sequence is genomically-integrated in a cell and is reacted with via Bxb1 -mediated recombination to insert an exogenous nucleic acid sequence comprising a start codon and promoter, which completes the protein-coding sequence of the landing pad nucleic acid sequence and allows expression thereof. In embodiments, the promoter is a constitutively active promoter, including any promoters described herein.

[0332] In embodiments, the selectable marker protein is an antibiotic resistance enzyme. In embodiments, the antibiotic resistance enzyme is for detoxifying an antibiotic that is toxic to eukaryotic cells. In embodiments, the antibiotic resistance enzyme is or comprises an enzyme for detoxifying hygromycin B, geneticin (G418), gentamicin, puromycin, blasticidin, neomycin, amphotericin B, actinomycin D, zeocin (bleomycin), or mycophenolic acid.

[0333] In embodiments, the antibiotic resistance enzyme is hygromycin B phosphotransferase. In embodiments, the hygromycin B phosphotransferase comprises about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 104. In embodiments, the hygromycin B phosphotransferase is or comprises the amino acid sequence of SEQ ID NO: 104.

[0334] In embodiments, the protein-coding nucleic acid sequence encodes hygromycin B phosphotransferase and comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 103. In embodiments, the protein-coding nucleic acid sequence encoding hygromycin B phosphotransferase is or comprises the nucleic acid sequence of SEQ ID NO: 103.

[0335] In embodiments, the landing pad nucleic acid molecule transcriptional terminator sequence comprises any transcriptional termination sequences described herein, including modifications thereof. ForDBl / 164937876.2 94Atorney Docket No.: KOM-003PC / 138774-5003 example, in non-limiting embodiments, the landing pad transcription terminator sequences is or comprises a transcription terminator sequence derived from a nucleic acid sequence of rabbit beta globin (rBG), e.g., having about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101. in embodiments, the rabbit beta globin sequence (rBG) is or comprises the nucleic acid sequence of SEQ ID NO: 101.

[0336] In embodiments, the landing pad transcriptional terminator sequence terminates transcription of the protein-coding nucleic acid sequence encoding a selectable marker protein. In embodiments, the transcriptional terminator comprises any transcriptional terminator described herein, including derivatives thereof. In embodiments, the transcriptional terminator sequence is or comprises one or more polyA sequences / signals (e.g., polyA tail), and / or a transcriptional terminator consensus sequence.

[0337] In embodiments, the landing pad nucleic acid molecule aft comprises any aft sequences described herein, including modifications thereof. For example, in non-limiting embodiments, the aff sequence is a bacterial attachment (attB) that has an ac core dinucleotide sequence and comprises about or at least about 70% sequence identity, about or at least about 75% sequence identity, about or at least about 80% sequence identity, about or at least about 85% sequence identity, about or at least about 90% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NOs: 1, 3, 5, and 89-95.

[0338] In embodiments, references herein to an “insertion sequence,” “gene of interest,” “protein of interest,” and variations thereof, refers to a transcribable / translatable element, including protein-coding and non-protein-coding sequences of interest (e.g., including sequences that are translated from an mRNA or pre-mRNA transcript transcribed from the insertion sequence).Cell Lines and Compositions

[0339] Described herein, in embodiments, are compositions comprising fusion proteins, nucleic acid molecules, plasmids, vectors (e.g., viral vectors, AAVs, non-viral vectors, etc.), and / or clonal cell populations produced by or used by methods herein. In embodiments, a cell line or cell population is “clonal” in that eachDBl / 164937876.2 95Atorney Docket No.: KOM-003PC / 138774-5003 cell in the cell line or cell population originates from a single cell, or is a population of cells with substantially the same desired genetic amendment(s), where each subsequent cell in the cell line or cell population is substantially genetically identical and maintains the genetic amendment(s). In embodiments, a “cell line” refers to a “clonal cell population,” and / or a population of cells that comprise substantially similar phenotype and genotype. In embodiments, the cell line has the same Bxb1 -mediated site-specific genomic insertion.

[0340] In embodiments, the clonal cell population (and compositions thereof) comprises one or more attB sites from Cas9-directed reverse transcription of one or more donor plasmid sequence introduced into one or more genomic loci. In such embodiments, the cell line is primed for insertion by one or more subsequent users or methods. In embodiments, such clonal cell populations represent off-the-shelf cell lines for recombinant protein production.

[0341] In embodiments, the clonal cell population (and compositions thereof) comprises one or more atiB / attP sequences compatible with a wild-type aft integrase sequence introduced into one or more genomic loci. In embodiments, the clonal cell population comprises an inducible hamster-specific caspase-9 fusion protein gene. In embodiments, the clonal cell population comprises a “landing pad” sequence, as described herein. In such embodiments, the cell line is primed for insertion by one or more subsequent users or methods, e.g., for negative / positive selection processes. In embodiments, such clonal cell populations represent off-the-shelf cell lines for recombinant protein production.

[0342] In embodiments, the clonal cell population (and compositions thereof) comprises one or more insertion sequences integrated into the one or more attB / attP sites from one or more Bxb1 integrases. In such embodiments, the cell line comprises one or more insertion sequences and may still undergo additional rounds of insertion within or adjacent to the first site by one or more subsequent users or methods (e.g., gene stacking). In embodiments, such clonal cell populations represent multiplexed off-the-shelf cell lines for recombinant protein production.

[0343] In embodiments, the clonal cell population comprises any cell type, e.g., as described herein.

[0344] In embodiments, nucleic acids herein (and compositions thereof) are lyophilized, or suspended in one or more excipients, including nuclease-free water, Tris buffer, salt (e.g., magnesium chloride), etc.

[0345] In embodiments, clonal cell populations herein (and compositions thereof) are suspended in one or more excipients, e.g., such as cell media (e.g., MEM, DMEM, RPMI 1640, Ham’s F-12K, Ham’s F-10, GMEM, BME, McCoy's 5A, M200, Leibovitz's L-15, or IMDM), fetal bovine serum (FBS), amino acids (L-DBl / 164937876.2 96Atorney Docket No.: KOM-003PC / 138774-5003 glutamate, non-essential amino acids (NEAA)), sodiumpyruvate, pH buffer (HEPES, etc.), NaCI, pH indicator (e.g., phenol red), hydrocortisone, dexamethasone, cell factors, (e.g., epidermal growth factor, insulin, etc.). In embodiments, clonal cell populations herein (and compositions thereof) are suspended in one or more cryoprotectant to protect cells for freeze-thaw cycles, such as DMSO, glycerol, sucrose, ethylene glycol (EG), propylene glycol (PG), and polyethylene glycol (PEG).Kits

[0346] In embodiments, enzymes, nucleic acids, and / or host cells of the present disclosure are assembled into a kit. In embodiments, the kit comprises enzymes, nucleic acids, and / or host cells in one or more formulations for use in one or more methods as described herein.

[0347] In embodiments, the kit comprises one or more vials (e.g., cryovials) of one or more clonal cell population, e.g., as described herein for off-the-shelf recombinant protein production. In embodiments, the kit comprises one or more vials (e.g., cryovials) of one or more nucleic acids (e.g., an inducible hamsterspecific caspase-9 fusion protein cassette, a donor DNA-specific cassette, a Bxb1 -expression cassette, etc.). In embodiments, the one or more nucleic acids are assembled into a plasmid, vector (e.g., viral or non-viral) and / or come with an excipient or reagent for contacting cells.

[0348] The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kits described herein may further comprise components needed for performing the manufacturing methods described herein. Each component of the kits, where applicable, may be provided in liquid form (e.g., in aqueous solution, a buffer, or cell media). In embodiments, some of the components are reconstitutable or otherwise processible (e.g., nucleic acids that reconstitute in nuclease-free water, or frozen cell stocks for seeding plates), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit.

[0349] In embodiments, the kits may optionally include instructions and / or promotion for use of the components provided. As used herein, "instructions" can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. As used herein, "promoted" includes all methods of doing business including methods of education, engineering instruction, scientific inquiry, discovery or development, academic research, manufacturing, chemical, cosmetic, and pharmaceutical industry activityDBl / 164937876.2 97Atorney Docket No.: KOM-003PC / 138774-5003 including sales, and any advertising or other promotional activity including written, oral, and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0350] The kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag.

[0351] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limiting of the remainder of the disclosure in anyway whatsoever.EXAMPLESExample 1: Mutant Bxb1 site-specific genomic integration at TRAC locus

[0352] This Example demonstrates using a donor DNA containing for comparing insertion efficiency of wild-type Bxb1 integrase to improved, hyperactive Bxb1 integrase derivatives using the wild-type attB for integration at the TRAC locus. A donor DNA (6.6 kbp in size) comprising a DNA sequence having the wildtype Bxb1 attP was constructed. Host cells were transfected with the donor DNA and three “helper” plasmids each encoding a hyperactive integrase. The first helper plasmid encoded a Bxb1 having hyperactive mutations. The second helper plasmid encoded a Bxb1 having hyperactive mutations and additional mutations for binding the left side of the TRAC target. The third helper plasmid encoded a Bxb1 having hyperactive mutations and additional mutations for binding the right side of the TRAC target. These three helpers are co-transfected with the donor plasmid containing an att site with a matching dinucleotide core to the TRAC target.

[0353] Insertion efficiency at the TRAC locus was measured using droplet digital polymerase chain reaction (ddPCR) probes that assesses the presence of the donor DNA at the TRAC locus and / or other locations in the genome. Such primers included a forward or reverse primer that is identical to or complementary with a genomic sequence at or near the target TRAC locus site and the other primer identical to or complementary with a sequence from the donor DNA.DBl / 164937876.2 98Atorney Docket No.: KOM-003PC / 138774-5003

[0354] As shown in Fig. 3, several Bxb1 mutants exhibited increased site-specific insertion efficiency at the TRAC locus compared to wild-type Bxb1. Table 10 summarizes the efficiency statistics shown graphically in Fig. 3.Table 7: Bxb1 insertion efficiencies at TRAC locus.DBl / 164937876.2 99Atorney Docket No.: KOM-003PC / 138774-5003

[0355] Next, nuclear localization sequences (NLSs) were tested for the ability to further improve mutant Bxb1 efficiency. A second set of experiments were performed, as described previously, to compare the ratio of insertion relative to a cognate Bxb1 lacking the NLS.

[0356] As shown in Fig. 4, several Bxb1 mutants with a NLS exhibited increased site-specific insertion efficiency at the TRAC locus relative to the cognate mutant Bxb1 lacking the NLS. This was also observed with the wild-type Bxb1 (wt Bxb1 vs. wt Bxb1 + NLS). Table 11 summarizes the efficiency statistics shown graphically in Fig. 4.Table 11 : Bxb1 insertion efficiencies at TRAC locus with NLS.

[0357] The data demonstrated, inter alia, that the mutant Bxb1 integrases exhibited an increased efficiency of site-specific insertion of the donor DNA at the target TRAC locus site(s) compared to the wild type Bxb1 integrase. The data demonstrated, inter alia, that the mutant Bxb1 integrases exhibited anDBl / 164937876.2 100Atorney Docket No.: KOM-003PC / 138774-5003 increased total amount of insertion of the donor DNA compared to the wild type Bxb1 integrase. The data demonstrated, inter alia, that each of the mutant Bxb1 integrases exhibited an increased efficiency of recombination with the addition of a NLS sequence, and the same was observed for wild-type Bxb1 .Example 2: Mutant Bxb1 site-specific genomic integration at ROSA26 locus

[0358] In this Example, Cas9-directed reverse transcription was used to insert an attB at the ROSA26 locus in HEK293T cells, as described in Hew, et al., “Directed evolution of hyperactive integrases for site specific insertion of transgenes,” Nucleic Acids Res. (2024) Vol. 52, No. 14: e64, the entire contents of which is incorporated by reference herein. Insertion efficiency at the ROSA26 locus of the 6.6 kbp donor DNA was measured using droplet digital polymerase chain reaction (ddPCR) using probes that assesses the presence of the donor DNA at the ROSA26 locus and / or other locations in the genome. Such primers included a forward or reverse primer that is identical to or complementary with a genomic sequence at or near the target ROSA26 locus site and the other primer identical to or complementary with a sequence from the donor DNA. Insertion efficiency was compared between wild-type Bxb1 and mutant Bxb1 , with and without nuclear localization sequences (NLSs).

[0359] As shown in Fig. 5, the efficiencies of Cas9-directed reverse transcription at the ROSA26 locus ranged from ~ 74% to 95%, with integration efficiencies (I nt) for each mutant outperforming the wild-type Bxb1. The inclusion of NLSs also improved both wild-type and mutant Bxb1 integration efficiency. Table 12 summarizes the efficiency statistics shown graphically in Fig. 5.Table 12: Bxb1 insertion efficiencies at ROSA26 locus with and without NLS.DBl / 164937876.2 101Atorney Docket No.: KOM-003PC / 138774-5003

[0360] As shown in Fig. 6, the integration efficiencies at ROSA26 using Cas9-directed reverse transcription with C-terminal NLSs were compared between select mutant Bxb1 recombinases and wild-type Bxb1. Each mutant outperformed the wild-type Bxb1. The C-terminal NLSs also improved the integration efficiency of wild-type Bxb1 and several Bxb1 mutants. Table 13 summarizes the efficiency statistics shown graphically in Fig. 6.Table 13: Bxb1 insertion efficiencies at ROSA26 locus with Cas9-directed reverse transcription with and without C-terminal NLS. Mutations are relative to SEQ ID NO: 12. npNLS = nucleoplasmin NLS KRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86).

[0361] As shown in Fig. 7, the integration efficiencies at ROSA26 using Cas9-directed reverse transcription with NLSs were compared between select mutant Bxb1 recombinases and wild-type Bxb 1 . Each mutant outperformed the wild-type Bxb1 . The NLSs also improved the integration efficiency of wild-type Bxb1 and several Bxb1 mutants. Table 14 summarizes the efficiency statistics shown graphically in Fig. 7.DBl / 164937876.2 102Atorney Docket No.: KOM-003PC / 138774-5003Table 14: Bxb1 insertion efficiencies at ROSA26 locus with Cas9-directed reverse transcription with and without NLS. Mutations are relative to SEQ ID NO: 12. bpNLS = bipartite NLS KRTADGSEFESPKKKRKV (SEQ ID NO: 85): npNLS = nucleoplasmin NLSKRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86).

[0362] As shown in Fig. 8, the integration efficiencies at ROSA26 using Cas9-directed reverse transcription with NLSs were compared between select mutant Bxb1 recombinases and wild-type Bxb1 . The NLSs also improved the integration efficiency of wild-type Bxb1 and several Bxb1 mutants. Table 15 summarizes the efficiency statistics shown graphically in Fig. 8.Table 15: Bxb1 insertion efficiencies at ROSA26 locus with Cas9-directed reverse transcription with and without NLS. Mutations are relative to SEQ ID NO: 12. bpNLS = bipartite NLS KRTADGSEFESPKKKRKV (SEQ ID NO: 85): npNLS = nucleoplasmin NLSDBl / 164937876.2 103Atorney Docket No.: KOM-003PC / 138774-5003KRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86). HisNLS = GSGSGSHHHHHHGSGPKKKRKV (SEQ ID NO: 88).DBl / 164937876.2 104Atorney Docket No.: KOM-003PC / 138774-5003

[0363] The data demonstrated, inter alia, that the mutant Bxb1 integrases exhibited an increased efficiency of site-specific insertion of the donor DNA at the target ROSA26 locus site(s) compared to the wild type Bxb1 integrase. The data demonstrated, inter alia, that the mutant Bxb1 integrases exhibited an increased total amount of insertion of the donor DNA compared to the wild type Bxb1 integrase. The data demonstrated, inter alia, that Bxb1 integrases (mutant or wild-type) exhibited an increased efficiency of recombination with the addition of a NLS sequence.Example 3; Mutant Bxb1 -mediated cell line construction for site-specific genomic integration and production of a recombinant protein

[0364] As shown in Fig. 11 , a Donorl knock-in construct was synthesized that incorporates two attB sites recognized by a Bxb 1 integrase. Between the two attB sites was a cassette that co-expressed GFP and a herpes simplex virus (HSV) thymidine kinase (TK) suicide gene (activatable by small molecule, ganciclovir). GFP was used to enrich cells with on-target genomic insertion and to verify that expression levels from the cassette site remain consistent over time. TK enabled the selection of on-target knock-in of the GOI. An on- target knock-in replaced the GFP / TK cassette with the GOI. Through ganciclovir treatment, parental cells and those with random insertion, both containing TK, were eliminated leaving only the cells with the correct insertion (e.g., as shown in Fig. 12).

[0365] The Donorl cassette design was created using a plasmid designed to insert at high-expressing genomic sites, such as the S100A locus utilizing CRISPR HDR with 300 bp homology arms. Each plasmid contains two attB sites with orthogonal core sequences to ensure correct orientation of recombination. The different central dinucleotides between the attB sites ensured compatibility with the attP sites on the Donor2 gene-of-interest (GOI). Several designs are possible:

[0366] Approach 1 : utilized the strong EF1 a promoter to drive expression of GFP and TK, separated by a T2A ribosome-skipping sequence. This allowed co-expression of both proteins without fusion, with GFP for monitoring expression and TK enabling the removal of cells without successful Donor2 GOI insertion after the second transfection.DBl / 164937876.2 105Atorney Docket No.: KOM-003PC / 138774-5003

[0367] Approach 2: this approach used GS selection to introduce a second P2A ribosome-skipping sequence connected to the glutamine synthetase (GS) selection gene. Using GS-deficient cells, only those successfully integrating the plasmid will survive in a glutamine-free medium. GS expression can be regulated by methionine sulfoximine (MSX) inhibition or by using GS knockout cells.

[0368] Approach 3: this approach used an alternative promoter strategy which employed a separate PGK promoter to drive GS gene expression, instead of linking GFP, TK, and GS with ribosome-skipping sequences. This design provides increased control over GS expression, potentially aiding in selection.

[0369] The Donor2 GOI design was inserted after the Donorl cassette was inserted into the cell line with stably integrated GFP and TK. The Donor2 was introduced into the cells via a second electroporation step. This step introduced the Donor2 GOI plasmid, containing two attP sites and the transgene along with mRNA encoding the hyperactive Bxb1 integrase into the cells. The integrase mediated precise integration at the Donorl locus, replacing both GFP and TK with the GOI. The Donorl construct backbone included a TK gene, allowing for ganciclovir selection to eliminate cells with unwanted backbone integration (e.g., at one or more sites outside of the desired location). Consequently, the final cells exclusively contained the GOI without any plasmid backbone.

[0370] To maximize recombinant protein expression (e.g., mA), several strategies are possible:

[0371] Strategy 1 : a dual promoter utilized with two promoters in a head-to-toe orientation (e.g., as shown in Fig. 11), each driving mAb heavy and light chain genes linked by T2A sequences to maximize protein output.

[0372] Strategy 2: stress response promoter or strong constitutive promoter, such as the Hspa5 promoter, creates a feedback loop for mAb productivity late in the culture period. The strong EF1 a promoter drives expression at all stages.

[0373] Strategy 3: a delayed expression method using a bidirectional doxycycline-inducible TRE promoter can delay GOI expression until cell expansion, benefiting cell health.

[0374] CRISPR HDR was used to insert GFP and a Thymidine Kinase suicide gene flanked by att sites to the S100A locus in CHO cells. Clones were generated that contained the intended insert which was verified by PCR (as shown in Fig. 13A) where products that contained the junction of the insert and genome confirmed site-specific insertion. The clonally expanded cells were >99% GFP positive, as shown in Fig. 13B.DBl / 164937876.2 106Atorney Docket No.: KOM-003PC / 138774-5003Control cells without the HDR landing pad insert did not produce PCR products or express GFP (e.g., as shown in the middle two lanes of the gel depicted in Fig. 13A and in the “neg control” of Fig. 13B).

[0375] After confirming GFP / TK knock-in, the clonal CHO cell line was then electroporated with mRNA encoding hyperactive Bxb1 integrase and a Donor2 plasmid carrying the expression cassette of the trastuzumab mAb under the Hspa5 promoter (e.g., as shown in Fig. 11). Integration was validated by loss of GFP and replacement with the mAb sequence. Fig. 14A shows GFP expression by flow cytometry which confirmed stable site-specific genomic integration of GFP and the suicide gene in CHO cells. Fig. 14B depicts that at GOI (~14 kb) was used in combination with the mutant Bxb1 described herein to knock-in the GOI where the GFP / suicide gene is present. As shown in Fig. 14C, negative selection using the thymidine kinase / GCV system removed non-integrated cells, resulting in a substantially homogenous site-specific genomically integrated cell line. As shown in Fig. 15, ELISA assays revealed a peak titer of up to about 0.7 g / L in shake flasks. Control CHO cells that did not receive an electroporation pulse (No EP Control) did not uptake mRNA did not express the GOI.

[0376] These data demonstrated, inter alia, that mutant Bxb1 integrases described herein are efficient for site-specific genomic integration to produce cell lines. Moreover, methods of gene stacking are useful for sequential site-specific genomic knock-in. Here, for example, a first negative selection cassette was site- specifically integrated at a safe harbor locus followed by a second round of insertion that was targeted within the suicide cassette that excised the suicide gene, replacing it with the gene-of-interest. This type of cell line production method resulted in a highly homogenous clonal cell population that achieved high expression titers of recombinant protein. tbaleTable 16a: Nucleic acid sequences of elements for positive / qenitive selection and cell line development.DBl / 164937876.2 107Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 108Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 109Atorney Docket No.: KOM-003PC / 138774-5003DBl / 164937876.2 110Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 111Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 112Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 113Atorney Docket No.: KOM-003PC / 138774-5003DBl / 164937876.2 114Atorney Docket No.: KOM-003PC / 138774-5003DBl / 164937876.2 115Atorney Docket No.: KOM-003PC / 138774-5003DBl / 164937876.2 116Atorney Docket No.: KOM-003PC / 138774-5003Example 4: Inducible hamster-specific caspase-9 fusion protein for cell selection.

[0377] CHO cells were transiently transfected with plasmid DNA encoding a bicistronic vector with a inducible hamster caspase-9 fusion protein and GFP separated by a T2A self-cleavage peptide. This ensured that expression of GFP was linked to the expression of the suicide gene in a 1 -to-1 stoichiometry. After transient transfection, the cells were incubated for 3 days with AP20187, which is a small molecule chemical inducer of the inducible hamster caspase-9 fusion. Control cells were transected with the thymidine kinase (TK) suicide gene that did not respond to AP20187. Table 16b provides the nucleic acid sequences and amino acid sequences of GFP, T2A peptides, and inducible hamster-specific caspase-9.Table 16b: Nucleic acid sequence and amino acid sequences of GFP, T2A peptide, and inducible hamsterspecific caspase-9 for positive / genitive selection and cell line development.DBl / 164937876.2 117Atorney Docket No.: KOM-003PC / 138774-5003DB1 / 164937876.2 118Atorney Docket No.: KOM-003PC / 138774-5003

[0378] As shown in the flow cytometry data of Fig. 16A, the inducible hamster caspase-9 fusion protein was successful in triggering a clear loss in cell viability from the presence of the small molecule inducer, AP20187 (as quantified in Fig. 16B). As shown in the flow cytometry data of Fig. 16C, the inducible hamster caspase-9 fusion protein was successful in triggering a clear loss of GFP from the presence of the small molecule inducer, AP20187 (as quantified in Fig. 16D). This was observed for all concentrations tested, from 0.5 nM to 10 nM, indicating the hamster caspase fusion protein is highly sensitive to induction. Bulk cell viability loss was also observed for other concentrations over 0.5 nM. Control cells transected with the thymidine kinase (TK) suicide gene that did not respond to AP20187 in either cell viability or GFP loss.

[0379] These data demonstrated, inter alia, that the inducible hamster-specific caspase-9 fusion proteins herein are useful for the positive or negative selection procedures, as described herein. For example, this construct is useful for use with Bxb1 (like in Example 3 in place of other suicide genes, such as TK). The inducible hamster caspase-9 fusion protein may be used over human analogs specifically for CHO cells due to the species agreement.DEFINITIONS

[0380] The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this disclosure belongs.

[0381] As used herein, “a,” “an,” or “the” can mean one or more than one.

[0382] Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.

[0383] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.DBl / 164937876.2 119Atorney Docket No.: KOM-003PC / 138774-5003

[0384] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”

[0385] In embodiments, as used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[0386] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[0387] In embodiments, the disclosure is directed to the following embodiments:

[0388] Embodiment 1. A method for generating a cell line comprising: (a) introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the single genomic locus, and wherein the one or more nucleic acids encodes: (i) one or more bacterial attachment (attB) sites and / or (ii) one or more phage attachment (attP) sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more integration sequences by one or more integrases; and (b) culturing a clonal cell population from the cell to generate the cell line.

[0389] Embodiment 2. The method of embodiment 1 , wherein the one or more nucleic acids encodes one or more one or more attB sites or one or more attP sites, optionally a single attB site or a single attP site.

[0390] Embodiment 3. The method of embodiment 1 or 2, wherein the one or more bacterial attachment (at B) sites comprises a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 1 , 3, and 5; and / or wherein the one or more bacterial attachment (attB) sites is compatible with one or more phage attachment (attP) sites, optionally comprising a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 2, 4, and 6.DBl / 164937876.2 120Atorney Docket No.: KOM-003PC / 138774-5003

[0391] Embodiment 4. The method of embodiment 1 or 2, wherein the one or more bacterial attachment (atiP) sites comprises a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 2, 4, and 6; and / or wherein the one or more bacterial attachment (attP) sites is compatible with one or more phage attachment (affB) sites, optionally comprising a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 1 , 3, and 5.

[0392] Embodiment 5. The method of any one of embodiments 1-4, wherein the one or more nucleic acids comprises one or more homology directed repair (HDR) insulator sequence and one or more attB site and / or atP site flanking one or more kill switch gene, one or one or more promoter, one or more 2A peptide sequence, one or more insertion sequence, and / or one or more polyA tail.

[0393] Embodiment 6. The method of embodiment 5, wherein the promoter comprises one or more of a mammalian gene promoter, viral promoter, inducible promoter, and bidirectional promoter.

[0394] Embodiment 7. The method of embodiment 6, wherein the promoter comprises one or more of a B29 promoter, CAG promoter, CD 14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, CMV promoter, desmin promoter, Ef1 a promoter, EGR1 promoter, elastase-1 promoter, elF2A1 promoter, endoglin promoter, FerH promoter, FerL promoter, fibronectin promoter, Flt-1 promoter, GAPDH promoter, GFAP promoter, GPU b promoter, GRP78 promoter, GRP94 promoter, HSP70 promoter, Hspa5p promoter, ICAM- 2 promoter, INF- promoter, Nphsl promoter, OG-2 promoter, PGK-1 promoter, ROSA promoter, SP-B promoter, SV40 promoter, SYN1 promoter, ubiquitin B promoter,, WASP promoter, 0-actin promoter, and - kin promoter.

[0395] Embodiment 8. The method of any one of embodiments 5-7, wherein the one or more 2A peptide comprises one or more of a T2A, P2A, E2A, and F2A peptide sequence.

[0396] Embodiment 9. The method of embodiment 8, wherein the one or more 2A peptide sequences comprises one or more amino acid sequence of SEQ ID NOs: 7-10.

[0397] Embodiment 10. The method of any one of embodiments 5-9, wherein the one or more 2A peptide sequences is located between two or more protein-coding sequences.

[0398] Embodiment 11. The method of any one of embodiments 5-10, wherein the kill switch gene comprises one or more of a suicide gene or molecular switch which induces cell death or apoptosis.DBl / 164937876.2 121Atorney Docket No.: KOM-003PC / 138774-5003

[0399] Embodiment 12. The method of any one of the preceding embodiments, wherein the introducing comprises using Cas9-directed reverse transcription, optionally by introducing one or more Cas9-directed reverse transcription plasmids.

[0400] Embodiment 13. The method of embodiment 12, wherein the Cas9-directed reverse transcription uses one or more of a nickase, reverse transcriptase, and Cas9-directed reverse transcription guide RNA (pegRNA) to introduce the one or more nucleic acids into the single genomic locus, optionally wherein the one or more Cas9-directed reverse transcription plasmids encodes one or more of the nickase, reverse transcriptase, and pegRNA.

[0401] Embodiment 14. The method of embodiment 13, wherein the nickase comprises a Cas9 nickase, optionally a Cas9 fusion protein.

[0402] Embodiment 15. The method of embodiment 13 or 14, wherein the reverse transcriptase comprises PE5.

[0403] Embodiment 16. The method of any one of embodiments 13-15, wherein the pegRNA comprises a structured RNA motif to prevent degradation and / or an optimized cr772 guide scaffold.

[0404] Embodiment 17. The method of any one of embodiments 13-16, wherein the Cas9-directed reverse transcription uses one or more dominant negative mutant of human mutL homolog 1 (MLH1), optionally wherein the one or more Cas9-directed reverse transcription plasmids encode the one or more dominant negative mutant of human MLH1.

[0405] Embodiment 18. The method of any one of embodiments 13-17, wherein the Cas9-directed reverse transcription is configured to introduce the one or more attB sites and / or attP sites into the single genomic locus without a double-strand DNA break.

[0406] Embodiment 19. The method of any one of embodiments 1-11 , wherein the introducing comprises using one or more zinc-finger nucleases (ZFNs), CRISPR / Cas endonucleases, transcription activator-like effector nucleases (TALENs), TALE-derived transcription factors, TALE repeat domain proteins, meganucleases, restriction enzymes, site-specific nucleases, and / or gene-editing systems, optionally selected from one or more of TALENs, ZFNs, RNase P RNA, RNase H, CRISPR / Cas, C2c1 , C2c2, C2c3, Cas9, Cpf1 , TevCas9, Archaea Cas9, CasY.1 , CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and any ortholog or homolog thereof, and further optionally wherein the one or more nucleic acids encodes the one or more zinc-finger nucleases (ZFNs), CRISPR / Cas endonucleases, transcriptionDBl / 164937876.2 122Atorney Docket No.: KOM-003PC / 138774-5003 activator-like effector nucleases (TALENs), TALE-derived transcription factors, TALE repeat domain proteins, meganucleases, restriction enzymes, site-specific nucleases, and / or gene-editing systems.

[0407] Embodiment 20. The method of embodiment 19, wherein the one or more ZFNs is configured to introduce the one or more attB sites and / or attP sites into the single genomic locus via homology directed repair.

[0408] Embodiment 21. The method of any one of the preceding embodiments, wherein the one or more attB sites and / or attP sites is introduced into the single genomic locus at a central dinucleotide sequence comprising guanine-thymine (GT).

[0409] Embodiment 22. The method of any one of the preceding embodiments, wherein the single genomic locus comprises an AAVS1 locus, ACTB locus, ACTB locus, ALB locus, albumin locus, B2M locus, CCR5 locus, CD38 locus, CFTR locus, COL7A1 locus, Factor IX locus, FANCA locus, GBA1 locus, GYS1 locus, Hippl 1 locus, Keppel-19 locus, MACO1 locus, OI6nne-18 locus, Pansio-1 locus, Rosa26 locus, S100A locus, SHS253 locus, Smn1 locus, S100A locus, TRAC locus, Xq22.1 locus, or a homologous or speciesequivalent thereof, or a homologous or species-equivalent thereof.

[0410] Embodiment 23. The method of any one of the preceding embodiments, wherein the cell comprises a mammalian cell, plant cell, insect cell, yeast cell, or bacterial cell, optionally a cell suitable for recombinant protein production.

[0411] Embodiment 24. The method of embodiment 23, wherein the mammalian cell comprises a Chinese hamster ovary (CHO) cell (CHO-K1 , CHO-DHB11 , CHO-DXB1 , CHO-S, CHO-DG44, CHO-M), human embryonic kidney (HEK293, HEK293T) cell, K562 human lymphoblast cell, U2OS human osteosarcoma cell, primary human fibroblasts (human dermal fibroblast (HDFa)) cell, baby hamster kidney (BHK) cell, Vero cell (Vero, Vero 76, Vero E6), human cervical carcinoma cell (HELA, 3T3), PERc6 cell, CAP cell, iPSCs, human embryonic stem cells (ESCs), or monkey kidney CV1 cell.

[0412] Embodiment 25. The method of any one of the preceding embodiments, wherein the cell is an auxotrophic cell, optionally a glutamine synthetase (GS) knock-out cell.

[0413] Embodiment 26. The method of any one of embodiments 1-24, wherein the cell is not an auxotrophic variant relative to a cognate wild-type cell.

[0414] Embodiment 27. The method of any one of the preceding embodiments, wherein the introducing further comprises using one or more of a lipid-based transfection reagent (cationic lipid-based reagent),DBl / 164937876.2 123Atorney Docket No.: KOM-003PC / 138774-5003 diethylaminoethyl (DEAE) -dextran, liposome, electroporation, sonoporation, chemical reagent (calcium phosphate), microinjection, or via a non-integrating episome or viral vector (AAV, lentivirus).

[0415] Embodiment 28. The method of any one of the preceding embodiments, wherein the clonal cell population is compatible with the one or more integrases to integrate at least two insertion sequences into the single genomic locus, optionally wherein the at least two insertion sequences are adjacent and / or operably linked within the genome of the cell.

[0416] Embodiment 29. The method of embodiment 28, wherein the clonal cell population is compatible with introducing two insertion sequences, three insertion sequences, four insertion sequences, or five or more insertion sequences.

[0417] Embodiment 30. The method of any one of the preceding embodiments, wherein the one or more integrases comprise one or more Bxb 1 integrases and / or one or more P hi C31 integrases.

[0418] Embodiment 31 . The method of embodiment 30, wherein the one or more integrases comprises one or more Bxb1 integrases comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12.

[0419] Embodiment 32. The method of embodiment 30 or 31 , wherein the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 154, 155, 156, 157, 158, 159, 231 , 232, 233, 234, 235, 236, 237, 257, 314, 316, 318, 321, 322, 323, and 325, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0420] Embodiment 33. The method of embodiment 32, wherei n the one or more mutations are selected from Y154W, Y154A, R155W, R155H, G156P, S157G, L158T, L158R, L158A, P159S, P159T, A234N, S231A, S231 L, S231 G, S231 R, S231Y, S231T, S231 H, A232G, A232S, A232R, A232T, A232V, A232Q, A232P, T233G, T233S, T233W, T233R, T233Y, T233D, T233N, T233H, T233Q, T233A, T233C, A234N, A234G, A234S, A234T, A234H, A234F, K236R, K236S, R237K, R237T, R237Q, R237N, R237C, R237V, D257K, F314M, F314L, F314N, F314R, F314K, G316R, G316W, G316S, G318H, G318P. G318S, G318N, G318P, G318K, G318R, H321Q, H321 K, H321 L, H321 R, H321Y, H321T, H321 S, H321W, P322A, P322R, P322G, R323L, R323G, R323Y, R323I, R325K, R325Q, and R325Y, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0421] Embodiment 34. The method of embodiment 30 or 31 , wherein the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 5, 14, 20, 24, 29, 35, 40, 45, 49, 50,DBl / 164937876.2 124Atorney Docket No.: KOM-003PC / 138774-500351, 60, 68, 69, 70, 73, 74, 78, 84, 86, 87, 100, 105, 116, 124, 157, 183, 197, 207, 208, 209, 229, 261, 267, 273, 287, 291, 333, 342, 343, 347, 361, 368, 375, 435, 449, 453, 462, 483, and 494, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0422] Embodiment 35. The method of embodiment 34, wherei n the one or more mutations are selected from V5I, D14N, E20K, E20Q, E24K, L29F, W35L, V40I, D45G, A49T, V50I, D51N, D51E, D51Y, N60S, E68K, E69D, Q70P, D73G, V74A, V74M, Y78H, Y78N, T84S, S86T, I87V, H100Y, V105I, T116P, A124S, S157G, E183L, P197T, R207Q, R208S, G209V, E229K, A261V, E267D, E273D, E273K, R287P, A291T, K333N, M342V, A343T, A347V, E361D, V368A, V375I, T435A, A449V, T453A, L462M, E483K, and R494Q, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0423] Embodiment 36. The method of embodiment 30 or 31 , wherein the one or more Bxb1 integrases comprise one or more amino acid mutations at a position selected from: 4, 5, 18, 24, 34, 36, 40, 42, 46, 51, 61, 62, 63, 67, 69, 79, 85, 87, 88, 89, 90, 92, 95, 99, 100, 105, 106, 110, 111, 119, 122, 130, 133, 137, 140, 145, 153, 156, 160, 164, 166, 174, 175, 178, 179, 181, 187, 189, 191, 203, 209, 218, 223, 229, 231, 239,248, 251, 254, 261, 264, 268, 272, 278, 280, 281, 282, 283, 285, 287, 288, 292, 295, 302, 306, 307, 311,313, 319, 321, 328, 331, 332, 333, 334, 347, 353, 355, 359, 360, 361, 362, 369, 370, 375, 380, 388, 397,398, 405, 409, 411, 414, 415, 416, 419, 425, 428, 434, 435, 444, 449, 453, 461, 462, 463, 466, 468, 476,479, 480, 483, 484, 487, 488, 489, 494, 496, and 499, relative to SEQ ID NO: 12, ora position corresponding thereto.

[0424] Embodiment 37. The method of embodiment 36, wherei n the one or more mutations are selected from L4I, G34D, D36A, V40I, V40A, E42K, D51N, L61F, R63K, F67S, E68K, E69A, I87V, I87L, H89G, Q92H, H95Y, D99N, H100N, V105A, H111 P, A119S, V122M, E133E, A145T, V179A, V179I, R181 K, V187I, V187A, H189N, H203Y, E229K, M239I, A248T, N251K, T254S, D257K, A261T, V264A, R272Q, A280T, T285A,A288V, A288T, A311V, R319K, R319G, H321P, H321N, F331S, P332H, H334R, A347E, V353I, D355N,D359N, A360T, R362K, A369P, A369E, A369T, A369S, V380I, T388M, A398S, R409H, A411V, A414V,A415S, R416K, A425T, E434G, R444L, T453I, T463I, V466M, G468D, L479I, Q480STQP, Q484K, delL488, R494S, H496N, and M499T, relative to SEQ ID NO: 12, or a position corresponding thereto.

[0425] Embodiment 38. The method of any one of embodiments 31-37, wherein the one or more Bxb1 integrases comprise a C-terminal deletion of one or more amino acids in the range of Q480-S500 of SEQ ID NO: 12.DBl / 164937876.2 125Atorney Docket No.: KOM-003PC / 138774-5003

[0426] Embodiment 39. The method of embodiment 38, wherein the C-terminal deletion comprises a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or all 21 amino acids of Q480-S500 relative to SEQ ID NO: 12.

[0427] Embodiment 40. The method of any one of embodiments 31 -39, wherein the one or more amino acid mutations comprises mutation of one or more of residues of Y154-P159; and / or wherein the one or more mutations alters specificity at -7 and / or -6 positions of a loop target DNA site compared to a wild-type Bxb1 of SEQ ID NO: 12.

[0428] Embodiment 41 . The method of embodiment 40, wherein the mutation comprises S157G.

[0429] Embodiment 42. The method of embodiment 40 or 41 , wherein the one or more Bxb1 integrases comprise a sequence from Y154-P159 selected from YRGSLS (SEQ ID NO: 13), WWGGTP (SEQ ID NO: 14), YRGGLP (SEQ ID NO:15), AHPGRT (SEQ ID NO: 16), and WRGGAS (SEQ ID NO: 17) relative to SEQ ID NO: 12, or a position corresponding thereto.

[0430] Embodiment 43. The method of any one of embodiments 40-42, wherein the one or more amino acid mutations alters the dinucleotide preference at position -7 and / or position -6, optionally altering the dinucleotide preference to one of adenine (A) and cytosine (C), adenine (A) and thymine (T), thymine (T) and cytosine (C), or thymine (T) and thymine (T).

[0431] Embodiment 44. The method of any one of embodiments 31-43, wherein one or more Bxb1 integrases comprise one or more amino acid mutations of residues S231-R237; and / or wherein the one or more mutations alters specificity at -11 , -10, and / or -9 positions of a 3 bp helix target site compared to a wildtype Bxb1 of SEQ ID NO: 12.

[0432] Embodiment 45. The method of embodiment 44, wherein L235 is not mutated.

[0433] Embodiment 46. The method of embodiment 44 or 45, wherein the one or more amino acid mutations comprises mutation of one or more residues of 231-234 and / or 236-237.

[0434] Embodiment 47. The method of any one of embodiments 44-46, wherein A234 is mutated to an asparagine (A234N), and / or wherein the mutation alters specificity at the -10 position to thymine (T) compared to a wild-type Bxb1 of SEQ ID NO: 12.DBl / 164937876.2 126Atorney Docket No.: KOM-003PC / 138774-5003

[0435] Embodiment 48. The method of any one of embodiments 44-47, wherein the one or more amino acid mutations comprises mutation of one or more residues of 231, 233, and 237; and / or wherein the mutation alters specificity at -10 and / or -9 positions of a 3 bp helix target site compared to a wild-type Bxb1 of SEQ ID NO: 12.

[0436] Embodiment 49. The method of any one of embodiments 44-48, wherein the Bxb1 integrase comprises 1 , 2, 3, 4, or 5 amino acids mutated between S231-R237 (inclusive) relative to SEQ ID NO: 12.

[0437] Embodiment 50. The method of embodiment 49, wherein the one or more Bxb1 integrases comprise a sequence from S231-R237 selected from AGGNLKR (SEQ ID NO: 18), LGTNLKR (SEQ ID NO: 19), SGTGLKK (SEQ ID NO: 20), SGSALKT (SEQ ID NO: 21), AAWALRR (SEQ ID NO: 22), GGRSLKR (SEQ ID NO: 23), SGYNLRR (SEQ ID NO: 24), SGWGLKK (SEQ ID NO: 25), SGWALRQ (SEQ ID NO: 26), SARALSR (SEQ ID NO: 27), RADTLRR (SEQ ID NO: 28), YSRNLKR (SEQ ID NO: 29), SRNGLRK (SEQ ID NO: 30), RGHALKN (SEQ ID NO: 31), GGSHLKR (SEQ ID NO: 32), TTRTLKR (SEQ ID NO: 33), AVQNLKR (SEQ ID NO: 34), RAAFLKK (SEQ ID NO: 35), RAWTLKC (SEQ ID NO: 36), RAWSLKR (SEQ ID NO: 37), HGWSLKV (SEQ ID NO: 38), HGCTLKR (SEQ ID NO: 39), YGSALKQ (SEQ ID NO: 40), SQWALKC (SEQ ID NO: 41), YPWSLRR (SEQ ID NO: 42), relative to SEQ ID NO: 12 or a position corresponding thereto.

[0438] Embodiment 51 . The method of any one of embodiments 31-50, wherein the one or more Bxb1 integrases comprise a mutation of D257K compared to a wild-type Bxb1 of SEQ ID NO: 12.

[0439] Embodiment 52. The method of any one of embodiments 31 -51 , wherein the one or more Bxb 1 integrases comprise one or more amino acid mutations of residues F314-R325; and / or wherein the mutation alters specificity at -19 through -12 positions of a DNA binding site compared to a wild-type Bxb1 of SEQ ID NO: 12.

[0440] Embodiment 53. The method of embodiment 52, wherein the Bxb1 residue P322 is maintained as a proline.

[0441] Embodiment 54. The method of embodiment 52 or 53, wherein the one or more amino acid mutations comprises mutation of one or more residues of F314, G316, G318, H321 , P322, R323, and R325.

[0442] Embodiment 55. The method of any one of embodiments 52-54, wherein the one or more Bxb 1 integrases comprise 1, 2, 3, 4, 5, 6, or 7 amino acids mutated between F314-R325 (inclusive) relative to SEQ ID NO: 12.DBl / 164937876.2 127Atorney Docket No.: KOM-003PC / 138774-5003

[0443] Embodiment 56. The method of embodiment 55, wherein the one or more Bxb1 integrases comprise a sequence from F314-R325 selected from MAGGHRKQALYR (SEQ ID NO: 43), MAGGPRKKRRYR (SEQ ID NO: 44), LARGSRKLALYR (SEQ ID NO: 45), NARGNRKRGRYR (SEQ ID NO: 46), LARGPRKRAGYK (SEQ ID NO: 47), RAWGKRKYAYYQ (SEQ ID NO: 48), KAWGSRKTRLYR (SEQ ID NO: 49), MARGGRKSAIYY (SEQ ID NO: 50), MASGSRKTAIYY (SEQ ID NO: 51), LARGRRKWARYR (SEQ ID NO: 52), and LARGSRKLALYR (SEQ ID NO: 53), relative to SEQ ID NO: 12, or a position corresponding thereto.

[0444] Embodiment 57. The method of any one of embodiments 31-56, wherein the one or more Bxb1 integrases comprise one or more mutations and / or deletions, and / or one or more combinations of mutations and / or deletions as described in Table 3 and / or Table 4.

[0445] Embodiment 58. The method of any one of embodiments 31-57, wherein the one or more Bxb 1 integrases comprise at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations.

[0446] Embodiment 59. The method of any one of embodiments 31-57, wherein the one or more Bxb 1 integrases comprise at least 90% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations.

[0447] Embodiment 60. The method of any one of embodiments 31-57, wherein the one or more Bxb1 integrases comprise at least 95% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or at least 20 mutations.DBl / 164937876.2 128Atorney Docket No.: KOM-003PC / 138774-5003

[0448] Embodiment 61 . The method of any one of embodiments 31-57, wherein the one or more Bxb 1 integrases comprise at least 96% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or 20 mutations.

[0449] Embodiment 62. The method of any one of embodiments 31-57, wherein the one or more Bxb 1 integrases comprise at least 97% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, or 15 mutations.

[0450] Embodiment 63. The method of any one of embodiments 31-57, wherein the one or more Bxb1 integrases comprise at least 98% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, or 10 mutations.

[0451] Embodiment 64. The method of any one of embodiments 31-57, wherein the one or more Bxb1 integrases comprise at least 99% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, or 5 mutations.

[0452] Embodiment 65. The method of any one of embodiments 1-30, wherein the Bxb1 integrase comprises: an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12; and one or more amino acid mutations at positions selected from L4, E42, R57, R63, V76, H111 , V122, V187, K313, R319, A341, D359, A369, A398, R416, A425, L479, and M499, relative to SEQ ID NO: 1.

[0453] Embodiment 66. The method of embodiment 65, wherein the Bxb1 integrase comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 12.

[0454] Embodiment 67. The method of embodiment 65, wherein the Bxb1 integrase comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 12.

[0455] Embodiment 68. The method of embodiment 65, wherein the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12.DBl / 164937876.2 129Atorney Docket No.: KOM-003PC / 138774-5003

[0456] Embodiment 69. The method of any one of embodiments 65-68, wherein the Bxb1 integrase comprises one or more amino acid mutations are selected from L4I, E42K, R57K, R63K, V76I, H111 P, V122M, V187, K313R, R319K, A341 D, D359A, A369P, A398S, R416K, A425T, L479I, and M499T relative to SEQ ID NO: 12.

[0457] Embodiment 70. The method of any one of embodiments 65-69, wherein the Bxb1 integrase further comprises one or more amino acid mutations selected from D36N, V40A, A49S, V74I, I87L or I87A or I87S, H89G, V175A, V179A, R287H, A288V, T453I, and H496N relative to SEQ ID NO: 12.

[0458] Embodiment 71. The method of any one of embodiments 65-70, wherein the Bxb1 integrase further comprises one or more amino acid mutations selected from V40I, D45G, I75V, H95Y, A119S, A280T, A311 V, E434G, and V466M relative to SEQ ID NO: 12.

[0459] Embodiment 72. The method of any one of embodiments 65-71 , wherein one or more of the following amino acids relative to SEQ ID NO: 1 are not mutated: A62, H100, Q191 , P195, G209, P295, L302, C307, L387, E419, S428, E483, and R487 relative to SEQ ID NO: 12.

[0460] Embodiment 73. The method of any one of embodiments 65-72, wherein the one or more amino acid mutations are selected from mutations at position V122 and / or position A369 relative to SEQ ID NO: 12.

[0461] Embodiment 74. The method of embodiment 73, wherein the mutation at position V122 relative to SEQ ID NO: 12 is V122M.

[0462] Embodiment 75. The method of embodiment 73, wherein the mutation at position A369 relative to SEQ ID NO: 12 is A369P.

[0463] Embodiment 76. The method of any one of embodiments 65-75, wherein the one or more amino acid mutations relative to SEQ ID NO: 12 are selected from a combination of positions selected from: V76 and V122; V76 and A369; I87 and V122; I87 and A369; H95 and V122; H95 and A369; V122 and E434; A369 and E434; V76, V122, and A369; I87, H95 and A369; I87, V122 and A369; I87, V122 and E434; I87, A369 and E434; H95, V122 and A369; H95, V122 and E434; H95, A369 and E434; V122, A369 and E434; I87, H95, V122 and E434; I87, H95, A369 and E434; and I87, V122, A369 and E434.

[0464] Embodiment 77. The method of embodiment 76, wherein the one or more mutations relative to SEQ ID NO: 12 are a combination selected from: V76I and V122M; V76I and A369P; I87L and V122M; I87L and A369P; H95Y and V122; H95Y and A369P; V122M and E434G; A369P and E434G; V76I, V122M, and A369P; I87L, H95Y and A369P; I87L, V122M and A369P; I87L, V122M and E434G; I87L, A369P and E434G;DBl / 164937876.2 130Atorney Docket No.: KOM-003PC / 138774-5003H95, V122M and A369P; H95Y, V122M and E434G; H95Y, A369P and E434G; V122, A369P and E434G; I87L, H95Y, V122M and E434G; I87L, H95Y, A369P and E434G; or I87L, V122, A369P and E434G.

[0465] Embodiment 78. The method of any one of embodiments 65-77, wherein the one or more amino acid mutations relative to SEQ ID NO: 12 are a combination of positions selected from: I87, A369, and E434; V122, A369, and E434; H95, A369, and E434; V122 and E434; V122, A369, E434, and V175; V76 and A119; A119, A369, and E434; I87, V175, and D359; I87, R57, and R63; I87 and A369; I87 and A425; R63 and V122; R63, V122, R319, and V40I; R63, A119, and M499; V122 and A369; H95 and A369; A49, 187, A396, and E434; R57, R63, 187, A396, and E434; D36, V122, A396, and E434; R57, R63 and H95; D36, 187, A396, and E434; I87, A341, A396, and E434; R57, R63, V122, A396, and E434; V122, R287, A396, and E434; I87, R287, A396, and E434; I87 and A341; I87, R287, A396, E434, and A341; V122, A396, E434, and A341; H95, and A341; A49, V122, A396, and E434; D45, A396, and E434; V122, R287, A341, A396, and E434; A49, A396, and E434; I87, A341, and R287; D36, A396, and E434; V122, D359, A369, and E434; R63 and I87; I87, D359, A369, and E434; V122, A369, A425, and E434; I87, A369, A425, and E434; V40, H95, A369, and E434; L4, H95, A369, and E434; H95, A369, E434, and V466; R63, V122, A369, and E434; V76, 187, and K313; R63, 187, and N194; I87 and D359; L4 and I87; V74 and V76; V122, A369, E434, and R319; I87, A369, E434, and R319; L4, V122, A369, and E434; I87 and A425; E42, V76, and I87; I87 and L479; H95, A369, A425, and E434; R63, I87, A369, and E434; I87 and R319; V76, A369, and E434; H95, A369, E434, and L479; V74, 175, and V76; H95, R319, A369, and E434; L4, 187, A369, and E434; V122, V175, D359, A369, and E434; R57, 187, A369, and E434; E42, H95, A369, and E434; H95, V179, A369, and E434; I75 and V76; L4 and V76; H95 and K313; V40, R63, and H89; A288, A311 , A398, R416, T453, H496, and E42; and H111 and E434.

[0466] Embodiment 79. The method of embodiment 78, wherein the one or more mutations relative to SEQ ID NO: 12 are a combination selected from: I87L, A369P, and E434G; V122M, A369P, and E434G; H95Y, A369P, and E434G; I87L and E434G; V122M and E434G; V122M, A369P, E434G, and V175A; V76I and A119S; D36N and A119S; A119S, A369P, and E434G; I87L, V175A, and D359A; I87L, R57K, and R63K; I87L and D36N; I87L and A369P; I87L and A425T; R63K and V122M; R63K, V122M, R319K, and V40I; R63K, A119S, and M499T; H95Y and E434G; V122M and A369P; H95Y and V466M; H95Y and A369P; A49S, I87L, A396P, and E434G; A49S and I87L; R57K, R63K, I87L, A396P, and E434G; D36N, V122M, A396P, and E434G; R57K, R63K and H95Y; D36N, I87L, A396P, and E434G; D36N and H95Y; I87L, A341D, A396P, and E434G; A49S and H95Y; R57K, R63K, V122M, A396P, and E434G; V122M, R287H, A396P, and E434G; I87L and R287H; I87L, R287H, A396P, and E434G; I87L and A341D; I87L, R287H, A396P,DBl / 164937876.2 131Atorney Docket No.: KOM-003PC / 138774-5003E434G, and A341D; V122M, A396P, E434G, and A341D; H95Y and A341D; A49S, V122M, A396P, and E434G; D45G, A396P, and E434G; V122M, R287H, A341D, A396P, and E434G; A49S, A396P, and E434G; I87L, A341D, and R287H; H95Y and R287H; D36N, A396P, and E434G; V122M, D359A, A369P, and E434G; R63K and I87L; I87L and V466M; I87L, D359A, A369P, and E434G; V122M, A369P, A425T, and E434G; I87L, A369P, A425T, and E434G; V40I, H95Y, A369P, and E434G; L4I, H95Y, A369P, and E434G; H95Y, A369P, E434G, and V466M; R63K, V122M, A369P, and E434G; V76I, I87L, and K313R; R63K, I87V, and N194D; I87L and D359A; I87L and L4I; V74I and V76I; V122M, A369P, E434G, and R319K; I87L, A369P, E434G, and R319K; L4I, V122M, A369P, and E434G; I87L and A425T; E42K, V76I, and I87L; I87L and L479I; H95Y, A369P, A425T, and E434G; V40I and I87L; R63K, I87L, A369P, and E434G; I87L and R319K; V76I, A369P, and E434G; H95Y, A369P, E434G, and L479I; V74I, I75V, and V76I; H95Y, R319K, A369P, and E434G; L4I, I87L, A369P, and E434G; V122M, V175A, D359A, A369P, and E434G; R57K, I87L, A369P, and E434G; E42K, H95Y, A369P, and E434G; H95Y, V179A, A369P, and E434G; I75V and V76I; I87L and V179A; L4I and V76I; H95Y and K313R; H95Y and A280T; V40A, R63K, and H89G; A288V, A311 V, A398S, R416K, T453I, H496N, and E42K; and H111P and E434G.

[0467] Embodiment 80. The method of embodiment 30, wherein the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12 and having the amino acid mutations I87L, A369P, and E434G relative to SEQ ID NO: 12.

[0468] Embodiment 81. The method of embodiment 30, wherein the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12 and having the amino acid mutations V122M, A369P, and E434G relative to SEQ ID NO: 12.

[0469] Embodiment 82. The method of embodiment 30, wherein the Bxb1 integrase comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12 and having the amino acid mutations H95Y, A369P, and E434G relative to SEQ ID NO: 12.

[0470] Embodiment 83. The method of embodiment 30, wherein the one or more PhiC31 integrases comprises: an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 54; and one or more amino acid mutations at positions selected from 1, 2, 12, 14, 18, 24, 32, 36, 41, 43, 44, 45, 51, 55, 74, 77, 96, 103, 107, 117, 153, 176, 188, 199, 200, 228, 230, 231, 235, 238, 240, 252, 255, 259, 262, 264, 266, 269, 274, 278, 302, 320, 322, 331, 333, 340, 344, 346, 347, 351, 355, 359, 362, 364, 378, 382, 393, 396, 397, 399, 406, 410, 424, 429, 431, 436, 438, 445, 448, 449, 450, 452, 457, 468, 475, 498,DBl / 164937876.2 132Atorney Docket No.: KOM-003PC / 138774-5003501, 505, 512, 516, 517, 520, 535, 536, 549, 551 , 552, 563, 580, 585, 586, 587, 590, 592, 600, 603, 604, 609, 616, and 621 , relative to SEQ ID NO 54, or a position corresponding thereto.

[0471] Embodiment 84. The method of embodiment 83, wherein the one or more amino acid mutations is selected from M1 E, M1V, D2V, D2M, S12N, E14G, S18N, D32A, D36A, V411, D44A, V51 M, 1153V, E176D, A199T, H228Y, P230S, F231 L, H240R, D252G, A255G, S269N, P274S, M278L, T302A, A333T, A333S,A333D, A340V, G344V, G344D, G346S, R347K, L351V, L351 Q, D362N, D362G, L364M, E378K, S396N,S396R, A397T, N406S, A410T, G429S, E431V, W438R, W448R, E449D, A450D, E452K, E475D, G505V,G505S, A516T, P535L, T536A, A580V, K586Q, D590G, D592G, T600S, V603I, V603A, A604S, P609S,A616V, and R621 L relative to SEQ ID NO 54, or a position corresponding thereto.

[0472] Embodiment 85. The method of embodiment 83 or 84, wherein the PhiC31 integrase comprises a N-terminal addition of one or more amino acids.

[0473] Embodiment 86. The method of embodiment 85, wherein the N-terminal addition comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, or about 50 amino acids relative to SEQ ID NO: 54.

[0474] Embodiment 87. The method of embodiment 85 or 86, wherein the N-terminal addition comprises a sequence selected from SEQ ID NOs: 55-67.

[0475] Embodiment 88. The method of any one of embodiments 83-87, wherein the one or more PhiC31 integrases comprise one or more mutations, N-terminal additions, and / or one or more combinations of mutations and / or N-terminal additions as described in Table 3, Table 6, and / or Table 7 relative to SEQ ID NO: 54.

[0476] Embodiment 89. The method of any one of embodiments 83-88, wherein the one or more PhiC31 integrases have at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations, e.g., as selected from Table 3, Table 6, and / or Table 7 relative to SEQ ID NO: 54.DBl / 164937876.2 133Atorney Docket No.: KOM-003PC / 138774-5003

[0477] Embodiment 90. The method of any one of embodiments 83-88, wherein the one or more PhiC31 integrases comprise at least 90% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, at least 20 mutations, at least 30 mutations, at least 40 mutations, or at least 50 mutations.

[0478] Embodiment 91 . The method of any one of embodiments 83-88, wherein the one or more PhiC31 integrases comprise at least 95% sequence identity to SEQ ID NO: 54 and having at least 1 mutation, at ...

Claims

Atorney Docket No.: KOM-003PC / 138774-5003CLAIMSWhat is claimed is:1 . A method for generating a cell line comprising:(a) introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the single genomic locus, and wherein the one or more nucleic acids encode:(i) one or more attB sites and / or(ii) one or more attP sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more insertion sequences by one or more integrases, wherein the one or more integrases comprises one or more mutant Bxb1 integrases comprising an amino acid sequence with one or more mutations relative to the amion acid sequence of SEQ ID NO: 12; and(b) culturing a cell population from the cell to generate the cell line.

2. A method for generating a cell line comprising:(a) introducing one or more nucleic acids at a single genomic locus in a cell, wherein the insertion is monoallelic at the single genomic locus, and wherein the one or more nucleic acids encodes:(i) one or more attB sites and / or one or more attP sites, and wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration of one or more insertion sequences by one or more integrases;(ii) one or more inducible kill switch genes wherein the one or more inducible kill switch genes encodes a protein which induces apoptosis in cells that do not receive an insertion sequence at the one or more attB sites and / or attP sites; and(iii) one or more homology directed repair (HDR) insulator sequences compatible with the single genomic locus, wherein the one or more HDR insulator sequences flanks the one or more attB sites and / or attP sites; wherein the one or more nucleic acids do not encode an antibiotic resistance gene or auxotrophic selection gene; and wherein the cell is not an auxotrophic variant relative to a cognate wild-type cell; and(b) culturing a cell population from the cell to generate the cell line,DBl / 164937876.2 172Atorney Docket No.: KOM-003PC / 138774-5003 wherein the one or more integrases comprises one or more mutant Bxb1 integrases comprising an amino acid sequence with one or more mutations relative to the amion acid sequence of SEQ ID NO: 12.

3. A method of negative cell selection to generate a cell line comprising:(a) introducing one or more nucleic acids into a population of cells at a single genomic locus, wherein the one or more nucleic acids comprises two or more attB sites and / or attP sites flanking an intervening DNA sequence, wherein the two or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration with one or more integrases, and wherein the population of cells comprises a genomically-integrated nucleic acid molecule encoding one or more inducible kill switch gene, wherein the one or more inducible kill switch comprises an inducible hamster-specific caspase-9 fusion protein having about or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 99;(b) culturing the population of cells under conditions suitable for recombinase-mediated cassette exchange by the one or more integrases between the genomically-integrated nucleic acid molecule encoding the inducible hamster-specific caspase-9 fusion protein and the two or more attB sites and / or attP sites of the one or more nucleic acids, such that the inducible hamster-specific caspase-9 fusion protein coding sequence is replaced by the intervening DNA sequence; and(c) inducing apoptosis in cells that did not undergo the recombinase-mediated cassette exchange based on expression of the inducible hamster-specific caspase-9 fusion protein to generate the cell line.

4. A method of positive cell selection to generate a cell line comprising:(a) introducing a donor DNA-specific nucleic acid molecule into a population of cells, each comprising a genomically-integrated landing pad nucleic acid molecule at a single genomic locus,(i) wherein the donor DNA-specific nucleic acid molecule comprises: a first nucleic acid sequence encoding a promoter, andDBl / 164937876.2 173Atorney Docket No.: KOM-003PC / 138774-5003 a second nucleic acid sequence encoding at least one start codon, at least one splice donor or splice acceptor, and one or more attB sites and / or attP sites, wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration with one or more integrases, and wherein the promoter is operably linked to drive expression from the start codon and the start codon is not linked to a protein-coding sequence; and(ii) wherein the genomically-integrated landing pad nucleic acid molecule comprises: one or more attB sites and / or attP sites, wherein the one or more attB sites and / or attP sites comprises a nucleic acid sequence suitable for integration with the one or more integrases; at least one splice donor or splice acceptor sequence; a protein-coding nucleic acid sequence, wherein the protein-coding sequence lacks a start codon, encodes a selectable marker protein, and is located downstream (3’) of the at least one splice donor or splice acceptor sequence and the aft sequence; and a transcriptional terminator sequence downstream of the protein-coding nucleic acid sequence;(c) culturing the population of cells under conditions suitable for recombinase-mediated cassette exchange by the one or more integrases, such that the promoter and the at least one start codon from the donor DNA-specific nucleic acid molecule becomes operatively linked to the protein-coding sequence of the genomically-integrated landing pad nucleic acid molecule to drive expression of the protein-coding sequence; and(d) selecting for the cells that underwent recombinase-mediated cassette exchange based on expression of the protein-coding sequence to generate the cell line.

5. The method of any one of claims 1-4, wherein the one or more attB sites, or two or more attB sites, comprises about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95, or is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95; and / or wherein the one orDBl / 164937876.2 174Atorney Docket No.: KOM-003PC / 138774-5003 more attB sites, or two or more attB sites, is compatible with one or more attP sites comprising about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97.

6. The method of any one of claims 1-5, wherein the one or more attP sites, or two or more attP sites, comprises about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% to the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97, or is or comprises the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 96-97; and / or wherein the one or more attP sites, or two or more attP sites, is compatible with one or more attB sites comprising about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 3, 5, and 89-95.

7. The method of any one of claims 1-6, wherein the one or more attB sites and / or attP, or two or more attB sites and / or attP sites is suitable for recombination at a central dinucleotide sequence comprising guanine-thymine (GT).

8. The method of claim 6 or 7, wherein the one or more attB sites and / or attP, or two or more attB sites and / or attP sites is suitable for recombination at a central dinucleotide sequence comprising adeninecytosine (AC).

9. The method of any one of claims 1 -8, wherein the one or more nucleic acids, the donor DNA-specific nucleic acid molecule, the genomically-integrated nucleic acid molecule, or the genomically- integrated landing pad nucleic acid molecule, comprises:(i) one or more homology directed repair (HDR) insulator sequences,(ii) one or more attB sites and / or attP sites,DBl / 164937876.2 175Atorney Docket No.: KOM-003PC / 138774-5003 optionally wherein the one or more homology directed repair (HDR) insulator sequences and / or one or more attB sites and / or attP sites flank one or more kill switch genes therebetween,(iii) one or one or more promoter,(iv) one or more 2A peptide sequence,(v) one or more insertion sequence, and / or(vi) one or more polyA tail or termination sequence.

10. The method of claim 9, wherein the promoter comprises one or more of a mammalian gene promoter, viral promoter, inducible promoter, and bidirectional promoter, optionally wherein the promoter comprises one or more of a B29 promoter, CAG promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, CMV promoter, desmin promoter, Ef1a promoter, EGR1 promoter, elastase-1 promoter, elF2A1 promoter, endoglin promoter, FerH promoter, FerL promoter, fibronectin promoter, Flt-1 promoter, GAPDH promoter, GFAP promoter, GPIIb promoter, GRP78 promoter, GRP94 promoter, HSP70 promoter, Hspa5p promoter, ICAM-2 promoter, INF- promoter, Nphsl promoter, OG-2 promoter, PGK-1 promoter, ROSA promoter, SP-B promoter, SV40 promoter, SYN1 promoter, ubiquitin B promoter, WASP promoter, 0-actin promoter, and 0-kin promoter.

11. The method of claim 9, wherein the one or more 2A peptide comprises one or more of a T2A, P2A, E2A, and F2A peptide sequence, optionally comprising a sequence motif of DXEXNPGP (SEQ ID NO: 11, where “X” denotes any amino acid), and further optionally wherein the one or more 2A peptide sequences comprises one or more amino acid sequence of SEQ ID NOs: 7-10.

12. The method of claim 11 , wherein the one or more 2A peptide sequences is located between two or more protein-coding sequences.

13. The method of any one of claims 1-12, wherein the kill switch gene comprises one or more of a molecular switch or suicide gene that encodes one or more protein that induces cell death or apoptosis.

14. The method of claim 13, further comprising inducing apoptosis via the one or more kill switch genes in cells that did not under integrase-mediated recombination, optionally wherein inducing apoptosisDBl / 164937876.2 176Atorney Docket No.: KOM-003PC / 138774-5003 comprises contacting the cell, or population of cells, with a small molecule, optionally selected from AP20187, AP1903, and rimiducid.

15. The method of any one of claims 1-14, wherein the introducing comprises using one or more zinc- finger nucleases (ZFNs), CRISPR / Cas endonucleases, transcription activator-like effector nucleases (TALENs), TALE-derived transcription factors, TALE repeat domain proteins, meganucleases, restriction enzymes, site-specific nucleases, and / or gene-editing systems to knock-in one or more nucleic acid sequences into a cellular genome, optionally comprising introducing into the cell, or the population of cells, one or more nucleic acid molecules encoding the one or more ZFNs, CRISPR / Cas endonucleases, TALENs, TALE-derived transcription factors, TALE repeat domain proteins, meganucleases, restriction enzymes, site-specific nucleases, and / or gene-editing systems, and further optionally selected from one or more of RNase P RNA, RNase H, C2c 1 , C2c2, C2c3, Cas9, Cpf1 , TevCas9, Archaea Cas9, CasY.1 , CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and any ortholog or homolog thereof.

16. The method of any one of claims 1-15, wherein the single genomic locus comprises an AAVS1 locus, ACTB locus, ACTB locus, ALB locus, albumin locus, B2M locus, CCR5 locus, CD38 locus, CFTR locus, COL7A1 locus, Factor IX locus, FANCA locus, GBA1 locus, GYS1 locus, Hippl 1 locus, Keppel-19 locus, MAC01 locus, OI6nne-18 locus, Pansio-1 locus, Rosa26 locus, S100A locus, SHS253 locus, Smn1 locus, S100A locus, TRAC locus, Xq22.1 locus, or a homologous or speciesequivalent thereof.

17. The method of any one of claims 1-16, wherein the cell, or the population of cells, comprises a mammalian cell, plant cell, insect cell, yeast cell, or bacterial cell, optionally a cell suitable for recombinant protein production.

18. The method of claim 17, wherein the cell, or the population of cells, comprise mammalian cells, optionally selected from Chinese hamster ovary (CHO) cells (CHO-K1 , CHO-DHB11 , CHO-DXB1, CHO-S, CHO-DG44, CHO-M), human embryonic kidney cells (HEK293, HEK293T), K562 human lymphoblast cells, U2OS human osteosarcoma cells, primary human fibroblasts (human dermal fibroblast (HDFa)), baby hamster kidney (BHK) cells, Vero cells (Vero, Vero 76, Vero E6), humanDBl / 164937876.2 177Atorney Docket No.: KOM-003PC / 138774-5003 cervical carcinoma cells (HELA, 3T3), PERc6 cells, CAP cells, iPSCs, human embryonic stem cells (ESCs), and monkey kidney CV1 cells, and further optionally comprise hamster cells selected from Chinese hamster ovary (CHO) cells, CHO-K1 cells, CHO-DHB11 cells, CHO-DXB1 cells, CHO-S cells, CHO-DG44 cells, and CHO-M cells.

19. The method of any one of claims 1-18, wherein the cell, or the population of cells, is an auxotrophic cell, optionally comprising a glutamine synthetase (GS) knock-out.

20. The method of any one of claims 1-18, wherein the cell, or the population of cells, is not an auxotrophic variant relative to a cognate wild-type cell.

21. The method of any one of claims 1-20, wherein the cell, or the population of cells, does not encode an antibiotic resistance gene.

22. The method of any one of claims 1-21 , wherein the introducing further comprises using one or more of a lipid-based transfection reagent (cationic lipid-based reagent), diethylaminoethyl (DEAE)- dextran, liposome, electroporation, sonoporation, chemical reagent (calcium phosphate), microinjection, or via a non-integrating episome or viral vector (AAV, lentivirus).

23. The method of any one of claims 1-22, wherein the cell line is compatible with the one or more integrases to integrate at least two insertion sequences, intervening DNA sequences, or proteincoding sequences, at the single genomic locus, optionally wherein the at least two insertion sequences, intervening DNA sequences, or protein-coding sequences are adjacent and / or operably linked within the genome of each cell of the cell line.

24. The method of claim 23, wherein the cell line is compatible with introducing two, three, four, or five or more insertion sequences, intervening DNA sequences, or protein-coding sequences, and optionally by using two, three, four, or five or more integrases, and optionally wherein each integration comprises using a distinct integrase.DBl / 164937876.2 178Atorney Docket No.: KOM-003PC / 138774-500325. The method of any one of claims 1-24, wherein the one of more insertion sequences, intervening DNA sequences, or protein-coding sequences, comprises a double-stranded DNA (dsDNA) molecule ranging in size from about 0.1 kb to about 50 kb or more.

26. The method of any one of claims 1-25, wherein the insertion sequences, intervening DNA sequences, or protein-coding sequences, comprises one or more promoter sequence, enhancer sequence, internal ribosome entry site (IRES), and 3’ polyadenine (polyA) sequence.

27. The method of any one of claims 1-26, wherein the one or more integrases comprises one or more Bxb1 integrase comprising an amino acid sequence that is at least 90% identical to amino acids 1- 480 of SEQ ID NO: 12; at least 90% identical to amino acids 1-488 of SEQ ID NO: 12; or at least 90% identical to SEO ID NO: 12.

28. The method of claim 27, wherein the one or more Bxb1 integrases comprises one or more amino acid mutations at a position selected from 4, 5, 14, 18, 20, 24, 29, 34, 35, 36, 40, 42, 45, 46, 49, 50, 51, 60, 61, 62, 63, 67, 68, 69, 70, 73, 74, 75, 76, 78, 79, 84, 85, 86, 87, 88, 89, 90, 92, 95, 99, 100, 105, 106, 110, 111, 116, 119, 122, 124, 130, 133, 137, 140, 145, 153, 156, 157, 158, 160, 164, 166, 174, 175, 178, 179, 181, 183, 187, 189, 191, 197, 203, 207, 208, 209, 218, 223, 229, 231, 232, 234, 236, 237239, 248, 251, 254, 257, 261, 264, 267, 268, 272, 273, 278, 280, 281, 282, 283, 285, 287,288, 291, 292, 295, 302, 306, 307, 311, 313, 314, 316, 318, 319, 321, 322, 323, 325, 328, 331, 332,333, 334, 342, 343, 347, 353, 355, 359, 360, 361, 362, 368, 369, 370, 375, 380, 388, 397, 398, 405,409, 411, 414, 415, 416, 419, 425, 428, 434, 435, 444, 449, 453, 461, 462, 463, 466, 468, 476, 479,480, 483, 484, 487, 488, 489, 494, 496, and 499, or a position corresponding thereto, relative to SEQ ID NO: 12.

29. The method of claim 27 or 28, wherein the one or more Bxb1 integrases comprises at least two amino acid substitutions at positions selected from (a) and (b), (b) and (c), or (a) and (c), wherein: (a) is 75, 76, 158, 232, 234, 236, 237, 257, 314, 316, 318, 322, 323, 325, or a position corresponding thereto,DBl / 164937876.2 179Atorney Docket No.: KOM-003PC / 138774-5003(b) is 5, 14, 20, 24, 29, 35, 40, 45, 49, 50, 51, 60, 68, 69, 70, 73, 74, 78, 84, 86, 87, 100, 105, 116,124, 183, 197, 207, 208, 209, 229, 261, 267, 273, 273, 287, 291, 333, 342, 343, 347, 361, 368, 375, 435, 449, 453, 462, 483, 494, or a position corresponding thereto, and(c) is 4, 18, 34, 36, 42, 46, 61, 62, 63, 67, 79, 85, 88, 89, 90, 92, 95, 99, 106, 110, 111, 119, 122,130, 133, 137, 140, 145, 153, 156, 157, 158, 160, 164, 166, 174, 175, 178, 179, 181, 187, 189, 191, 203, 218, 223, 231, 239, 248, 251, 254, 264, 268, 272, 278, 280, 281, 282, 283,285, 288, 292, 295, 302, 306, 307, 311, 313, 319, 321, 328, 331, 332, 334, 353, 355, 359,360, 362, 369, 370, 380, 388, 397, 398, 405, 409, 411, 414, 415, 416, 419, 425, 428, 434,444, 461, 463, 466, 468, 476, 479, 480, 484, 487, 488, 489, 496, and / or 499, or a position corresponding thereto.

30. The method of any one of claims 27-29, wherein the one or more Bxb 1 integrases comprises one or more amino acid mutations selected from L4I, V5I, D14N, E20K, E20Q, E24K, L29F, G34D, W35L, D36A, V40I, V40A, E42K, D45G, A49T, V50I, D51N, D51E, D51Y, N60S, L61F, R63K, F67S, E68K, E69A, E69D, Q70P, D73G, V74A, V74I, V74M, I75V, V76I, Y78H, Y78N, T84S, S86T, I87V, I87L, H89G, Q92H, H95Y, D99N, H100N, H100Y, V105A, V105I, H111 P, T116P, A119S, V122M, A124S, A145T, S157G, T166I, V179A, V179I, R181K, E183L, V187I, H189N, P197T, H203Y, R207Q, R208S, G209V, E229K, A232G, A232S, A232R, A232T, A232V, A232P, A232Q, T233G, T233W, T233R, T233Y, T233D, T233G, T233N, T233H, T233S, T233Q, T233A, T233C, A234N, A234G, A234S, A234T, A234H, A234F, A234S, K236R, K236S, R237K, R237Q, R237N, R237V, R237C, M239I, A248T, N251K, T254S, D257K, A261T, A261V, V264A, E267D, R272Q, E273D, E273K, A280T, T285A, R287P, A288V, A288T, A291T, A311V, K313R, F314M, F314L, F314N, F314R,F314K, G316R, G316W, G318H, G318P, G318S, G318N, G318K, R319K, R319G, H321P, H321Q,H321 K, H321L, H321R, H321Y, H321T, H321S, P322A, P322R, P322G, P322L, R323L, R323G,R323Y, R323I, R325K, R325Q, R325Y, F331S, P332H, K333N, H334R, M342T, M342V, A343T,A347E, A347V, V353I, D355N, D359N, A360T, E361D, R362K, V368A, A369P, A369E, A369T,A369S, V375I, V380I, T388M, A396P, A398S, R409H, A411V, A414V, A415S, R416K, A425T, E434G, T435A, R444L, A449V, T453I, T453A, L462M, T463I, V466M, G468D, L479I, Q480STOP, E483K, Q484K, R487R, del L488 (del L488 frameshift), R494S, R494Q, H496N, and / or M499T, or a position corresponding thereto, relative to SEQ ID NO: 12.DBl / 164937876.2 180Atorney Docket No.: KOM-003PC / 138774-500331. The method of any one of claims 27-30, wherein one or more of the following amino acids are not mutated relative to SEQ ID NO: 12: V5, S18, E24, V40, V46, D51 , A62, E69, R79, R85, R88, L90, Q92, S106, A110, A130, E133, 1137, R140, K153, G156, P160, L164, L174, V175, P178, V187, Q191 , G209, A218, R223, S231 , N251, P268, L278, A280, E281, L282, V283, R287, P292, P295, L302, V306, C307, K313, S328, K333, D359, E361, G370, V375, R397, A405, A414, E419, A425, S428, T435, R461 , V466, G468, F476, E483, G489, and R494, or a position corresponding thereto.

32. The method of any one of claims 27-31 , wherein the one or more Bxb1 integrases comprises the mutation S157G.

33. The method of any one of claims 27-31 , wherein S157 is not mutated.

34. The method of any one of claims 27-33, wherein the one or more Bxb1 integrases comprise one or more mutations and / or deletions, and / or one or more combinations of mutations and / or deletions as described in Table 3 and / or Table 4.

35. The method of any one of claims 27-34, wherein the one or more Bxb1 integrases comprise:(i) at least 95% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or at least 20 mutations;(ii) at least 96% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, at least 15 mutations, at least 16 mutations, at least 17 mutations, at least 18 mutations, at least 19 mutations, or 20 mutations;(iii) at least 97% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6DBl / 164937876.2 181Atorney Docket No.: KOM-003PC / 138774-5003 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, or 15 mutations;(iv) at least 98% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, or 10 mutations; or(v) at least 99% sequence identity to SEQ ID NO: 12 and having at least 1 mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, or 5 mutations.

36. The method of any one of claims 26-35, wherein the one or more Bxb1 integrases comprises one or more nuclear localization sequences and / or one or more stabilization sequences, optionally fused at the N-terminus, at the C-terminus, at both the N-terminus and C-terminus, or within the integrase sequence.

37. The method of claim 36, wherein the one or more nuclear localization sequences comprises a sequence selected from: PKKKRKV (SEQ ID NO: 68), NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 69); PAAKRVKLD (SEQ ID NO: 70), RQRRNELKRSF (SEQ ID NO: 71); NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 72), RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 73), VSRKRPRP (SEQ ID NO: 74), PPKKARED (SEQ ID NO: 75), PQPKKKPL (SEQ ID NO: 76), SALIKKKKKMAP (SEQ ID NO: 77), DRLRR (SEQ ID NO: 78), PKQKKRK (SEQ ID NO: 79), RKLKKKIKKL (SEQ ID NO: 80), REKKKFLKRR (SEQ ID NO: 81), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 82), RKCLQAGMNLEARKTKK (SEQ ID NO: 83), DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 84), KRTADGSEFESPKKKRKV (SEQ ID NO: 85),KRPAATKKAGQAKKKKGGGGSGGGGSGSKRPAATKKAGQAKKKK (SEQ ID NO: 86), GSHHHHHHGSGPKKKRKV (SEQ ID NO: 87), and GSGSGSHHHHHHGSGPKKKRKV (SEQ ID NO: 88).

38. The method of claims 37, wherein the one or more stabilization sequences is or comprises about 5 amino acids to about 300 amino acids in length, optionally wherein the one or more stabilizationDBl / 164937876.2 182Atorney Docket No.: KOM-003PC / 138774-5003 sequences is or comprises the amino acid sequence of any one of SEQ ID NOs: 106-110, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes thereto.

39. The method of any one of claims 36-38, wherein the one or more Bxb1 integrases further comprising fusion with one or more DNA-binding domains, optionally wherein the one or more DNA-binding domains comprises one or more TAL domains, one or more Cas proteins, and / or one or more zinc finger proteins (ZFPs), optionally wherein the one or more Bxb1 integrases is a fusion with about 1 to about 30 ZFP domains.

40. A cell line produced by the method of any one of claims 1 -39.

41. A method for generating one or more recombinant proteins comprising:(a) providing a cell line of claim 40, wherein the cell line comprises one or more insertion sequences, intervening DNA sequences, or protein-coding sequences, which encode the one or more recombinant proteins; and(b) culturing the cell line under conditions suitable to generate the one or more recombinant proteins.

42. The method of claim 41, wherein the one or more recombinant proteins comprises a therapeutic protein, chimeric antigen receptor (CAR), optionally an antibody or antibody-format protein, therapeutic enzyme, fusion protein, secretory protein, protein hormone, and / or protein toxin or antitoxin.

43. The method of claim 41 or 42, further comprising recovering, isolating, and / or purifying the one or more recombinant proteins from the cell line lysate and / or culture media.

44. An inducible hamster-specific caspase-9 fusion protein comprising an amino acid sequence having about or at least about 90%, about or at least about 92%, about or at least about 94%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 99, or the amino acid sequence of SEQ ID NO: 99.DBl / 164937876.2 183Atorney Docket No.: KOM-003PC / 138774-500345. A nucleic acid molecule comprising a polynucleotide sequence encoding the inducible hamsterspecific caspase-9 fusion protein of claim 44.

46. A donor DNA-specific nucleic acid molecule comprising: a first nucleic acid sequence encoding a promoter; and a second nucleic acid sequence encoding at least one start codon, at least one splice donor or splice acceptor sequence, and at least one att sequence, and wherein the promoter is operably linked to drive expression from the start codon and the start codon is not linked to a protein-coding sequence.

47. A landing pad nucleic acid molecule comprising: an aft sequence; at least one splice donor or splice acceptor sequence; a protein-coding nucleic acid sequence, wherein the protein-coding sequence lacks a start codon, encodes a selectable marker protein, and is located downstream of the at least one splice donor or splice acceptor sequence and the aft sequence; and a transcriptional terminator sequence downstream of the protein-coding nucleic acid sequence.DBl / 164937876.2 184

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