System for targeted nucleic acid integration

Abstract

The present invention relates to a polynucleotide for integrating a target nucleic acid into a genome of a cell, the polynucleotide comprising: a) the target nucleic acid; b) one or more flippase recognition target (FRT) sites flanking the target nucleic acid; c) one or more homology arms flanking the one or more FRT sites and / or the target nucleic acid; and d) one or more single guide RNA (sgRNA) recognition sites flanking the one or more homology arms and / or the one or more FRT sites and / or the target nucleic acid. There is synergistic enhancement of integration efficiency obtained by combining FLPe and CRISPR / Cas9. The present invention also relates to a system for integrating a target nucleic acid into a genome of a cell. In another aspect, the present invention relates to a method of integrating a target nucleic acid into a genome of a cell.

Description

DESCRIPTIONTITLE OF THE INVENTION: SYSTEM FOR TARGETED NUCLEIC ACID INTEGRATIONFIELD OF THE INVENTION

[0001] The invention generally relates to the field of genetic engineering. In particular, the invention relates to systems and methods for targeted gene integration.BACKGROUND OF THE INVENTION

[0002] The efficiency of targeted integration of transgenes into a pre-determined genomic locus in mammalian cells is often very low, limiting the use of mammalian cells for screening of large size libraries of proteins, DNA regulatory elements and non-coding RNA elements.

[0003] Previous studies reported that FLP and Cre recombinases were able to achieve 1 % to 6% targeted gene integration in CHO cells. However, this low efficiency of targeted gene integration limits the size of protein, DNA and RNA libraries that can be screened. While CRISPR / Cas9 programmable nuclease could achieve around 10% targeted gene integration, a drawback was the unavoidable off-target integration.

[0004] There is thus a need for a method of targeted gene integration that overcomes the drawbacks of the prior art. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides a polynucleotide for integrating a target nucleic acid into a genome of a cell, the polynucleotide comprising: a) the target nucleic acid; b) one or more flippase recognition target (FRT) sites flanking the target nucleic acid; c) one or more homology arms flanking the one or more FRT sites and / or the target nucleic acid; and d) one or more single guide RNA (sgRNA) recognition sites flanking the one or more homology arms and / or the one or more FRT sites and / or the target nucleic acid.

[0006] In one embodiment, the one or more FRT sites comprises two FRT sites.

[0007] In one embodiment, the one or more homology arms comprises a 5’ homology arm and a 3’ homology arm.

[0008] In one embodiment, the 5’ homology arm is homologous to an intron A sequence.

[0009] In one embodiment, the 3’ homology arm is homologous to a selectable marker sequence.

[0010] In one embodiment, the one or more single guide RNA recognition sites comprises two single guide RNA recognition sites.

[0011] In one embodiment, the target nucleic acid is selected from the group consisting of a gene, a DNA regulatory element or a non-coding RNA.

[0012] In one aspect, there is provided a vector comprising the polynucleotide as described herein.

[0013] In one aspect, there is provided a system for integrating a target nucleic acid into a genome of a cell, the system comprising: a) the cell comprising the genome engineered to comprise a landing pad, wherein the landing pad comprises a region comprising a sgRNA recognition site and wherein the region is flanked by a first FRT site and a second FRT site; b) a targeting vector comprising the polynucleotide as described herein, wherein the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad are configured to be cleaved by a CRISPR / Cas system; c) a first helper vector encoding a FLP recombinase; and d) a second helper vector encoding Cas9 and a sgRNA, wherein the sgRNA is configured to target the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad, wherein introduction of the targeting vector, the first helper vector and the second helper vector into the cell results in integration of the target nucleic acid at the landing pad of the genome.

[0014] In one embodiment, the one or more FRT sites of the polynucleotide and the first and second FRT sites of the landing pad are recognized by a FLP recombinase.

[0015] In one embodiment, each of the one or more homology arms is homologous to either an upstream sequence adjacent to the first FRT site of the landing pad or a downstream sequence adjacent to the second FRT site of the landing pad.

[0016] In one embodiment, the one or more homology arms comprises a 5’ homology arm and a 3’ homology arm, wherein the 5’ homology arm is homologous to the upstream sequence adjacent to the first FRT site of the landing pad and wherein the 3’ homology arm is homologous to the downstream sequence adjacent to the second FRT site of the landing pad.

[0017] In one embodiment, the one or more sgRNA recognition sites of the polynucleotide is substantially identical to the sgRNA recognition site of the landing pad.

[0018] In one embodiment, the target nucleic acid replaces the region of the landing pad flanked by the first and second FRT sites during integration of the target nucleic acid at the landing pad.

[0019] In one embodiment, the region of the landing pad flanked by the first and second FRT sites comprises a selectable marker gene prior to integration of the target nucleic acid.

[0020] In one embodiment, the sgRNA recognition site of the landing pad is located between the first and second FRT sites.

[0021] In one aspect, there is provided a method of integrating a target nucleic acid into a genome of a cell, comprising: a) inserting a landing pad into the genome of the cell, wherein the landing pad comprises a region comprising a sgRNA recognition site and wherein the region is flanked by a first FRT site and a second FRT site; and b) delivering to the cell: i) a targeting vector comprising the polynucleotide as described herein, wherein the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad are configured to be cleaved by a CRISPR / Cas system; ii) a first helper vector encoding a FLP recombinase, and ill) a second helper vector encoding Cas9 and a sgRNA, wherein the sgRNA is configured to target the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad, thereby resulting in integration of the target nucleic acid at the inserted landing pad of the genome.

[0022] In one aspect, there is provided a method of expressing a target polypeptide, the method comprising: a) integrating a target nucleic acid encoding the target polypeptide into a genome of a cell according to the method as described herein; and b) culturing the cell under suitable culture conditions such that the target polypeptide is expressed.

[0023] In one embodiment, the FLP recombinase is an enhanced FLP (FLPe) recombinase.

[0024] In one aspect, there is provided a use of the polynucleotide as described herein, the vector as described herein, the system as described herein or the method as described herein, for screening one or more of a protein library, a DNA library and a RNA library.

[0025] In one embodiment, the cell is a mammalian cell.

[0026] In one embodiment, the mammalian cell is a Chinese hamster ovary (CHO) cell and wherein the landing pad is located within Chr 9:57120324-57211782 of the genome of the CHO cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0028] Fig. 1 shows a schematic presentation for generation of MegaFLP (A) and BxB1 (B) master clones. ChiP, a chimeric promoter consisting of murine cytomegalovirus (CMV) enhancer and human CMV core promoter; Intron A, human CMV intron A; EGFP, cDNA encoding enhanced green fluorescence protein; IRESm, mutated encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES); Zeo, zeocin resistant gene cDNA; pA, simian virus 40 (SV40) polyadenylation signal; FRT and FRT3, Wild-type and mutated flippase recognition target sites; (ATG-)Pur, puromycin N-acetyl-transferase cDNA without start codon; Hyg, hygromycin resistant gene; FLPe, enhanced flippase recombinase cDNA; l-Scel, l-Scel meganuclease; AttP and AttPm, wild type and mutated BxB1 attachment sites; 5’HA and 3’HA, 5’ and 3’ homology arms; sgRNA-E, single guide RNA (targeting EGFP); arrowhead (A or V) indicates cutting site targeted by l-Scel or single guide RNA; Cas9 / sgRNA-E, Cas9 and single guide RNA (targeting EGFP).

[0029] Fig. 2 shows an evaluation of the efficiencies of targeted integration: FLP recombinase, BxB1 integrase, CRISPR / Cas9, l-Scel meganuclease, and combinations. (A) Schematic representation of targeted integration achieved through various technologies. ChiP, a chimeric promoter consisting of murine cytomegalovirus (CMV) enhancer and human CMV core promoter; Intron A, human CMV intron A; IRES, wild-type encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES); pA, simian virus 40 (SV40) polyadenylation signal; BpA, BGH polyadenylation signal sequence; FRT and FRT3, Wild-type and mutated flippase recognition target sites; (ATG-)Pur, puromycin N-acetyl-transferase cDNA without start codon; Bia, blasticidin resistant gene; arrowhead (▲ or ▼) indicates cutting site targeted by l-Scel or single guide RNA; Hyg, hygromycin resistant gene; DsRed, cDNA encoding red fluorescence protein; 5’HA and 3’HA, 5’ and 3’ homology arms. (B) Schematic representation of targeting vectors (pTarget) and helper vectors (pHelper). mCMV, murine CMV enhancer and promoter; FLPe, enhanced flippase recombinase cDNA; BxB1 , cDNA encoding BxB1 integrase; l-Scel, cDNA encoding l-Scel meganuclease; AttP and AttPm, wild type and mutated BxB1 attachment sites; U6, a type III RNA polymerase III promoter; CBh, chicken |3- actin hybrid promoter; sgRNA-H, single guide RNA (targeting hygromycin); hSpCas9, humanized S. pyogenes Cas9. (C) Levels of integration efficiency achieved through various technologies. The level of integration efficiency was determined as the percentage of DsRed cells in the transfected master cells. Each data point represents the average and standard deviation of measurements from two transfections using independently prepared plasmids.

[0030] Fig. 3 shows an evaluation of essential components required for obtaining high integration efficiency provided by combination of FLPe recombinase and CRISPR / Cas9. (A) Dot plot analysis of DsRed expressing cells in the transfected cells by flow cytometry. (B)Level of integration efficiency as determined by the percentage of DsRed cells in the transfected cells. Each data point represents the average and standard deviation of measurements from two transfections. (C) Changes in viability of the transfected cells under puromycin selection. (D) Dot plot analysis of DsRed expressing cells in the recovered stable pools. FRT and FRT3, Wild-type and mutated flippase recognition target sites; DsRed, cDNA encoding red fluorescence protein; 5’HA and 3’HA, 5’ and 3’ homology arms; sgRNA-H, single guide RNA targeting hygromycin; arrowhead (▼) indicates cutting site targeted by single guide RNA. The MegaFLP master cells were co-transfected with different targeting vectors and helper vectors. The percentage of DsRed expressing cells in the transfected cells were analyzed at day 4 by using flow cytometry. The transfected cells were then subjected to selection by using puromycin. The changes in viabilities during selection were determined. When the viabilities recovered to over 95%, the recovered stable pools were analyzed again for DsRed expression using flow cytometry.

[0031] Fig. 4 shows an evaluation of the combinatorial effect of FLPe recombinase and CRISPR / Cas9 on alternative FRT mutants and integration site. FRT15 and FRT14, mutated flippase recognition target sites; DsRed, cDNA encoding red fluorescence protein; NPT, neomycin resistant gene; Zeo, zeocin resistant gene; 5’HA and 3’HA, 5’ and 3’ homology arms; sgRNA-N, single guide RNA targeting NPT; arrowhead (▼) indicates cutting site targeted by single guide RNA. The master cells were co-transfected with different targeting vectors and helper vectors. The percentage of DsRed expressing cells in the transfected cells were analyzed at day 4 by using flow cytometry.

[0032] Fig. 5 shows the impact of homologous arm length on efficiency of targeted integration. (A) Schematic representation of targeting vectors containing different lengths of homologous arms. FRT3 and FRT, mutated and wild type flippase recognition target sites; DsRed, cDNA encoding red fluorescence protein; sgRNA-H, single guide RNA targeting Hyg; arrowhead (▼) indicates cutting site targeted by sgRNA-H; Hyg, hygromycin resistant gene; DsRed, cDNA encoding red fluorescence protein; 5’HA, 5’HA1952, 5’HA2952, 5’ homology arms with 961 bp, 1952 bp and 2952 bp, respectively; 3’HA, 3’HA1080, 3’HA1952 and 3’HA2952, 3’ homologous arms with 564 bp, 1080 bp, 1952 bp and 2952 bp, respectively. (B) Efficiency of targeted integration for different targeting vectors. The master cells were cotransfected with different targeting vectors, with and without helper vectors expressing FLPe and Cas9 / sgRNA. The percentage of DsRed expressing cells in the transfected cells were analyzed at day 4 by using flow cytometry.DETAILED DESCRIPTION OF THE INVENTION

[0033] The inventors initially developed a Chinese Hamster Ovary (CHO) targeted integration platform for high throughput screening of antibody libraries. This platform included three key components: 1) a CHO master clone containing a single copy landing pad in a genomic site that provides stable and high-level gene expression, 2) a targeting vector which allows simultaneous display and secretion of antibodies, and 3) a helper vector expressing recombinase FLPe. Integration of single-copy targeting vectors carrying different antibody genes into the landing pad in each master cell is achieved through recombinase-mediated cassette exchange (RMCE). The integration efficiency of this initial platform was low (~1%), which limits the size of antibody library that can be transfected into the master cells. Integration efficiency determines the size of antibody library that can be screened. For instance, with the integration efficiency of 1 %, transfection of ~107master cells allows expression of 1 x105IgGs. As such, application of the platform is limited to screening of small size antibody libraries.

[0034] To enhance the screening capacity of this platform, the inventors optimized the targeting vector and explored alternative targeted integration technologies including CRISPR / Cas9, meganucleases and BxB1 integrase for obtaining increased integration efficiency. FLP and BxB1 achieves targeted integration through RMCE, while l-Scel meganuclease and CRISPR / Cas9 enable targeted integration via homology-directed repair (HDR) pathway. These different targeted integration technologies were evaluated individually and in combination for integration efficiency in the CHO master cell line. Compared to FLPe which gave around 0.9% integration efficiency, BxB1 , l-Scel and CRISPR / Cas9 enhanced integration efficiency to 7.3%, 1.9% and 8%, respectively. Surprisingly, combination of FLPe and CRISPR / Cas9 enhanced integration efficiency to 14.6%, indicating synergistic interaction happened between the two technologies. Further studies indicated that obtaining such a high level of integration efficiency also requires the targeting vector to have one or two FLPe recognition site(s) (FRT(s)), one or two homologous arm(s) and one or two sgRNA targeting sequence(s) flanking the gene of interest. In addition, the combination of FLPe and CRISPR / Cas9 could maintain high accuracy of targeted gene integration and contributed to a shorter recovery timeline as compared to FLPe or CRISPR / Cas9 alone. The inventors confirmed the synergistic enhancement obtained by FLPe and CRISPR / Cas9 is independent of FRT mutants and integration sites. Interestingly, combination of ‘FLPe and IScel’ or ‘BxB1 and CRISPR / Cas9’ did not have combinatorial effects for enhancing integration efficiency, although l-Scel also use HDR pathway, which is the same as CRISPR / Cas9, to accomplishtargeted integration. These results indicate that the synergistic enhancement of integration efficiency obtained by combining FLPe and CRISPR / Cas9 is unique to these two enzymes.

[0035] It has been shown that the combination of FLPe and CRISPR / Cas9 enhances the efficiency of targeted integration of transgenes into CHO genome when the gene of interest in the targeting vector is flanked by one or two FRT site(s), homologous arm(s) and sgRNA targeting sequence(s). Where the gene of interest in the targeting vector is flanked by two FRT sites, the FRT sites are preferably heterospecific to avoid self-recombination.

[0036] It is unexpected that combination of FLPe and Cas9-sgRNA exhibited synergistic enhancement of targeted integration efficiency. I-Scel and Cas9 both use HDR pathway for targeted integration. However, combination of FLPe and l-Scel and combination of BxB1 and Cas9-sgRNA had no combinatorial effects for enhancing integration efficiency. This result indicates that synergistic enhancement of integration efficiency cannot be obtained by combining any recombinases and nucleases. Therefore, the combination of FLPe and Cas9- sgRNA is inventive and non-obvious.

[0037] It would be generally appreciated by a person skilled in the art that the present invention can be applied for screening of any protein libraries as well as DNA libraries (e.g. promoters, untranslated regions, IRES) and non-coding RNA libraries (e.g. siRNA and sgRNA).

[0038] In one aspect, the present disclosure provides a polynucleotide for integrating a target nucleic acid into a genome of a cell, the polynucleotide comprising: a) the target nucleic acid; b) one or more flippase recognition target (FRT) sites flanking the target nucleic acid; c) one or more homology arms flanking the one or more FRT sites and / or the target nucleic acid; and d) one or more single guide RNA (sgRNA) recognition sites flanking the one or more homology arms and / or the one or more FRT sites and / or the target nucleic acid.

[0039] The term “polynucleotide” as used herein refers to a nucleotide sequence of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either singlestranded or double-stranded. In one example, the polynucleotide is double-stranded. Nonlimiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, exons, introns, cDNA, recombinant polynucleotides, plasmids, and vectors.

[0040] In one embodiment, the target nucleic acid is selected from the group consisting of a gene, a DNA regulatory element or a non-coding RNA.

[0041] The term “target nucleic acid” may refer to a target gene, a DNA regulatory element (such as a promoter, IRES or untranslated region (UTR)) or a non-coding RNA (such as siRNA or sgRNA). The term “target gene” as used herein refers to a nucleotide sequence which encodes a polypeptide of interest and which is capable of being translated to produce theencoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. In one example, the target gene may encode a polypeptide of interest, or a fragment, derivative, or variant thereof. In one example, the “target gene” encodes an antibody or an antigen-binding fragment thereof.

[0042] The term “flippase recognition target (FRT) site” as used herein refers to a nucleotide sequence, in particular a defined stretch of nucleotides, i.e. a defined length of a nucleotide sequence, which can be recognized by a flippase (FLP) recombinase. The FRT site is necessary for allowing, together with a FLP recombinase, a targeted integration by recombination and defines the location of such a recombination.

[0043] In one embodiment, the one or more FRT sites comprises two FRT sites.

[0044] In one example, the methods herein involve a recombinase comprising a flippase (FLP) recombinase. In one example, the FLP recombinase is capable of mediating integration by recombination of nucleotide sequences flanked by FRT site(s) - recombinase-mediated cassette exchange (RMCE). During RMCE, cross-over events occur between one or two or more flanking FRT site(s): either at the 5' and / or the 3' end of the cassette to be exchanged. Non-limiting examples of a FLP recombinase include wild type FLP, FLP variants such as enhanced FLP (FLPe), or wild type FLP and FLP variants with optimized codons for mammalian cells. Preferably, the FLP recombinase is an enhanced FLP recombinase (FLPe). In one example, a FRT site comprises a 34 bp sequence that consists of two palindromic sequences of 13 bp each flanking an 8 bp spacer. In such example, the FLP may bind to the 13 bp palindromic sequences and mediates DNA break, exchange and ligation within the 8 bp spacer. In one example, a FRT site comprises a 48 bp sequence. In one example, a FRT site comprises a wild-type FRT sequence. In one example, a FRT site comprises a mutant FRT sequence which can be, but not limited to, a FRT1 , FRT2, FRT3, FRT4, FRT5, FTR10, FRT11, FRT12, FRT13, FRT14, FRT15, FRT16 sequence (See Schlake T, et al. (1994), Biochemistry, 33, 12746-12751, and Turan S, et al. J. Mol. Biol. (2010) 402, 52-69). In one example where two or more flanking FRT sites are present, the FRT sites may comprise wild-type FRT sequence(s) only, mutant FRT sequence(s) only, or a combination thereof. In another example where a pair of flanking FRT sites is present, the FRT sites may be heterospecific (to avoid self-recombination) and may comprise a wild-type FRT and a mutant FRT sequence or a pair of heterospecific mutant FRT sequences. For example, each of the one or more FRT sites of the polypeptide as described herein comprises a wild-type FRT sequence or a mutant FRT sequence, and each FRT site of the landing pad comprises a wild-type FRT sequence or a mutant FRT sequence. In one example, the mutant FRT sequence is a FRT3 sequence. Both the wild-type and the mutant FRT sequences can mediate FLP-dependent recombination. The whole system, also called "FRT / FLP" is well known in the art.

[0045] In one embodiment, the one or more homology arms comprises a 5’ homology arm and a 3’ homology arm.

[0046] In one embodiment, the 5’ homology arm is homologous to an intron A sequence.

[0047] In one embodiment, the 3’ homology arm is homologous to a selectable marker sequence.

[0048] In one embodiment, the one or more single guide RNA recognition sites comprises two single guide RNA recognition sites.

[0049] The term “single guide RNA (sgRNA) recognition site” as used herein refers to a target nucleotide sequence to which a single guide RNA (sgRNA) is designed to recognize (e.g., by having sufficient complementarity), where hybridization between a single guide RNA (sgRNA) recognition site sequence and a single guide RNA sequence promotes the formation of a CRISPR complex. For the purpose of this disclosure, the term “single guide RNA (sgRNA) recognition site” may also be referred to as “sgRNA targeting sequence” or “cutting site targeted by single guide RNA” or “sgRNA cutting site”. A non-limiting example of the sgRNA recognition site sequence is listed in the table of sequence listing below.

[0050] A “single guide RNA (sgRNA)” refers to an RNA that directs sequence-specific binding of a CRISPR complex to the target single guide RNA (sgRNA) recognition site. Typically, a single guide RNA comprises at least a guide sequence that has sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence. The single guide RNA may be composed of single-stranded RNA or encoded in a DNA polynucleotide. In one example, the single guide RNA is encoded in a DNA polynucleotide which is transcribed into a sgRNA sequence when introduced in the host cell. The sgRNA sequences can be designed using various variety of algorithms and computer programs that are available, and one such method can involve using Benchling (https: / / benchling.com / ) which uses the scoring method for on- and off-target from Doench, Fusi et al. and Hsu et al studies. A non-limiting example of the sgRNA sequence is listed in the table of sequence listing below.

[0051] In one example, the methods described herein involves a nuclease system comprising a CRISPR / Cas system. In general, "CRISPR / Cas system" used herein refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Gas”) genes. CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats”. A Cas protein, such as Cas9, is a nuclease discovered in bacteria that can bind and cut DNA at specific sequences by means of a singleguide RNA (sgRNA). Several CRISPR / Cas systems are known in the art (See Liu Z, et al. Application of different types of CRISPR / Cas-based systems in bacteria. Microbial CellFactories (2020) 19:172). In one example, the CRISPR / Cas system comprises a CRISPR / Cas9 system. In examples that include CRISPR / Cas nuclease systems, the Cas endonuclease may be encoded by a nucleotide sequence (e.g., DNA or RNA) or a protein.

[0052] The term “flanking” as used herein refers to a first nucleotide sequence that is located at either a 5’ or 3’ end, or both ends of a second nucleotide sequence. The flanking nucleotide sequence can be adjacent to or at a defined distance from the second nucleotide sequence. There is no specific limit of the length of a flanking nucleotide sequence. For example, a flanking sequence can be a few base pairs or a few thousand base pairs.

[0053] The term “homologous” as used herein in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same (“identical”) or have a specified percentage of amino acid residues or nucleotides that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm (e.g., by a BLAST alignment, or any other algorithm known to persons of skill), or alternatively, by visual inspection. For example, two sequences can be about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% homologous. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. For example, sequence homology may be measured using the BLAST program which is available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). For the purpose of this disclosure, the term “homologous arm” may also be referred to as “homology arm” and they can be used interchangeably.

[0054] The term “substantially identical” as used herein in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identity, or at least 99% identity (e.g., as determined using any of the above described sequence comparison algorithms or programs or alternatively, by visual inspection).

[0055] In one example, the polynucleotide described herein is a polynucleotide for integrating a target nucleic acid at a landing pad in the genome of a cell, and the polynucleotide comprises: a) the target nucleic acid, b) a pair of flippase recognition target (FRT) sites flanking the target nucleic acid, c) a pair of 5’ and 3’ homology arms (HA) flanking the pair of FRT sites, and d) one or more single guide RNA (sgRNA) recognition site(s) flanking the pair of 5’ and 3’ homology arms, wherein the 5’ homology arm is homologous to an upstream sequence adjacent to a first FRT site of the landing pad and the 3’ homology arm is homologous to a downstream sequence adjacent to a second FRT site of the landing pad, wherein the one or more sgRNA recognition site(s) defined in d) is (are) substantially identical to a sgRNArecognition site located in the landing pad of the genome, and wherein the one or more sgRNA recognition site(s) defined in d) and the sgRNA recognition site located in the landing pad are targeted for cleaving by a CRISPR / Cas system. In one example, the upstream sequence to which the 5’ homology arm is homologous comprises intron A sequence. The 5’ homology arm may be 30 bases to 10 kb in length. In one example, the 5’ homology arm is 961 b in length. The 3’ homology arm may be 30 bases to 10 kb in length. In one example, the 3’ homology arm is 1kb in length. In one example, the downstream sequence to which the 3’ homology arm is homologous comprises a selectable marker sequence and polyadenylation signal sequence with length of 1080 bp, optionally wherein the selectable marker sequence is a puromycin-resistant gene sequence without the ATG codon.

[0056] In one aspect, there is provided a vector comprising the polynucleotide as described herein.

[0057] In one aspect, there is provided a system for integrating a target nucleic acid into a genome of a cell, the system comprising: a) the cell comprising the genome engineered to comprise a landing pad, wherein the landing pad comprises a region comprising a sgRNA recognition site and wherein the region is flanked by a first FRT site and a second FRT site; b) a targeting vector comprising the polynucleotide as described herein, wherein the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad are configured to be cleaved by a CRISPR / Cas system; c) a first helper vector encoding a FLP recombinase; and d) a second helper vector encoding Cas9 and a sgRNA, wherein the sgRNA is configured to target the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad, wherein introduction of the targeting vector, the first helper vector and the second helper vector into the cell results in integration of the target nucleic acid at the landing pad of the genome.

[0058] The term “landing pad” as used herein refers to an integration target site in a host cell’s genome that facilitates insertion of an exogenous nucleotide sequence that comprises a sequence or part thereof that does not originate from a host cell which may include, but not limited to, a recombinant polynucleotide. The term “recombinant DNA” includes a DNA molecule that is created by combining genetic material from two or more different sources. The host cell may be a mammalian cell or a microbial cell. In one example, the “landing pad” is an integration target site that has been engineered into a host cell’s genome (e.g. a CHO genome) to facilitate further insertion (integration) of another exogenous nucleotide sequence. The landing pad may be located near or adjacent or within a specific locus of the genome of the host cell. In one example, the landing pad is located near or adjacent or within a specific locus of the genome of a mammalian cell. A non-limiting example of a mammalian cell is aChinese hamster ovary (CHO) cell. For example, the specific locus can be any position in the genome that is known to have increased rates of recombination in the field of integration and / or cloning methods. In one example, the specific locus is within CHO genome scaffold_9: 57120324-57211782 and the landing pad can be located near er adjacent or within this exemplified locus. For example, the landing pad can be at any position near or adjacent or within spanning positions numbered 57120324-57211782 of chromosome 9 of the CHO genome. In yet another example, the specific locus is between a pair of upstream and downstream DNA sequences, and the landing pad can be located near or adjacent or within this exemplified locus. The upstream DNA sequence can comprise, but are not limited to, nucleotide sequences that are at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleotide sequence of SEQ ID NO:1. The downstream DNA sequence can comprise, but are not limited to, nucleotide sequences that are at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 2. It is to be understood that the exact location of the landing pad in the genome with respect to the above exemplified positions for facilitating further / subsequent insertion or integration of an exogenous nucleotide sequence is not essential. Also, whether a landing pad located near or adjacent or within a specific locus of the genome facilitates further / subsequent integration of an integrated exogenous gene can be determined in accordance with standard procedures well known in the art or methods exemplified herein.

[0059] In one example, a landing pad comprises recombinase and / or nuclease target recognition sites which are flanked by landing pad homology sequences used for integration of an exogenous nucleotide sequence.

[0060] In one embodiment, the one or more FRT sites of the polynucleotide and the first and second FRT sites of the landing pad are recognized by a FLP recombinase.

[0061] In one embodiment, each of the one or more homology arms is homologous to either an upstream sequence adjacent to the first FRT site of the landing pad or a downstream sequence adjacent to the second FRT site of the landing pad.

[0062] In one embodiment, the one or more homology arms comprises a 5’ homology arm and a 3’ homology arm, wherein the 5’ homology arm is homologous to the upstream sequence adjacent to the first FRT site of the landing pad and wherein the 3’ homology armis homologous to the downstream sequence adjacent to the second FRT site of the landing pad.

[0063] In one embodiment, the one or more sgRNA recognition sites of the polynucleotide is substantially identical to the sgRNA recognition site of the landing pad.

[0064] In one embodiment, the target nucleic acid replaces the region of the landing pad flanked by the first and second FRT sites during integration of the target nucleic acid at the landing pad.

[0065] In one embodiment, the region of the landing pad flanked by the first and second FRT sites comprises a selectable marker gene prior to integration of the target nucleic acid.

[0066] In one embodiment, the sgRNA recognition site of the landing pad is located between the first and second FRT sites.

[0067] The term “vector” as used herein refers to a nucleic acid construct capable of carrying another nucleotide sequence and / or expressing another nucleotide sequence to which it is linked when introduced into a host cell. The vector may include, but not limited to, a vector as a self-replicating nucleic acid structure as well as a vector incorporated into the genome of a host cell into which it has been introduced. In one example, the vector can be an expression vector which directs the expression of nucleic acids to which it is operatively linked. In one example, a vector can comprise the following components including, but not limited to, one or more replication origins, one or more promoters, one or more genes of interest, and one or more selectable markers.

[0068] The term “targeting vector” as used herein refers to a vector that is capable of carrying one or more nucleotide sequences used for integration thereof at an integration target site or landing pad of a host cell. In one example, a targeting vector may lack a promoter and / or polyadenylation signal sequences.

[0069] A “helper vector” as used herein provides helper function(s) to facilitate integration of the nucleotide sequences expressed by the targeting vector. For example, the vector providing helper function(s) may express one or more elements and / or genes necessary for integration mediated in the presence of recombinases and / or nucleases. In one example, the helper vector can comprise the following components including, but not limited to, one or more promoters, a gene encoding enhanced flippase recombinase, and polyadenylation (polyA) signal sequences. In another example, the helper vector can comprise the following components including, but not limited to, one or more promoters, one or more single guide RNA sequences, a gene encoding cas9, and polyadenylation (polyA) signal sequences.

[0070] The term “master cell clone” as used herein refers to a host cell comprising exogenous nucleotide sequences which is preferably stably integrated, in its genome. In oneexample, the exogenous nucleotide sequences of the master cell clone include one or more integration target sites or landing pads, enabling a further site specific integration of other exogenous nucleotide sequences, thus, enabling a predetermined localized and directed integration of nucleotide sequences of interest at a desired location in a host cell's genome. Thus, in one example, a master cell clone is capable of targeted integration of one or more genes encoding sequence of interest. In one example, the targeted integration is mediated in the presence of recombinase and / or nuclease systems. In one example, the targeted integration is mediated in the presence of FRT / FLP and CRISPR / cas systems. Preferably, such a process introduces just one functional copy of a gene coding sequence, preferably just one copy of a gene coding sequence of interest at a predetermined locus.

[0071] The term “selectable marker” as used herein refers to a gene that allows cells carrying the gene to be specifically selected for or against, in the presence of a corresponding selection agent. For example, but not by way of limitation, a selectable marker can allow the host cell transformed with the selectable marker gene to be positively selected for in the presence of the gene; a non-transformed host cell would not be capable of growing or surviving under the selective conditions. Selectable markers can be positive, negative or bifunctional. Positive selectable markers can allow selection for cells carrying the marker, whereas negative selectable markers can allow cells carrying the marker to be selectively eliminated. A selectable marker can confer resistance to a drug or compensate for a metabolic or catabolic defect in the host cell. Preferably, the selectable marker is an antibiotic resistance gene or a reporter gene. Antibiotic genes include, but not limited to hygromycin-resistant and puromycin-resistant genes.

[0072] Beyond facilitating a selection in the presence of a corresponding selection agent, a selectable marker can alternatively provide a gene encoding a molecule normally not present in the cell, such as a reporter gene which can include, but not limited to, green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells harboring such a gene can be distinguished from cells not harboring this gene, e g., by the detection of the fluorescence emitted by the encoded polypeptide.

[0073] The term “antibody” used herein is to be interpreted in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half antibodies, and antibody fragments so long as they exhibit a desired antigen-binding activity.

[0074] The term “antigen binding molecule” used herein refers to an antibody, an antibody fragment or other protein construct, such as a domain.

[0075] In one aspect, there is provided a method of integrating a target nucleic acid into a genome of a cell, comprising: a) inserting a landing pad into the genome of the cell, wherein the landing pad comprises a region comprising a sgRNA recognition site and wherein the region is flanked by a first FRT site and a second FRT site; and b) delivering to the cell: i) a targeting vector comprising the polynucleotide as described herein, wherein the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad are configured to be cleaved by a CRISPR / Cas system; ii) a first helper vector encoding a FLP recombinase, and iii) a second helper vector encoding Cas9 and a sgRNA, wherein the sgRNA is configured to target the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad, thereby resulting in integration of the target nucleic acid at the inserted landing pad of the genome.

[0076] In one aspect, there is provided a method of expressing a target polypeptide, the method comprising: a) integrating a target nucleic acid encoding the target polypeptide into a genome of a cell according to the method as described herein; and b) culturing the cell under suitable culture conditions such that the target polypeptide is expressed.

[0077] In one embodiment, the FLP recombinase is an enhanced FLP (FLPe) recombinase.

[0078] In one aspect, there is provided a use of the polynucleotide as described herein, the vector as described herein, the system as described herein or the method as described herein, for screening one or more of a protein library, a DNA library and a RNA library.

[0079] In one embodiment, the cell is a mammalian cell.

[0080] In one embodiment, the mammalian cell is a Chinese hamster ovary (CHO) cell and wherein the landing pad is located within Chr 9:57120324-57211782 of the genome of the CHO cell.

[0081] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features,modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0082] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0083] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0084] MATERIAL AND METHODS

[0085] Cell culture and media for maintenance of master clone

[0086] The CHO master clones were grown in a protein-free medium (maintenance media) consisting of 50% HyQ PF (GE Healthcare Life Sciences) and 50% CD CHO (Thermo Fisher Scientific) supplemented with 1g / L sodium carbonate (Sigma), 6 mM glutamine (Sigma), 0.05% Pluronic F-68 (Thermo Fisher Scientific), and 600 pg / mL hygromycin in a humidified Kuhner shaker (Adolf Kuhner AG) with 8% CO2 at 37°C. Routine subculture was conducted every 3 to 4 days by seeding cells at density of 3x105cells / mL in 15 mL of fresh medium in 125 mL shake flasks (Corning). Cell density and viability were determined by trypan blue exclusion method on Vi-Cell XR viability analysers (Beckman Coulter).

[0087] Vector construction

[0088] The helper vectors expressing FLPe, BxB1 integrase and l-Scel meganuclease were synthesized by Genscript. The sequence of FLPe gene was from the pCAGGS-FLPe vector (Gene Bridges). Single guide RNA (sgRNA) sequence inserts targeting EGFP or Hyg were cloned into pX330-U6-Chimeric BB-CBh-hSpCas9 vector (Addgene, #42230) to generate helper Cas9-sgRNA vector. The sgRNA sequences were designed using Benchling (https: / / benchling.com / ) which use the scoring method for on- and off-target from Doench, Fusi et al. and Hsu et al studies.

[0089] Transfections

[0090] The CHO master cells were co-transfected with an appropriate targeting vector and one or two helper vectors using Amaxa SG Cell Line 4D-Nucleofector X Kit and program FF-137 (Lonza). In each transfection, 1*107cells were transfected with 5 g of pDonor vector and 5 pg of pHelper vector in circular format. In the case of two pHelper vectors, 2.5 pg of each vector will be used. The transfected cells were then re-suspended in 2 mL of maintenance media preloaded in 6-well suspension culture plates and incubated in the static IncuSafe incubators (Sanyo). After 24 h post- transfection, they were collected by centrifugation (100xg, 5 min) and re-suspended in 20 mL of protein-free maintenance medium (without antibiotics) in 125 mL shake flasks in the humidified Kuhner shaker (Adolf Kuhner AG) with 8% CO2 at 37°C. Seven days later, the transfected cells were subjected to flow cytometry analysis.

[0091] Flow cytometry

[0092] For analysis of integration efficiency, transfected cells in shake flasks were prepared in phosphate-buffered saline (PBS). The percentage of DsRed expressing in 10,000 transfected cells were analysed using MACSQuantX Flow Cytometer (Miltenyi Biotec). Integration efficiency was determined by calculating the percentage of DsRed-positive cells in the total number of cells.

[0093] EXAMPLES

[0094] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0095] Example 1 : Generation of master clones for evaluating the efficiency of various targeted integration technologies

[0096] To compare the efficiency of different technologies for targeted integration, two master clones were generated: 1) a MegaFLP master clone, and 2) a BxB1 master clone (Figure 1). The MegaFLP master clone allows for targeted integration through FLPe recombinase, l-Scel meganuclease, and CRISPR / Cas9 individually or in combination (Figure 2A). The BxB1 master clone allows for targeted integration through BxB1 integrase and CRISPR / Cas9 individually or in combination (Figure 2A). Both master clones were derived from the same primary master clone and have a single copy of the landing pad located at the same genomic locus, eliminating any impact of different genomic sites on the efficiency of targeted integration. The primary master clone was generated by transfection of a landing pad vector into CHO cells, followed by southern blot screening to identify clones with single copy integration. The landing pad vector used a chimeric promoter (ChiP) and the human CMV intron A to express EGFP and zeocin-resistant genes. These two genes were linked by an IRES mutant (IRESm) and transcribed as one transcript. The two FLPe recombinase recognition targets, FRT3 and FRT, are positioned to flank the EGFP-Zeo expression unit,allowing future cassette exchange. An impaired puromycin resistant gene ((ATG-)Puro), which start codon is removed, is placed downstream of FRT for selecting correct cassette exchange. To generate the MegaFLP master clone, a targeting vector FRT3-Hyg-FRT was designed to carry a hygromycin resistant gene (Hyg) and an l-Scel cutting site, flanked by FRT3 and FRT. This FRT3-Hyg-FRT targeting vector was co-transfected with a helper vector expressing FLPe into the primary master clone. The transfected cells were subsequently selected in medium containing hygromycin. As the Hyg targeting vector lacked a promoter, only cells in which the EGFP was replaced with the Hyg cassette survived the selection. Subsequently, 40 clones were isolated and screened for correct cassette exchange and single copy integration. To generate the BxB1 master clone, a helper vector expressing Cas9 and a sgRNA targeting GFP (sgRNA-E) was designed and co-transfected with a AttP-Hyg-AttPm targeting vector carrying Hyg, flanked by one pair of heterospecific BxB1 recognition sites (AttP and AttPm) into the primary master clone. In the AttP-Hyg-AttPm targeting vector, Intron A was placed upstream of AttP, and (ATG-)Puro was placed downstream of AttPm. These sequences served as homologous arms (HA) to aid CRISPR-mediated knock-in. In addition, two cutting sites, with the same sequence as the one targeted by sgRNA-E, were added into the targeting vector to enhance the targeted integration efficiency. Following co-transfection of the Cas9- sgRNA vector and AttP-Hyg-AttPm vector into the primary master clone, 40 clones were isolated and screened for correct and single copy integration into the landing pad.

[0097] Example 2: Evaluating the targeted Integration efficiencies of FLPe recombinase, BxB1 integrase, CRISPR / Cas9, l-Scel meganuclease, and combinations

[0098] The efficiencies of FLPe recombinase, CRISPR / Cas9 and l-Scel meganuclease were evaluated individually and in combination for targeted integration in the MegaFLP master clone. Similarly, the BxB1 integrase and CRISPR / Cas9 were evaluated individually and in combination in the BxB1 master clone (Figure 2A and 2B). To assess the efficiency of various technologies, a set of targeting vectors was designed, each containing a DsRed gene and an IRES element. The targeting vectors for recombinase-mediated cassette exchange (RMCE) contained FRT3 / FRT or AttP / AttPm flanking the DsRed-IRES sequence. In addition to FRT3 / FRT or AttP / AttPm, the targeting vectors for nuclease-mediated cassette exchange (NMCE) and combination of recombinase and nuclease also included the upstream homologous arms (5’HA) and downstream homologous arms (3’HA), along with flanking sgRNA or l-Scel cutting sites. The 5’HA sequence matched intron A, while the 3’HA covered the sequence of puromycin resistance gene from the 4th to the 557th base. The sgRNA cutting sites corresponded to the DNA sequence targeted by the sgRNA-H for Hyg. For the MegaFLP master clone, the targeting vectors were co-transfected with either FLPe expressing vector,Cas9-sgR NA vector, l-Scel expressing vector or their combination. Forthe BxBI master clone, the targeting vectors were co-transfected with either BxB1 expressing vector, Cas9-sgRNA vector, or their combination. Since the targeting vectors lacked a promoter and polyadenylation signal, DsRed expression occurred only upon integration into the landing pads. The transfected cells were collected at day 4, and analyzed by flow cytometry to quantify the percentage of red-colored cells (DsRed expressing), indicating the targeted integration efficiency (Figure 2C). When FLPe, Cas9-sgRNA, and l-Scel were used individually, the integration efficiencies at day 4 were 0.9%, 8.0%, and 1.9%, respectively, in the MegaFLP master clone.

[0099] For the BxB1 master clone, using BxB1 and Cas9-sgRNA alone resulted in integration efficiencies of 7.3% and 2.9% at day 4, respectively. Surprisingly, the 'FLPe + Cas9' combination significantly increased the integration efficiency to 14.3% at day 4. However, the 'FLPe+ IScel' or 'BxB1 + Cas9' combinations did not improve the integration efficiency. These results suggest a synergistic interaction between FLPe and Cas9-sgRNA.

[0100] Example 3: Essential components necessary for enhancing the targeted integration efficiency mediated by the combination of FLP / FRT and Cas9 / sgRNA

[0101] The combination of FLPe and Cas9 / sgRNA-H demonstrated a synergistic enhancement of integration efficiency when applied to the targeting vector pTargetF3F-H5H3- DCg(H) containing FLPe recognition sites (FRT and FRT3), homologous arms (5’HA and 3’HA), and double sgRNA-H cutting sites (DC). To examine the underlying reasons for this phenomenon and the essential components involved, five additional targeting vectors were constructed: one with FRT / FRT3, 5’HA and 3’HA but having only one sgRNA cutting site (pTargetF3F-H5H3-SCg(H)), one with FRT / FRT3, DC but only having only 5’HA (pTargetF3F- H5-DCg(H)), one with 5’HA, 3’HA and DC but only having FRT3 (pTargetF3-H5H3-BPA- DCg(H)), one with FRT / FRT3 and HA but lacking DC (pTargetF3F-H5H3), and the other with HA and DC but lacking FRT / FRT3 (pTargetH5H3-DCg(H)). These five targeting vectors were assessed alongside the targeting vector pTargetF3F-H5H3-DCg(H), as well as the targeting vector with only FRT / FRT3 (pTargetF3F), for their efficiency of targeted integration in the MegaFLP master clone (Figure 3). For simplicity, pTargetF3F-H5H3-DCg(H) is abbreviated as F3FH5H3D, pTargetF3F-H5H3-SCg(H) is abbreviated as F3FH5H3S, pTargetF3F-H5- DCg(H) is abbreviated as F3FH5D, pTargetF3-H5H3-BPA-DCg(H) is abbreviated as F3H5H3D, pTargetF3F-H5H3 is abbreviated as F3FH5H3, pTargetH5H3-DCg(H) is abbreviated as H5H3D, and pTargetF3F is abbreviated as F3F, respectively. Each targeting vector was co-transfected with either the helper vector expressing FLPe, the helper vector expressing Cas9 / sgRNA-H, or the combination of both helper vectors. Flow cytometry wasconducted to analyze the efficiency of targeted integration in the transfected cells on day 4. Subsequently, puromycin selection was employed to select for accurate cassette exchange. Once the viability of the transfected cells recovered over 95%, the percentage of DsRed- expressing cells was determined again using flow cytometry.

[0102] Consistent with the results observed in the previous experiment, the combination of FLPe and Cas9 / sgRNA-H demonstrated a remarkable boost in integration efficiency when applied to the targeting vector F3FH5H3D. It achieved an efficiency of 12.1 %, surpassing the individual efficiencies of FLPe (1.4%) and Cas9 / sgRNA-H (4.6%) alone (Figure 3A and 3B). Removing one sgRNA cutting site from the targeting vector did not affect the integration efficiency achieved by using FLPe and Cas9 / sgRNA-H alone and their combination compared to the use of F3FH5H3D targeting vector. Applying FLPe, Cas9 / sgRNA-H and the combined approach on the vector F3FH5H3S resulted in efficiencies of 1.0%, 3.9% and 11.3%, respectively, showcasing a continued synergistic enhancement from the combination of FLPe and Cas9 / sgRNA-H. However, removing one homologous arm, e g., 3’HA, from the targeting vector resulted in slightly lower integration efficiency for Cas9 / sgRNA-H alone (2.8% vs. 4.6%) and the combination of FLPe and Cas9 / sgRNA-H (9.7% vs. 12.1%), while having no impact on the use of FLPe alone (1.3% vs. 1.4%) compared to the F3FH5H3D targeting vector. In contrast, removing one FLPe recognition site from the targeting vector resulted in slightly increased integration efficiency for Cas9 / sgRNA-H alone (9.1% vs. 4.6%) and the combination of FLPe and Cas9 / sgRNA-H (14.8% vs. 12.1 %), while reducing integration efficiency for FLPe alone (0.4% vs. 1.4%) compared to the F3FH5H3D targeting vector. Although the combination of FLPe and Cas9 / sgRNA-H still led to a synergistic enhancement of integration efficiency in the targeting vector F3FH5H3 with DC removed, the removal of DC resulted in a significant reduction in integration efficiency compared to the targeting vector F3FH5H3D, both for Cas9 / sgRNA-H alone (0.3% vs. 4.6%) and the combination of FLPe and Cas9 / sgRNA-H (2.6% vs. 12.1%). Nonetheless, the removal of DC had no impact on the integration efficiency from FLPe alone (1.2% vs. 1.4%). For the targeting vector H5H3D with FRT / FRT3 removed, FLPe alone did not result in targeted integration, as expected. When Cas9 / sgRNA-H alone or the combination of FLPe and Cas9 / sgRNA-H was employed on the targeting vector H5H3D, similar integration efficiencies of 4.3% to 5% were observed, suggesting that integration primarily occurred through the action of Cas / sgRNA in both conditions. When analyzing the targeting vector F3F with both HA and DC removed, FLPe alone yielded an integration efficiency of 1.5%, comparable to the efficiencies obtained with FLPe alone on the targeting vector F3FH5H3 and F3FH5H3D. This suggests that HA and DC had no impact on FLPe- mediated targeted integration. As expected, Cas9 / sgRNA-H alone did not lead to targetedintegration when applied to the targeting vector F3F, as the presence of HA is necessary for Cas9 / sgRNA-mediated integration. Furthermore, no increase in integration efficiency was observed when the combination of FLPe and Cas9 / sgRNA-H was employed on the targeting vector F3F. The integration efficiency level obtained from the combination (1.2%) was similar to that achieved with FLPe alone (1.5%), indicating that integration primarily occurred through the action of FLPe in this condition. These results emphasize the crucial role of all three components (FRT3 / FRT, HA(s), and sgRNA cutting site(s)) in the targeting vector for achieving a high synergistic enhancement of integration efficiency through the combination of FLPe and Cas9 / sgRNA-H. It is worth noting that although a synergistic enhancement of integration efficiency also occurred when applying the combination of FLPe and Cas9 / sgRNA- H to the targeting vector F3FH5H3, which contained FRT / FRT3 and HA, the magnitude of integration efficiency was significantly lower compared to that obtained from the targeting vector FHD, which contained all three components.

[0103] Reducing the recovery timeline is crucial for efficient generation of stably transfected cell lines. A high integration efficiency plays a vital role in achieving this goal by increasing the number of transfected cells with the targeting vector integrated into the chromosome, enabling their survival during selection. Similar profiles of recovery were observed between F3FH5H3D and F3FH5H3S targeting vectors under different conditions. Applying the combination of two helper vectors expressing FLPe and Cas9 / sgRNA-H on F3FH5H3D and F3FH5H3S demonstrated the highest integration efficiency of 12.1% and 11.3% at day 4, respectively, outperforming other transfection conditions (Figure 3A and 3B). As expected, these transfected cells exhibited the shortest recovery timeline, with viabilities dropping to their lowest level of approximately 60% at day 7 after undergoing puromycin selection at day 4, followed by a rapid recovery to 95% by day 11 (Figure 3C). In contrast, applying FLPe and Cas9 / sgRNA-H alone on the F3FH5H3D or F3FH5H3S targeting vector resulted in lower integration efficiency and slower recovery period of 14 days. The pools transfected with the targeting vector F3FH5D with 3’HA removed exhibited similar recovery profile to those transfected with F3FH5H3D for FLPe alone and the combination of FLPe and Cas9 / sgRNA-H, respectively. However, the lowest level of viabilities dropped to 7.8% at day 7 for Cas9 / sgRNA-H clone compared to 35% obtaining by applying Cas9 / sgRNA-H alone on F3FH5H3D, corresponding to the reduced targeted integration efficiency of 2.8% at day 4 obtained by applying Cas9 / sgRNA-H alone on the F3FH5D targeting vector. Similarly, the pools transfected with the targeting vector F3H5H3D with FRT removed exhibited similar recovery profile as those transfected with F3FH5H3D for FLPe alone and the combination of FLPe and Cas9 / sgRNA-H, while the pools generated using Cas9 / sgRNA-H clone hadincreased lowest level of viability at day 7 and reduced recovery timeline of 11 days, corresponding to the increased targeted integration efficiency of 9.7% at day 4. Among the pools co-transfected with the targeting vector H5H3D and different helper vectors, those cotransfected with the helper vector expressing FLPe alone did not survive the selection, as no integration occurred under this condition. Interestingly, the pools co-transfected with the helper vector expressing Cas9 / sgRNA-H alone or the combination of FLPe and Cas9 / sgRNA-H took 18 days to recover, despite having higher integration efficiencies than those transfected with the targeting vectors F3FH5H3 and F3F. The pools transfected with the targeting vector F3FH5H3 and F3F displayed a recovery period of 14 days, except for the condition where Cas9 / sgRNA-H was applied alone on the targeting vector F3F, which resulted in no integration and failure to survive the selection. The lowest viabilities for these transfected pools were also observed at day 7, ranging from 20% to 33%, which were lower than those observed for the condition of applying the combination of FLPe and Cas9 / sgRNA-H on the targeting vector F3FH5H3D and F3FH5H3S.

[0104] The recovered stable pools were analyzed to determine the percentage of DsRed- expressing cells and the homogeneity of DsRed expression levels (Figure 3D). Since the targeting vectors lack promoters and polyadenylation signals, only cells with correct integration of the targeting vectors into the landing pads would express DsRed and the puromycin-resistant gene, allowing their survival in puromycin-containing medium during selection. Given that the targeting vectors integrate into the same genomic sites in the targeted cells, the expression level of DsRed should be relatively consistent among different cells in a stable pool when accurate integration happens. All stable pools generated by co-transfecting the targeting vector F3FH5H3D with either FLPe or Cas9 / sgRNA-H alone, or their combinations, showed over 95% of cells expressing DsRed at similar levels (Figure 3D). Over 95% of cells expressing DsRed were also observed in all stable pools generated using the F3FH5H3S vector, which lacks one sgRNA cutting site, across the three conditions. In the case of the targeting vector F3FH5D with one 3’HA removed, pools generated using FLPe alone and the combination of FLPe and Cas9 / sgRNA-H had over 95% of cells expressing DsRed cells. However, when using Cas9 / sgRNA-H alone, the percentage dropped 49%. In the case of the targeting vector F3H5H3D, the pools generated using FLPe alone, Cas9 / sgRNA-H alone, and their combination had DsRed expressing cells of 62.4%, 69.2% and 83.3%, respectively. In the case of the targeting vector F3FH5H3 with DC removed, pools generated using FLPe alone also exhibited over 95% of cells expressing DsRed at similar levels. However, when using Cas9 / sgRNA-H alone or the combination of FLPe and Cas9 / sgRNA-H on FH, the percentages dropped to 84% and 91%, respectively, indicating asignificant number of colorless cells with no DsRed expression. Analyzing the stable pools generated by co-transfection of the targeting vector H5H3D, which had FRT / FRT3 removed, with either Cas9 / sgRNA-H alone or the combination of FLPe and Cas9 / sgRNA-H revealed that more than half of the cells were colorless. When the targeting vector F3F with both HA and DC removed was used, over 95% of cells in the pools co-transfected with FLPe alone showed consistent DsRed expression levels, similar to those observed when using FLPe alone on the targeting vectors F3FH5H3D, F3FH5H3S, F3FH5D and F3FH5H3. However, when the combination of FLPe and Cas9 / sgRNA-H was used, the percentage dropped to 80%. These results suggested that Cas9 / sgRNA-H mediated integration is not accurate. The mechanism by which colorless cells survived the selection is not yet clear. One possible reason is that Cas9 / sgRNA-H caused double-strand breaks in the hygromycin-resistant gene, resulting in a frame shift upon recombination and activation of the puromycin-resistant gene. Interestingly, the inclusion of FRT / FRT3 in the targeting vectors seemed to alleviate this issue, as indicated by the increased percentage of DsRed-expressing cells in the stable pools generated using the targeting vector F3FH5H3 compared to those generated using H5H3D. Inclusion of FRT / FRT3 and DC in the targeting vector further minimized the proportion of colorless cells to less than 5% in the stable pools, regardless of the types of helper vectors used. Similarly, the inclusion of FRT / FRT3 and one sgRNA cutting site in the targeting vector also minimized the proportion of colorless cells to 5% or less than 5% in the stable pools, regardless of the types of helper vectors used.

[0105] Example 4: Combination of FLP / FRT and Cas9 / sgRNA enhances the targeted integration efficiency on alternative FRT mutants and integration site

[0106] To confirm that the synergistic enhancement of integration efficiency obtained by the combination of FLPe and Cas9 / sgRNA is applicable to targeting vectors containing different FRT mutants and landing pads in different integration sites, a primary master clone was developed by transfecting a landing pad vector into CHO cells, followed by Southern blot screening to identify clones with single copy integration. The landing pad vector has the same structure as that for the generation of the MegaFLP master clone except that FRT3 / FRT were replaced with FRT15 / FRT14, EGFP-Zeo was replaced with DsRed-blasticidin (Bla)-resistant genes, and the impaired puromycin-resistant gene (ATG-Pur) was replaced with the zeocin- resistant gene (ATG-Zeo). To generate the FRT15 / FRT14 master clone, a targeting vector FRT15-NPT-FRT14 was designed to carry a neomycin-resistant gene (NPT) flanked by FRT15 and FRT14. This FRT15-NPT-FRT14 targeting vector was co-transfected with a helper vector expressing FLPe into the primary master clone. The transfected cells were subseguently selected in medium containing G418. As the NPT targeting vector lacked apromoter, only cells in which the DsRed was replaced with the NPT cassette survived the selection. Subsequently, 40 clones were isolated and screened for correct cassette exchange and single copy integration.

[0107] To assess the efficiency of targeted integration obtained using FLPe alone, Cas9 / sgRNA alone, and their combination for the newly developed master clone, two targeting vectors were designed (Figure 4). The targeting vector for evaluating FLPe contained a DsRed gene and an IRES element flanked by FRT15 / FRT14. In addition to FRT15 / FRT14, the targeting vector for Cas9 / sgRNA and the combination of FLPe and Cas9 / sgRNA also included the upstream homologous arms (5’HA) and downstream homologous arms (3’HAZ), along with flanking sgRNA cutting sites. The 5’HA sequence matched intron A, while the 3’HAZ covered the sequence of the zeocin resistance gene from the 4th to the 370th base. The sgRNA cutting sites corresponded to the DNA sequence targeted by the sgRNA-N for NPT. The integration efficiencies for FLPe alone, Cas9 / sgRNA alone, and their combination were evaluated by co-transfection of the FRT15 / FRT14 master clone with the targeting vectors and an FLPe expressing vector, a Cas9-sgRNA vector, or their combination. The transfected cells were collected on day 4 and analyzed by flow cytometry to quantify the percentage of red- colored cells (DsRed expressing), indicating the targeted integration efficiency. When FLPe and Cas9 / sgRNA-N were used individually, the integration efficiencies on day 4 were 0.14% and 2.21 %, respectively. In contrast, the combination of FLPe and Cas9 / sgRNA-N enhanced the integration efficiency to 3.83%.

[0108] These results, together with previous findings, demonstrated that the synergistic enhancement obtained by the combination of FLPe and Cas9 / sgRNA is applicable to different FRT mutants and integration sites.

[0109] Example 5: Optimizing length homologous arms for enhancing efficiency of targeted integration efficiency

[0110] The 5' homologous arm (5’HA) and 3' homologous arm (3’HA) in the targeting vector pTargetF3F-H5H3-DCg(H) have 961 bp and 564 bp, respectively. These arms match the intron A and truncated puromycin resistance gene in the landing pad vector. This design lacks a promoter, polyadenylation signal, and a complete puromycin resistance gene, ensuring that only cells with correct integration of the targeting vector into the landing pad express DsRed and puromycin resistance gene, allowing them to survive selection in puromycin-containing medium. To further enhance the efficiency of targeted integration, new targeting vectors were constructed with the 5’HA remaining the same length of 961 bp but increasing the length of the 3’HA to 1080 bp, 1952 bp, and 2952 bp, respectively. Vectors were also constructed with the 3’HA remaining the same length of 564 bp while increasing the length of the 5’HA to 1952bp and 2952 bp, respectively (Figure 5A). To minimize the chance of random integration leading to gene expression, the newly designed vectors were created without a promoter or polyadenylation signal sequence. For instance, the targeting vectors containing 3’HA1080, 3’HA1952, and 3’HA2952 do not have a promoter but do have a polyadenylation signal sequence, while the targeting vectors containing 5’HA1952 and 5’HA2952 have a promoter but lack a polyadenylation signal sequence. Each of these targeting vectors was transfected into the MegaFLP master cells, both with and without helper vectors expressing FLPe and Cas9 / sgRNA. The percentage of DsRed-expressing cells in the transfected populations was analyzed on day 4 using flow cytometry (Figure 5B). When the 5’HA length remained the same at 961 bp, increasing the length of the 3’HA from 564 bp to 1080 bp doubled the percentage of red cells from 11 % to 21% in the transfected pools. Further increasing the length of the 3’HA to 1952 bp and 2952 bp did not result in a further increase, but rather a decrease, in the percentage of red cells compared to that obtained from the targeting vector containing 3’HA1080. The red cells in these pools should be due to targeted integration, as no red cells were detected in transfected master cells with the same targeting vectors but without helper vectors. Increasing the length of the 5’HA while keeping the 3’HA the same length at 564 bp also resulted in an increased percentage of red cells in the transfected pools. However, some red cells in these transfected pools were likely due to random integration, as nearly 5% of red cells were also detected in master cells transfected with the same targeting vectors without helper vectors.

[0111] These results indicate that the targeting vector containing the 5’HA and 3’HA1080 is the most effective, as it provides high efficiency of targeted integration while avoiding gene expression from randomly integrated targeting vectors.

[0112] Table 1 : Sequence listing

[0113] While embodiments of the invention have been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

CLAIMS1. A polynucleotide for integrating a target nucleic acid into a genome of a cell, the polynucleotide comprising: a) the target nucleic acid; b) one or more flippase recognition target (FRT) sites flanking the target nucleic acid; c) one or more homology arms flanking the one or more FRT sites and / or the target nucleic acid; and d) one or more single guide RNA (sgRNA) recognition sites flanking the one or more homology arms and / or the one or more FRT sites and / or the target nucleic acid.

2. The polynucleotide of claim 1 , wherein the one or more FRT sites comprises two FRT sites.

3. The polynucleotide of claim 1 or 2, wherein the one or more homology arms comprises a 5’ homology arm and a 3’ homology arm.

4. The polynucleotide of claim 3, wherein the 5’ homology arm is homologous to an intron A sequence.

5. The polynucleotide of claim 3 or 4, wherein the 3’ homology arm is homologous to a selectable marker sequence.

6. The polynucleotide of claim any one of claims 1 to 5, wherein the one or more single guide RNA recognition sites comprises two single guide RNA recognition sites.

7. The polynucleotide of any one of claims 1 to 6, wherein the target nucleic acid is selected from the group consisting of a gene, a DNA regulatory element or a non-coding RNA.

8. A vector comprising the polynucleotide of any one of claims 1 to 7.

9. A system for integrating a target nucleic acid into a genome of a cell, the system comprising:a) the cell comprising the genome engineered to comprise a landing pad, wherein the landing pad comprises a region comprising a sgRNA recognition site and wherein the region is flanked by a first FRT site and a second FRT site; b) a targeting vector comprising the polynucleotide of any one of claims 1 to 7, wherein the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad are configured to be cleaved by a CRISPR / Cas system; c) a first helper vector encoding a FLP recombinase; and d) a second helper vector encoding Cas9 and a sgRNA, wherein the sgRNA is configured to target the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad, wherein introduction of the targeting vector, the first helper vector and the second helper vector into the cell results in integration of the target nucleic acid at the landing pad of the genome.

10. The system of claim 9, wherein the one or more FRT sites of the polynucleotide and the first and second FRT sites of the landing pad are recognized by a FLP recombinase.

11. The system of claim 9 or 10, wherein each of the one or more homology arms is homologous to either an upstream sequence adjacent to the first FRT site of the landing pad or a downstream sequence adjacent to the second FRT site of the landing pad.

12. The system of claim 11 , wherein the one or more homology arms comprises a 5’ homology arm and a 3’ homology arm, and wherein the 5’ homology arm is homologous to the upstream sequence adjacent to the first FRT site of the landing pad and wherein the 3’ homology arm is homologous to the downstream sequence adjacent to the second FRT site of the landing pad.

13. The system of any one of claims 9 to 12, wherein the one or more sgRNA recognition sites of the polynucleotide is substantially identical to the sgRNA recognition site of the landing pad.

14. The system of any one of claims 9 to 13, wherein the target nucleic acid replaces the region of the landing pad flanked by the first and second FRT sites during integration of the target nucleic acid at the landing pad.

15. The system of any one of claims 9 to 14, wherein the region of the landing pad flanked by the first and second FRT sites comprises a selectable marker gene prior to integration of the target nucleic acid.

16. The system of claim 16, wherein the sgRNA recognition site of the landing pad is located between the first and second FRT sites.

17. A method of integrating a target nucleic acid into a genome of a cell, the method comprising: a) inserting a landing pad into the genome of the cell, wherein the landing pad comprises a region comprising a sgRNA recognition site and wherein the region is flanked by a first FRT site and a second FRT site; and b) delivering to the cell: i)a targeting vector comprising the polynucleotide of any one of claims 1 to 7, wherein the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad are configured to be cleaved by a CRISPR / Cas system; ii) a first helper vector encoding a FLP recombinase, and iii) a second helper vector encoding Cas9 and a sgRNA, wherein the sgRNA is configured to target the one or more sgRNA recognition sites of the polynucleotide and the sgRNA recognition site of the landing pad, thereby resulting in integration of the target nucleic acid at the inserted landing pad of the genome.

18. A method of expressing a target polypeptide, the method comprising: a) integrating a target nucleic acid encoding the target polypeptide into a genome of a cell according to the method of claim 17; and b) culturing the cell under suitable culture conditions such that the target polypeptide is expressed.

19. The system of any one of claims 9 to 16 or the method of claim 17 or 18, wherein the FLP recombinase is an enhanced FLP (FLPe) recombinase.

20. Use of the polynucleotide of any one of claims 1 to 7, the vector of claim 8, the system of any one of claims 9 to 16 and 19, or the method of any one of claims 17 to 19, for screening one or more of a protein library, a DNA library and a RNA library.

21. The polynucleotide of any one of claims 1 to 7, the vector of claim 8, the system of any one of claims 9 to 16 and 19, the method of any one of claims 17 to 19, or the use of claim 20, wherein the cell is a mammalian cell.

22. The polynucleotide of any one of claims 1 to 7 and 21 , the vector of claim 8 and 20, the system of any one of claims 9 to 16, 19 and 21, the method of any one of claims 17 to 19 and 21, or the use of claim 20 or 21 , wherein the mammalian cell is a Chinese hamster ovary (CHO) cell and wherein the landing pad is located within Chr 9:57120324- 57211782 of the genome of the CHO cell.

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