Methods and compositions for improving precision of endonuclease-mediated genome editing

Abstract

Disclosed are methods and compositions for reducing concatemer formation and formation of other artifacts during insertion of donor nucleic acid into a genome. Applications to genome editing and genome-engineered call therapy are also disclosed.

Description

METHODS AND COMPOSITIONS FOR IMPROVING PRECISION OF ENDONUCLEASE-MEDIATED GENOME EDITINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application Serial No. 63 / 605,082 filed on December 1, 2023, and to U.S. provisional application Serial No. 63 / 699,524 filed on September 26, 2024.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] None.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 30, 2024, is named CBI054_30_SL.xml and is 107,155 bytes in size.FIELD OF THE INVENTION

[0004] The invention relates to the field of genome engineering and more specifically, to an improved method of CRISPR-mediated transgene insertion.BACKGROUND OF THE INVENTION

[0005] Genome editing is a method of modifying the genome of a living cell in order to impart desired properties on the cells and its progeny. One type of genome editing involves inserting a DNA fragment at a specific location in the cellular genome. Earlier attempts to insert DNA cassettes at targeted loci relied on sporadic homologous DNA recombination between a DNA cassette flanked by homology arms and the complementary DNA sequence in the genome. More recently developed technology utilizes endonucleases that cut the cellular DNA at a precise location where gene insertion is desired in order to increase the frequency of HDR events and subsequently the intended integration events. Endonucleases capable of precise cleavage of complex eukaryotic genomes include rare-cutting endonucleases or nucleic acid sequence-guidedendonucleases with long recognition sequences. Zinc finger nucleases (ZFNs), (TALENs) and CRISPR endonucleases are examples of recently discovered endonucleases capable of being programmed to cleave a predetermined nucleotide sequence in the genome. ZFNs, TALENs, and CRISPR endonucleases have been successfully used to engineer human cells both ex vivo and in vivo for therapeutic purposes.

[0006] Since its discovery in 2012, the CRISPR-Cas mediated genetic engineering has found many uses in animal and plant cells. Sequence-guided CRISPR nucleases are capable of cleaving the genome of a cell at a precise location to accomplish gene disruption (gene knock-out) or insertion of a new gene sequence (gene knock-in). CRISPR-based genome modification has proven to be safer and more accurate than the previous generation methods utilizing integrating viruses and random DNA insertion.

[0007] An endonuclease-mediated insertion of a transgene is accomplished by cutting the genome at a desired locus followed by insertion of a DNA cassette which provides a genomic edit (such as a correction of a mutation) or a transgene. If the DNA cassette is flanked by homology arms, the insertion is accomplished primarily by homology-directed DNA repair (HDR). The intended HDR outcome is an insertion of a single copy of the cassette into the targeted locus. Problematically, multiple copies (concatemers) of the transgene are often inserted instead. Concatemer insertion is a commonly observed byproduct that occurs at a significant frequency during CRISPR-Cas mediated gene insertion (see Skryabin B., et al., (2020) Pervasive head-to- tail insertion of DNA templates mask desired CRISPR-Cas9-mediated genome editing events, Science Advances 6.eaax2941, and Tai, P.W.L, et al., (2018) Adeno-associated virus genome population sequencing analysis achieved full vector genome resolution and reveals human-vector chimeras, Molecular therapy, methods and clinical development, 9:130.)

[0008] There is a need for a method of reducing concatemer formation in order to increase the precision of genome editing to among other goals, increase safety and potency of various therapeutic modalities developed using endonucleases-mediated genome editing.

[0009] Another challenging aspect of genome editing is multiplex transgene insertion (knock-in). Currently, multiplex knock-in requires preparing a separate viral construct (e.g., an AAV-based construct) and a separate viral particle for each knock-in step. Viral constructs and viruses are costly to make, and multiplex or serial viral infections negatively impact cell health (Fulop, T., et al. (2013) Human T cell aging and the impact of persistent viral infections. Frontiersin Immunology, 4:271). Additionally, successive electroporations to deliver the genome editing reagents for each transgene knock-in step is detrimental to the cells (Batista, T., et al. (2021) Cell death due to electroporation -A review. Bioelectrochemistry 141 : 107871). There is an unmet need for a robust method of inserting multiple transgenes into the same cellular genome.SUMMARY OF THE IN VENITON

[0010] In one embodiment, the invention is a nucleic acid construct for delivery of a donor nucleic acid into a genome of a target cell, the construct comprising: at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; at least one cleavage site for a sequence-guided endonuclease, the cleavage site located outside of the homology arms relative to the donor nucleic acid; and a vector backbone. In some embodiments, at least one cleavage site for the sequence- guided endonuclease comprises a sequence substantially identical to the target sequence in the genome of the cell, and wherein the sequence-guided endonuclease capable of cleaving the construct is also capable of cleaving the target site in the genome. In some embodiments, the at least one donor nucleic acid comprises in the 5’-3’ direction: a first donor nucleic acid flanked by a first set of first and second homology arms capable of hybridizing to a first target sequence in the genome of the cell, and a second donor nucleic acid flanked by a second set of first and second homology arms capable of hybridizing to a second target sequence in the genome of the cell, wherein one cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid. In some embodiments, the construct comprises two or more cleavage sites for a sequence-guided endonuclease, wherein at least two cleavage sites are for different sequence-guided endonucleases. In some embodiments, the construct comprises three cleavage sites for a sequence-guided endonuclease, wherein the first cleavage site is located between the vector backbone and the first homology arm of the first donor nucleic acid; the second cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid; and the third cleavage site is located between the second homology arm of the second donor nucleic acid and the vector backbone. In some embodiments, the endonuclease is a CRISPR endonuclease, and the cleavage site comprises a protospacer and a protospacer adjacent motif (PAM). In some embodiments, the protospacer is substantially identical to a sequence in the target sequence in thegenome of the cell, e.g., to a sequence in a gene selected from the group consisting of PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, GISH, CBLB, 2B4, TRAC, GUTA, DNMT3A, DNMT3L, FAS, TET1, TET2, TET3, CD155, RASA2, SOCS1, SOCS3 and B2M. In some embodiments, the protospacer is substantially identical to a sequence in the PDCD1 gene and comprises or consists essentially of SEQ ID NO: 22. In some embodiments, the protospacer is substantially identical to a sequence in the TRAC gene and comprises or consists essentially of SEQ ID NO: 10. In some embodiments, the protospacer is substantially identical to a sequence in the B2M gene and comprises or consists essentially of SEQ ID NO: 5. In some embodiments, the protospacer is substantially identical to a sequence in the CISH gene and comprises or consists essentially of SEQ ID NO: 20.

[0011] In some embodiments, the CRISPR endonuclease is a Type V CRISPR endonuclease, and the PAM consists of a sequence selected from 5’-TTN-3’, 5’-TTTN-3’ and 5’- TTTV-3’. In some embodiments, the CRISPR endonuclease is a Type II CRISPR endonuclease, and the PAM consists of a sequence selected from 5'-NGG-3', 5'-NGGNG-3’, 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3’. In some embodiments, the CRISPR endonuclease is a Type I (CRISPR CASCADE) endonuclease, and the PAM consists of a sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5 ’-GAG-3’, 5 ’-C AG-3’, 5’-GTG-3’, 5’-TAA- 3’, 5’-TGG-3’, 5’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5’-ATA-3’, 5’-TAG-3’, and 5’-TTG-3’. In some embodiments, the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALEN recognition sequences each comprising 18-30 nucleotides separated by a spacer comprising 12-25 nucleotides. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18 nucleotides long ZFN recognition sequences each comprising 9-18 nucleotides separated by a spacer comprising 4-18 nucleotides.

[0012] In some embodiments, the vector is a plasmid vector, or a viral vector selected from adenovirus (AdV), adeno-associated virus (AAV) and a lentivirus.

[0013] In some embodiments, the donor nucleic acid comprises an expression cassette. In some embodiments, the expression cassette comprises a protein coding sequence encoding a protein selected from the group consisting of CD47, a chimeric antigen receptor (CAR), a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a portion of an MHC Class I molecule and a fusionincluding an MHC Class I molecule. In some embodiments, the construct comprises SEQ ID NO: 31, or SEQ ID NO: 29, or SEQ ID NO: 28. In some embodiments, the expression cassette comprises a promoter selected from the EFla promoter, the PGK1 promoter, the MND promoter, the Ubc promoter, the CAG promoter, the CaMKIIa promoter, the 0-Actin promoter, the 0- interferon promoter, the hsp70 promoter, the MMTV-LTR promoter, the RSV-LTR promoter the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. In some embodiments, the expression cassette encodes a functional RNA, e.g., a functional RNA is selected from the group consisting of micro-RNA, small inhibitory RNA (siRNA), small hairpin RNA (shRNA), and guide RNA for a CRISPR endonuclease (NATNA). In some embodiments, the donor nucleic acid comprises one, two or three stop codons wherein the two or three stop codons are in different translation frames, In some embodiments, the construct comprises SEQ ID NO: 19. In some embodiments, the donor nucleic acid comprises an splice site selected from the group consisting of a donor splice site, an acceptor splice site and a donor-acceptor splice site. In some embodiments, the construct comprises a sequence selected from the group consisting of SEQ ID NO: 15-18 and 30.

[0014] In one embodiment, the invention is a composition comprising the nucleic acid construct comprising at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; at least one cleavage site for a sequence-guided endonuclease, the cleavage site located outside of the homology arms relative to the donor nucleic acid; and a vector backbone. In some embodiments, the composition further comprises one or more sequence-guided endonucleases capable of cleaving the one or more sequence-guided endonuclease recognition sites in the nucleic acid construct. In some embodiments, the sequence-guided endonuclease is a CRISPR endonuclease, and the composition further comprises a nucleic acid targeting nucleic acid (NATNA) capable of binding the protospacer and promoting cleavage by the CRISPR endonuclease.

[0015] In one embodiment, the invention is a cell comprising the nucleic acid construct comprising at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; at least one cleavage site for a sequence-guided endonuclease, the cleavage site located outside of thehomology arms relative to the donor nucleic acid; and a vector backbone. In some embodiments, the cell is a bacterial cell, a plant cell, a human cell, or a non-human animal cell. In some embodiments, the human cell is in vitro. In some embodiments, the human cell is not a human germline cell or a human totipotent cell.

[0016] In one embodiment, the invention is a method for integrating a donor nucleic acid sequence into a cellular genome in a population of cells resulting in a reduced number of cells comprising concatemers of the donor nucleic acid in the cellular genome, the method comprising introducing into the cells of a population of cells the nucleic acid construct comprising at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; at least one cleavage site for a sequence- guided endonuclease, the cleavage site located outside of the homology arms relative to the donor nucleic acid; and a vector backbone, and further introducing into the cells the sequence-guided endonuclease capable of cleaving the sequence-guided endonuclease recognition sites in the nucleic acid construct.

[0017] In some embodiments, the method further comprises integration of at least a portion of the donor nucleic acid into the target cell’s chromosome. In some embodiments, only one copy of at least a portion of the donor nucleic acid integrates into the genomes of cells of a population of cells. In some embodiments, a number of cells comprising concatemers of the donor nucleic acid integrated into the genome is reduced relatively to the number of cells comprising concatemers of the donor nucleic acid integrated into the genome where the donor nucleic acid is introduced via a nucleic acid construct lacking any cleavage sites for the sequence-guided endonuclease. In some embodiments, the method further comprises integration of one or more additional donor nucleic acids into the cellular genome. In some embodiments, the at least one donor nucleic acid in the nucleic acid construct comprises in the 5’-3’ direction: a first donor nucleic acid flanked by a first set of first and second homology arms capable of hybridizing to a first target sequence in the genome of the cell, and a second donor nucleic acid flanked by a second set of first and second homology arms capable of hybridizing to a second target sequence in the genome of the cell, wherein one cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid. In some embodiments, the nucleic acid construct comprises two or more cleavage sites for a sequence- guided endonuclease, wherein at least two cleavage sites are for different sequence-guidedendonucleases. In some embodiments, the nucleic acid construct comprises three cleavage sites for a sequence-guided endonuclease, wherein the first cleavage site is located between the vector backbone and the first homology arm of the first donor nucleic acid; the second cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid; and the third cleavage site is located between the second homology arm of the second donor nucleic acid and the vector backbone. In some embodiments, the sequence-guided endonuclease is a CRISPR endonuclease, and the cleavage site comprises a protospacer and a protospacer adjacent motif (PAM). In some embodiments, the protospacer is substantially identical to a sequence in the target sequence in the genome of the cell, e.g., a sequence in a gene selected from the group consisting of PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, GISH, CBLB, 2B4, TRAC, CIITA, DNMT3A, DNMT3L, FAS, TETl, TET2, TET3, CD155, RASA2, SOCS1, SOCS3 and B2M. In some embodiments, the protospacer is substantially identical to a sequence in the PDCD1 gene and comprises or consists essentially of SEQ ID NO: 22. In some embodiments, the protospacer is substantially identical to a sequence in the TRAC gene and comprises or consists essentially of SEQ ID NO: 10. In some embodiments, the protospacer is substantially identical to a sequence in the B2M gene and comprises or consists essentially of SEQ ID NO: 5. In some embodiments, the protospacer is substantially identical to a sequence in the CISH gene and comprises or consists essentially of SEQ ID NO: 20.

[0018] In some embodiments, the CRISPR endonuclease is a Type V CRISPR endonuclease, and the PAM consists of a sequence selected from 5’-TTN-3’, 5’-TTTN-3’ and 5’- TTTV-3’. In some embodiments, the CRISPR endonuclease is a Type II CRISPR endonuclease, and the PAM consists of a sequence selected from 5'-NGG-3', 5'-NGGNG-3’, 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3’. In some embodiments, the CRISPR endonuclease is a Type I (CRISPR CASCADE) endonuclease, and the PAM consists of a sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5 ’-GAG-3’, 5 ’-C AG-3’, 5’-GTG-3’, 5’-TAA- 3’, 5’-TGG-3’, 5’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5’-ATA-3’, 5’-TAG-3’, and 5’-TTG-3’. In some embodiments, the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALEN recognition sequences each comprising 18-30 nucleotides separated by a spacer comprising 12-25 nucleotides. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18nucleotides long ZFN recognition sequences each comprising 9-18 nucleotides separated by a spacer comprising 4-18 nucleotides.

[0019] In some embodiments, the vector is a plasmid vector, or a viral vector selected from adenovirus (AdV), adeno-associated virus (AAV) and a lentivirus.

[0020] In some embodiments, the donor nucleic acid comprises an expression cassette. In some embodiments, the expression cassette comprises a protein coding sequence encoding a protein selected from the group consisting of CD47, a chimeric antigen receptor (CAR), a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a portion of an MHC Class I molecule and a fusion including an MHC Class I molecule. In some embodiments, the expression cassette comprises SEQ ID NO: 31, or SEQ ID NO: 29, or SEQ ID NO: 28. In some embodiments, the expression cassette comprises a promoter selected from the EFla promoter, the PGK1 promoter, the MND promoter, the Ubc promoter, the CAG promoter, the CaMKIIa promoter, the P-Actin promoter, the -interferon promoter, the hsp70 promoter, the MMTV-LTR promoter, the RSV-LTR promoter the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. In some embodiments, the expression cassette encodes a functional RNA. In some embodiments, the functional RNA is selected from the group consisting of micro-RNA, small inhibitory RNA (siRNA), small hairpin RNA (shRNA), and guide RNA for a CRISPR endonuclease (NATNA). In some embodiments, the donor nucleic acid comprises one, two or three stop codons wherein the two or three stop codons are in different translation frames. In some embodiments, the donor nucleic acid comprises SEQ ID NO: 19. In some embodiments, the donor nucleic acid comprises a splice site selected from the group consisting of a donor splice site, an acceptor splice site and a donor-acceptor splice site. In some embodiments, the nucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NO: 15-18 and 30.

[0021] In some embodiments, the method further comprises detecting sustained expression of a protein or RNA encoded by the expression cassette. In some embodiments, the population of cells is a population of bacterial cells, plant cells, human cells, or non-human animal cells. In some embodiments, the human cells are present zz? vitro. In some embodiments, the human cells are present in vivo but are not human germline cells or human totipotent cells and the method does not alter genetic identity of a human being. In some embodiments, the human cell is an immune cellselected from a T cell, a T cell precursor, a B cell, a B cell precursor, a macrophage, and a macrophage precursor.

[0022] In one embodiment, the invention is a method of manufacturing the nucleic acid construct, the method comprising linking together at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; at least one cleavage site for a sequence-guided endonuclease, the cleavage site located outside of the homology arms relative to the donor nucleic acid; and a vector backbone. In some embodiments, the method comprises linking in the 5’-3’ direction: a first donor nucleic acid flanked by a first set of first and second homology arms capable of hybridizing to a first target sequence in the genome of the cell, and a second donor nucleic acid flanked by a second set of first and second homology arms capable of hybridizing to a second target sequence in the genome of the cell, wherein one cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid. In some embodiments, the construct comprises two or more cleavage sites for a sequence-guided endonuclease, wherein at least two cleavage sites are for different sequence-guided endonucleases. In some embodiments, the construct comprises three cleavage sites for a sequence-guided endonuclease, wherein the first cleavage site is located between the vector backbone and the first homology arm of the first donor nucleic acid; the second cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid; and the third cleavage site is located between the second homology arm of the second donor nucleic acid and the vector backbone. In some embodiments, the sequence-guided endonuclease is a CRISPR endonuclease, and the cleavage site comprises a protospacer and a protospacer adjacent motif (PAM). In some embodiments, the protospacer is substantially identical to a sequence in the target sequence in the genome of the cell, e.g., to a sequence in a gene selected from the group consisting of PDCD1, CTLA-4, LAGS, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, CISH, CBLB, 2B4, TRAC, CIITA, DNMT3A, DNMT3L, FAS, TET1, TET2, TET3, CD155, RASA2, SOCS1, SOCS3 and B2M. In some embodiments, the protospacer is substantially identical to a sequence in the PDCD1 gene and comprises or consists essentially of SEQ ID NO: 22. In some embodiments, the protospacer is substantially identical to a sequence in the TRAC gene and comprises or consists essentially of SEQ ID NO: 10. In some embodiments, the protospacer is substantially identical to a sequence in the B2M gene and comprises or consistsessentially of SEQ ID NO: 5. In some embodiments, the protospacer is substantially identical to a sequence in the CISH gene and comprises or consists essentially of SEQ ID NO: 20. In some embodiments, the CRISPR endonuclease is a Type V CRISPR endonuclease, and the PAM consists of a sequence selected from 5’-TTN-3’, 5’-TTTN-3’ and 5’-TTTV-3’. In some embodiments, the CRISPR endonuclease is a Type II CRISPR endonuclease, and the PAM consists of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'- NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3’. In some embodiments, the CRISPR endonuclease is a Type I (CRISPR CASCADE) endonuclease, and the PAM consists of a sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5 ’-GAG-3’, 5 ’-C AG-3’, 5’-GTG-3’, 5 ’-PAAS’, 5’-TGG-3’, 5’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5’-ATA-3’, 5’-TAG-3’, and 5’-TTG-3’. In some embodiments, the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALEN recognition sequences each comprising 18-30 nucleotides separated by a spacer comprising 12-25 nucleotides. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18 nucleotides long ZFN recognition sequences each comprising 9-18 nucleotides separated by a spacer comprising 4-18 nucleotides.

[0023] In some embodiments, the vector is a plasmid vector, or a viral vector selected from adenovirus (AdV), adeno-associated virus (AAV) and a lentivirus.

[0024] In some embodiments, the donor nucleic acid comprises an expression cassette. In some embodiments, the expression cassette comprises a protein coding sequence encoding a protein selected from the group consisting of CD47, a chimeric antigen receptor (CAR), a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a portion of an MHC Class I molecule and a fusion including an MHC Class I molecule. In some embodiments, the expression cassette comprises SEQ ID NO: 31, or SEQ ID NO: 28, or SEQ ID NO: 29. In some embodiments, the expression cassette comprises a promoter selected from the EFla promoter, the PGK1 promoter, the MND promoter, the Ubc promoter, the CAG promoter, the CaMKIIa promoter, the P-Actin promoter, the P-interferon promoter, the hsp70 promoter, the MMTV-LTR promoter, the RSV-LTR promoter the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. In some embodiments, the expression cassette encodes a functional RNA. In some embodiments, thefunctional RNA is selected from the group consisting of micro-RNA, small inhibitory RNA (siRNA), small hairpin RNA (shRNA), and guide RNA for a CRISPR endonuclease (NATNA). In some embodiments, the donor nucleic acid comprises one, two or three stop codons wherein the two or three stop codons are in different translation frames. In some embodiments, the donor nucleic acid comprises SEQ ID NO: 19. In some embodiments, the donor nucleic acid comprises an splice site selected from the group consisting of a donor splice site, an acceptor splice site and a donor-acceptor splice site. In some embodiments, the construct comprises a sequence selected from the group consisting of SEQ ID NO: 15-18 and 30.

[0025] In one embodiment, the invention is a method of making a cell comprising a donor nucleic acid integrated into the genome of the cell, the method comprising introducing into the cell a nucleic acid construct comprising at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; at least one cleavage site for a sequence-guided endonuclease, the cleavage site located outside of the homology arms relative to the donor nucleic acid; and a vector backbone. In some embodiments, the cell is selected from the group consisting of a bacterial cell, a plant cell, a human cell, or a non-human animal cell. In some embodiments, the human cell is present in vitro. In some embodiments, the human cell is present in vivo but is not a human germline cell or a human totipotent cell and the method does not alter genetic identity of a human being. In some embodiments, the human cell is an immune cell selected from a T cell, a T cell precursor, a B cell, a B cell precursor, a macrophage, and a macrophage precursor.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGURE 1 is a diagram of DNA cassette insertion with and without concatemer formation.

[0027] FIGURE 2 is a diagram of an AAV construct with endonuclease cleavage sites.

[0028] FIGURE 3 is a diagram of a plasmid construct with endonuclease cleavage sites.

[0029] FIGURE 4 is a detailed diagram of cleavage site options in an AAV construct.

[0030] FIGURE 5 is a diagram of an exemplary engineered Cast 2a cleavage site in the human genome and in an AAV construct. Figure discloses SEQ ID NOS 32-33, respectively, in order of appearance.

[0031] FIGURE 6 is a diagram of an AAV construct with two Casl2a cleavage sites. Figure discloses SEQ ID NOS 34 and 33, respectively, in order of appearance.

[0032] FIGURE 7 is a diagram of an AAV construct with two Cas9 cleavage sites. Figure discloses SEQ ID NOS 35-36, respectively, in order of appearance.

[0033] FIGURE 8A and FIGURE 8B show reduction in imperfect insertions and concatemer insertions respectively in human T cells at the B2M locus.

[0034] FIGURE 9 is a diagram of the homology arms (left and right) that include a Casl2a target site.

[0035] FIGURE 10 is a diagram of a single AAV producing a single site-specific insertion event (prior art).

[0036] FIGURE 11 is a diagram of one STACK approach where two site specific insertion events are accomplished with a single AAV vector, and a Cas target site is present in the AAV vector allowing the separation of the two HDR templates.

[0037] FIGURE 12 is a diagram of an alternative STACK approach where the AAV vector contains two different Cas target sites.

[0038] FIGURE 13 is a diagram of an alternative STACK approach where the AAV vector contains three Cas target sites.

[0039] FIGURE 14: is a diagram of an alternative STACK approach where two AAV vectors each contain Cas target sites allowing the separation of the four HDR templates. In this example, two of the HDR templates (B and B’) have homology arms capable of hybridizing to the same insertion sites so that B and B’ can be inserted as alternative alleles of the same locus.DETAILED DESCRIPTION OF THE INVENTION

[0040] Definitions

[0041] The following definitions are provided to aid in understanding of the disclosure. Unless defined in this section, technical and scientific terms used in this disclosure have the meaning commonly understood by a person of ordinary skill in the art. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual , 4thEd. Cold Spring Harbor Lab. Press (2012).

[0042] The term “concatemer” refers to a continuous stretch of nucleic acid that contains two or more tandem copies of the same sequence. For example, a concatemer containing multiplecopies of the donor nucleic acid can occur in the genome of a cell when the donor nucleic acid is introduced into the cell.

[0043] The terms “CRISPR” (clustered regularly interspaced short palindromic repeats), “Cas” (CRISPR-associated protein) “CRISPR-Cas” and “CRISPR system” refer to the genome editing tool derived from prokaryotic organisms and comprising a nucleic acid guide molecule and a sequence-specific nucleic acid-guided endonuclease capable of cleaving a target nucleic acid strand at a site complementary to a sequence in the nucleic acid guide. Properties of various CRISPR systems are reviewed in Makarova et al., (2011) Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 9(6): 467-477.

[0044] The term “endonuclease cleavage site” refers to a combination of all the nucleic acid sequences necessary for the endonuclease to recognize, bind and cleave the nucleic acid. For some endonucleases (e.g., CRISPR Cas), the recognition and binding sequence is separate from the sequence that is cleaved. For other endonucleases (e.g., Type II restriction endonucleases) the cleaved sequence falls within the recognition sequence. The CRISPR / Cas endonuclease cleavage site comprises a protospacer which is a binding site for a nucleic acid targeting nucleic acid (NATNA) and a protospacer adjacent motif (PAM).

[0045] The term “AAV” refers to adeno-associated virus and includes human AAV types 1-13 and various animal AAV types including human serotypes AAV3, AAV5, AAV 8 and AAV9 suitable for liver cells, and AAV6 suitable for immune cells including T cells and NK cells.

[0046] The term “inverted terminal repeat” or “ITR” refers to a terminal repeat sequence that forms a hairpin structure. Viral ITR functions include enabling replication, integration, and provirus rescue. In some embodiments, the ITR is a naturally-occurring AAV ITR (e.g., ITR derived from any of the AAV 1-13). In some embodiments, the ITR is a synthetic ITR or a modified AAV ITR that is able to form a hairpin and perform the essential functions of the wildtype ITR. In some embodiments, the ITR has regions of complementary palindromic sequences which can anneal and allow the formation of a T or Y structure, (see U.S. Patent No. 5,478,745).

[0047] The term “donor nucleic acid” and “donor DNA” refer to the nucleic acid (and DNA) fragment that is intended to be inserted into the genome of a cell. Donor nucleic acid sequences may be combined with vector nucleic acid sequences to form a construct useful for delivery of the donor nucleic acid into the target cell. The construct may contain nucleic acid elements that are not part of the vector and not part of the donor DNA. For example, endonucleasecleavage sites described herein are not part of the AAV backbone. Likewise, the endonuclease cleavage sites are not intended to be inserted into the cellular genome (i.e., are not part of the donor DNA.)

[0048] The term “flanking” in relation to nucleic acid sequences refers to sequences located outside and both sides of another sequence. For example, FIGURE 3 shows homology arms flanking the transgene cassette. Flanking sequences can themselves be flanked by another sequence. For example, FIGURE 3 shows exonuclease cleavage sites flanking the homology arms which in turn, are flanking the transgene cassette. The term “flanking” is not intended to mean immediately adjacent. For example, FIGURE 2 shows endonuclease cleavage sites flanking the entire AAV construct sequence and located adjacent the inverted terminal repeat (ITR) of the virus. In FIGURE 2, the endonuclease cleavage sites are flanking the transgene although they are not immediately adjacent to it.

[0049] The terms “perfect insertion” and “perfect integration” refer to insertion of a single complete copy of the donor nucleic acid without any vector sequences into the genome of a cell into which the donor nucleic acid has been delivered.

[0050] The terms “partial insertion” and “partial integration” refer to insertion of only a part of the donor nucleic acid (with or without vector sequences) into the genome of a cell into which the donor nucleic acid has been delivered.

[0051] The terms “imperfect insertion” and “imperfect integration” refer to any insertion event other than the perfect insertion (perfect integration) event. Imperfect insertions include insertion of a partial or complete single copy of the donor nucleic acid but with some vector sequences and insertion of a concatemer of the donor nucleic acid with or without any vector sequences.

[0052] The terms “target site,” “target sequence” and “target nucleic acid” are used interchangeably to refer to a nucleic acid sequence in a locus (site) in a genome of a cell that is targeted for cleavage by an endonuclease and optionally, a subsequent transgene insertion. While insertions occur at a single location (e.g., between two nucleotides), a longer sequence is needed to direct an endonuclease to a particular target sequence in the genome. In case of a CRISPR / Cas endonuclease, the target sequence directing the endonuclease to a particular locus in the genome is a protospacer capable of hybridizing to a spacer in a nucleic acid targeting nucleic acid (NATNA) associated with the CRISPR / Cas endonuclease in a nucleoprotein complex (NPC), anda protospacer adjacent motif (PAM). One of skill in nucleic acid chemistry would appreciate that the formation of a hybrid between two nucleic acids (e.g., between a protospacer in the target nucleic acid and a spacer in the NATNA) does not require perfect complementarity and depends on reaction conditions such as temperature and ionic strength of the reaction mixture and the length of the hybridizing nucleic acids. For example, two nucleic acids (e.g., protospacers) that are substantially identical but less than 100% identical can hybridize to the same NATNA under certain conditions. Similarly, a NATNA having a spacer that is substantially complementary but less than 100% complementary to a protospacer in the target nucleic acid can hybridize to the protospacer under certain conditions.

[0053] The term “homology arms” refers to nucleic acid sequences built into a transgene delivery construct and flanking a transgene in the construct that are capable of hybridizing to a sequence located 5’ (upstream) and 3’ (downstream) of the desired transgene insertion site in the genome. For this reason, it can be said that homology arms are capable of hybridizing to the target sequence in the genome. If a CRISPR / Cas endonuclease is used for transgene insertion, one or both homology arms may comprise a protospacer or a sequence substantially similar to the protospacer or a part of a protospacer, or a part of a sequence substantially similar to the protospacer. For example, the protospacer or a sequence substantially similar to the protospacer may be contained in one homology arm or be split between the two homology arms.

[0054] The term “construct” refers to an engineered nucleic acid formed by combining elements from different sources. For example, a construct may comprise vector nucleic acid sequences (“backbone”) derived from for example, a plasmid, a virus, or an artificial chromosome, and insert nucleic acid sequences derived for example from a eukaryotic organism such as a human. A construct may retain certain biological properties of the vector (e.g., self-replication or the ability to be packaged into a viral particle) while retaining and propagating the insert sequence. For example, a construct may comprise nucleic acid sequences of AAV sufficient for packaging the construct into an AAV particle and an insert comprising a coding sequence of a human gene.

[0055] The terms “sequence guided endonuclease” and “different sequence guided endonuclease” refer to sequence-guided endonucleases capable of cleaving different target nucleic acid sequences. In the case of CRISPR / Cas endonucleases, the endonuclease is present in the form of a nucleoprotein complex (NPC) that includes an endonuclease protein complexed with a nucleic acid targeting nucleic acid (NATNA). The term “different sequence guided endonucleases” mayrefer to two NPCs that include identical endonuclease proteins (e.g., two Cas9 proteins or two Casl2a proteins) but different NATNAs comprising different spacers. Such two NPCs will bind and cleave two different target sequences thereby acting as two different sequence guided endonucleases.

[0056] The bacterial CRISPR system (clustered regularly interspaced short palindromic repeats) and associated Cas (CRISPR-associated) nucleases along with other rare-cutting or sequence-guided endonucleases have become a widely used genome editing tool. A nucleic acid guide molecule (CRISPR RNA or crRNA) guides an endonuclease to cleave a target nucleic acid strand having complementarity to the nucleic acid guide. For example, CRISPR mediated knock- in of a transgene is accomplished by cutting the genome at a desired locus with a CRISPR endonuclease followed by insertion of the transgene. The transgene is commonly introduced as a nucleic acid sequence (e.g., an expression cassette) flanked by homology arms. By comparison, in restriction endonuclease-based genetic engineering the transgene is flanked by restriction endonuclease cleavage sites and restriction endonucleases are used to excise the transgene from the plasmid. In case of traditional use of rare-cutting endonucleases such as CRISPR, it is he homology arms that mediate the homology-directed DNA repair (HDR) resulting in insertion of a single copy of the transgene into the targeted locus. In practice, CRISPR-mediated gene insertion frequently results in unwanted concatemerization (tandem insertion of multiple full or partial copies) (FIGURE 1). Another common artifact of endonuclease-mediated transgene insertion is insertion of some vector nucleic acids into the genome along with the transgene (“partial vector insertion” or “backbone insertion”). Backbone insertions are especially undesirable in eukaryotic cells because they introduce viral sequences, bacterial plasmid sequences and even antibiotic resistance genes. Yet another artifact is tandem insertion of sequences from two or more distinct donor DNA nucleic acids (e.g., transgenes) delivered to the cell simultaneously.

[0057] The instant disclosure teaches modifications of the nucleic acid construct used to deliver donor DNA, where the modifications reduce the formation of concatemers and other artifacts. In broadest terms, the elements added to the nucleic acid construct promote cleavage of concatemers and other artifacts into monomers of the donor nucleic acid.

[0058] It is well known in the art to use an endonuclease (e.g., a restriction endonuclease) to excise donor DNA prior to any subsequent step (including insertion into a cellular genome).More recently, WO2017118598 disclosed that CRISPR Cas9 can be used to excise donor DNA from a plasmid vector, and WO2019238772 disclosed that CRISPR Casl2a expressed in a cell can promote complementarity-directed end joining (CDEJ) recombination between two Cast 2a target sites cleaved by Casl2a (i.e., the same Casl2a cleaves both targets).

[0059] By comparison, the instant disclosure teaches an improvement of adding endonuclease cleavage sites to the nucleic acid delivery construct that already has homology arms capable of mediating HDR-directed insertion of donor DNA. While insertion of donor nucleic acid occurs without the improvement, with the improvement, the accuracy of insertion is greatly increased. Not intending to be bound by a particular theory, the inventors propose that the observed reduction in concatemer and other artifact formation results from cleaving concatemers and other artifacts formed at all stages of the donor nucleic acid insertion process. Concatemers present in the delivery construct (e.g., a virus, plasmid, or linear DNA), concatemers formed in the cell prior to integration into the genome, as well as concatemers integrated into the genome are all capable of being cleaved by the instant method. Similarly, other artifacts (e.g., partial vector insertions into the genome of the cell) are all also capable of being cleaved by the instant method. For brevity, the improvement is referred to herein as a “concatemer reduction method,” while the method is capable of reducing several additional artifacts.

[0060] Currently, each site-specific knock-in of a transgene requires designing and manufacturing a separate viral construct and a separate viral particle (e.g., an AAV-based construct and an AAV capsid forming a viral particle). Viruses such as AAV are costly to make as a GMP raw material and repeated or multiplex viral infections negatively impact cell health. In the context of a product comprising multiple transgene insertions (e.g., two, three, four or more insertions), the feasibility and cost of manufacturing separate lots of AAV for each transgene present a challenge. Once the separate AAVs are made, accomplishing all transfections resulting in robust rates of transgene knock-in and expression while having infected cells maintain viability, proliferative capabilities, and desired functions is a significant limitation in the field of genome editing and cell therapy.

[0061] Disclosed herein is the Sequential Template AAV Cas Knock-In method (STACK) and a STACK donor nucleic acid delivery construct (“a STACK construct”). In some embodiments, the construct comprises two or more donor nucleic acids and further comprises one or more CRISPR / Cas nuclease cleavage sites that enable separating the individual donor nucleicacids for integration into the genome. In some embodiments, each donor nucleic acid comprises homology arms capable of hybridizing to nucleic acids in the target genome near the desired insertion site (target site). Without reliance on a particular theory, it is thought that the homology arms act as templates for homology-directed repair (HDR) and bias the repair away from nonhom ologous end joining (NHEJ) pathway towards the HDR pathway thereby promoting efficient insertion of at least a portion of the donor nucleic acid into the desired insertion site. A donor nucleic acid comprising one or more homology arms is referred to as an “HDR template.”

[0062] The STACK design does not only pertain to delivery of a transgene (KI), but also can be used for gene knockout (KO). To achieve silencing of a gene, the donor nucleic acid in the STACK construct instead of an expression cassette comprises a cassette comprising a combination of stop codons in all three reading frames (referred to as a “stop cassette” or “3x stop”). Integration of a stop cassette will increase the likelihood of introducing of a premature stop codon into the coding sequence of a gene and gene silencing. In some embodiments, the stop cassette is present as a part of an HDR template, e.g., is flanked by homology arms directing the stop cassette to the gene to be silenced. As with transgene insertions, the homology arms would bias the repair away towards HDR and ensure efficient integration of the stop cassette and gene silencing. In some embodiments, the donor nucleic acid in a STACK construct comprises a donor splice site. Integration of a donor splice site leads to an alternative splicing of the mRNA transcribed from a gene resulting in a non-functional protein or a protein with an alternative function. In some embodiments, the donor nucleic acid in a STACK construct comprises a donor-acceptor site. Integration of a donor-acceptor site leads to an alternative splicing of the mRNA transcribed from a gene resulting in a non-functional protein or a protein with an alternative function,

[0063] Further disclosed herein are methods of genome editing comprising transfecting a cell with a STACK construct and further contacting the cell with components of CRISPR / Cas nucleoprotein complexes (NPCs) or with fully assembled NPCs wherein the NPCs cleave the genome of the cell at each target site and further cleave the construct into two or more individual HDR templates, wherein the released HDR templates promote insertion of at least a portion of each donor nucleic acid into each of the cleaved target sites. In some embodiments, at least one of the HDR templates comprises an expression cassette. In some embodiments, at least one of the HDR templates comprises a stop cassette. In some embodiments, the construct comprises at leasttwo HDR templates wherein the first HDR template comprises an expression cassette and the second HDR template comprises a stop cassette.

[0064] In some embodiments, the invention is a method for introducing a donor nucleic acid sequence into a cell with reduced occurrence of concatemers of the donor nucleic acid in the cellular genome. In some embodiments, the method comprises introducing into a cell the nucleic acid construct comprising a donor nucleic acid sequence and at least one cleavage site for a sequence-guided endonuclease (located in the construct outside of the donor nucleic acid), and further introducing into the cell an endonuclease capable of cleaving the endonuclease recognition sites in the nucleic acid construct.

[0065] The donor nucleic acid is a nucleic acid to be inserted (partially or completely) into the genome of a cell. The nucleic acid construct used to deliver the donor nucleic acid into the cell is described in more detail elsewhere in this disclosure. The donor nucleic acid may comprise an expression cassette, i.e., a protein coding sequence or a sequence coding for a functional RNA, e.g., micro-RNA, RNAi, shRNA, and guide RNA for a CRISPR endonuclease. In some embodiments, the donor nucleic acid is introduced into the genome of a cell for the purpose of integrating the expression cassette and enabling expression of the gene contained therein. This process is referred to as a gene knock-in (KI). In some embodiments, the donor nucleic acid is introduced into the genome of a cell for the purpose of disrupting a gene present in the genome at the integration site of the donor nucleic acid. This process is referred to as a gene knock-out (KO). In some embodiments, the donor nucleic acid is introduced into the genome of a cell for the purpose of changing the DNA sequence to correct an existing mutation or insert an intentional change in nucleic acid sequence. In some embodiments, gene knock-in occurs simultaneously with another gene’s knock-out.

[0066] In some embodiments, the donor nucleic acid used in the method comprises one or two homology arms having a sequence capable of hybridizing to a sequence adjacent to a desired integration site and capable of promoting homology directed repair (HDR) that would facilitate insertion of at least a portion of the donor DNA (e.g., an expression cassette) into the integration site. In some embodiments, the homology arms are between 10 and 100 base pairs long. In some embodiments, the homology arms are between 100 and 1000 base pairs long. In some embodiments, the homology arms are about 500-550 base pairs long. In some embodiments, thehomology arms are single or double stranded stretches of nucleic acids. Although the homology arms facilitate HDR, the presence of the homology arms does not prevent the formation of concatemers (one or more copies of the donor nucleic acid integrated in tandem) and other artifacts such as insertion of vector sequences.

[0067] The endonuclease cleavage sites present in the construct correspond to the endonuclease used in the step of concatemer reduction method. The properties of various suitable endonucleases and their corresponding cleavage sites are described in more detail elsewhere in this disclosure. Briefly, in some embodiments, the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18 nucleotides long ZFN recognition sequences separated by a 4-18 nucleotides long spacer. In some embodiments, the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALEN recognition sequences, each 18-30 nucleotides long, separated by a 12-25 nucleotide long spacer. In some embodiments, the endonuclease is a CRISPR endonuclease, and the cleavage site comprises a NATNA recognition sequence (protospacer) and a protospacer adjacent motif (PAM). In some embodiments, the endonuclease is a Fanzor endonuclease, and the cleavage site comprises an omega-RNA recognition sequence and a target adjacent motif (TAM). In some embodiments, the endonuclease is a Homing endonuclease (also known as a meganuclease), and the cleavage site comprises a recognition sequence of 14-40 bp.

[0068] In some embodiments, the method further comprises introducing into the cell the endonuclease whose cleavage site is present in the construct. In some embodiments, the same endonuclease is used to catalyze incorporation of the donor nucleic acid into the cellular genome. In some embodiments, a different endonuclease is used to catalyze incorporation of the donor nucleic acid into the cellular genome and the method further comprises introducing into the cell such different endonuclease. In some embodiments, the method further comprises introducing into the cell the NATNA required by the endonuclease for cleavage. In some embodiments, the endonuclease and the NATNA form a nucleoprotein complex prior to being introduced into the cell.

[0069] In some embodiments, the method comprises introducing into the cell the nucleic acid construct comprising the donor nucleic acid and further comprising at least one cleavage site for sequence-guided endonuclease and further intruding into the cell the sequence-guided endonuclease capable of cleaving the cellular genome at the desired integration site for the donornucleic acid and further capable of cleaving the cleavage sites in the nucleic acid construct (before or after integration into the cellular genome) so that the occurrence of concatemers or imperfect integrations of the donor nucleic acid in the cellular genome is reduced. In some embodiments, the cleavage sites for the sequence-guided endonuclease comprise sequences substantially identical to the target sequence in the genome of the cell so that the same sequence-guided endonuclease is capable of cleaving the construct and the target site in the genome.

[0070] In some embodiments, the method further comprises incubating the cell under the conditions suitable for enzymatic processes resulting in insertion of the donor DNA into the cellular genome.

[0071] In some embodiments, the method further comprises assessing the cell for successful insertion of the donor DNA into the cellular genome. The assessing can be performed by any nucleic acid analysis methods including fluorescent in situ hybridization (FISH), PCR, dPCR, ddPCR and nucleic acid sequencing.

[0072] In some embodiments, the method comprises introducing into a cell a nucleic acid which is a Sequential Template AAV Cas Knock-in (STACK) construct or “construct”, the construct comprising a viral vector nucleic acid and more than one donor nucleic acid (e.g., two, three, or more donor nucleic acids) and further comprising one or more CRISPR / Cas endonuclease cleavage sites (e.g., one, two, three, or more CRISPR / Cas endonuclease cleavage sites) wherein at least one cleavage site in the construct comprises a sequence substantially identical to a target sequence in the genome targeted by the construct, so that the endonuclease cleaving the construct is also capable of cleaving the target site in the genome as shown in FIGURES 11, 12, 13 or 14. The construct further comprises homology arms capable of hybridizing to nucleic acid sequences adjacent to the cleavage site in the target genome (e.g., flanking cleavage site in the target genome) and promoting homology-directed repair (HDR). The donor nucleic acid comprising homology arms is an HDR template. The method further comprises introducing into the cell various components of CRISPR / Cas nucleoprotein complexes (NPCs), including precursors of NPC components or a fully assembled NPC, under conditions suitable for assembly of active NPCs in the cell, cleavage of CRISPR / Cas endonuclease cleavage sites in the construct and in the cellular genome, releasing of the HDR templates and insertion of at least a portion of each donor nucleic acid into the genome of the cell.

[0073] In some embodiments, at least one of the HDR templates may comprise an expression cassette. In some embodiments, at least one of the HDR templates may comprise a stop cassette. In some embodiments, the construct comprises at least two HDR templates wherein the first HDR template comprises an expression cassette and the second HDR template comprises a stop cassette.

[0074] In some embodiments, the invention is method comprising introducing into a cell a composition comprising a STACK construct, the construct comprising a viral vector nucleic acid and more than one donor nucleic acid (e.g., two, three, or more donor nucleic acids) and further comprising one or more sequence-guided endonuclease cleavage sites, wherein at least one cleavage site in the construct is identical to at least one cleavage site in the target genome targeted by the construct. In some embodiments, the method further comprises introducing into the cell one or more sequence-guided endonucleases or precursors thereof (e.g., the mRNA encoding the endonuclease). In some embodiments, the sequence-guided endonucleases are CRISPR / Cas endonucleases, and the method further comprises introducing into the cell one or more components of a CRISPR / Cas nucleoprotein complex (NPC) or a precursor of such components (e.g., mRNA) or a fully assembled NPC.

[0075] In some embodiments, the method further comprises integration of at least a portion of a donor nucleic acid flanked by homology arms acid into the cleavage site in the target genome. In some embodiments, the donor nucleic acid in the construct comprises an expression cassette and the integration results in the expression of a transgene selected from CD47, a chimeric antigen receptor (CAR) a cytokine, a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a cytokine receptor, a cytokine-cytokine receptor fusion, a portion of an MHC Class I molecule, and a fusion including an MHC Class I molecule (e.g., a fusion of B2M and an MHC class I molecule such as HLA-E, HLA-G or HLA-F). In some embodiments, the donor nucleic acid in the construct comprises a stop cassette and the integration results in silencing of a gene selected from PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, GISH, CBLB, 2B4, TRAC, GUTA, DNMT3A, DNMT3L, FAS, TETl, TET2, TET3, CD155, RASA2, SOCS1, SOCS3, and B2M.

[0076] In some embodiments, the invention is a method of modifying genomes of cells. The method of the invention is not limited to a cell type as long as the cell possesses thecomponents of at least one DNA recombination pathway, for example, homology-directed repair (HDR) pathway. In cell may be a prokaryotic cell or a eukaryotic cell. The eukaryotic cell may be of plant, fungal or animal origin. In some embodiments, the cell is a unicellular organism, i.e., a bacterium or a unicellular eukaryote, including a pathogenic organism of said category. In some embodiments, the cell is derived from a multi-cellular organism and is a cultured cell. In some embodiments, the cell is a part of a multi-cellular organism and is subjected to the genome modification in situ, e.g., in the organ of a subject or a patient in need of a therapeutic intervention that involves genome modification. In some embodiments, the cell is human cell which is not a human germline cell. In some embodiments, the cell is human cell which is not a totipotent cell.

[0077] In some embodiments, the cell is present in vitro. In some embodiments, the cell is present in vivo. In some embodiments, the cell is a human cell present in vivo wherein the cell is not a human germline cell. In some embodiments, the cell is a human cell present in vivo or in vitro wherein the cell is not a totipotent cell.

[0078] In some embodiments, the cell is an immune cell. In some embodiments, the cell is autologous, i.e., isolated from a patient for in vitro genome modification and reinfusion into the patient. In some embodiments, the cell is allogeneic, i.e., isolated from a donor individual, such as a healthy human donor of either gender. The cells may be isolated using standard techniques. For example, lymphocytes can be isolated from blood, including peripheral blood and cord blood, or from lymphoid organs such as the thymus, bone marrow, lymph nodes, and mucosal-associated lymphoid tissues (MALT). Techniques for isolating lymphocytes from such tissues are well known in the art, see, e.g., Smith, J.W. (1997) Apheresis techniques and cellular immunomodulation, Ther. Apher. 1:203-206. The isolated lymphocytes may be characterized in terms of specificity, frequency and function or enriched for specific subsets of cells, such as T cells or NK cells. For example, the isolated lymphocyte population can be enriched for specific subsets of T cells, such as CD4+, CD8+, CD25+, or CD62L+. (See, e.g., Wang et al., Mol. Therapy - Oncolytics (2016) 3: 16015) or for NK cells (CD56+phenotype). T cells can be activated using soluble CD3 / 28 activators, or magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies.

[0079] In some embodiments, the cell is differentiated in vitro into a desired cell type. For example, NK cells for genome modification into CAR-NK cells can be produced by differentiating stem cells such as embryonic stem cell (ESCc) and induced pluripotent stem cells (iPSCs). See e.g., T. Cheng (ed.), Hematopoietic Differentiation of Human Pluripotent Stem Cells,SpringerBriefs in Stem Cells, DOI 10.1007 / 978-94-017-7312-6_5, Hermanson, el al., Chapter 5, Human Pluripotent Stem Cells as a Renewable Source of Natural Killer Cells:, Woll, etal., (2016) Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity, Blood, 113(24) 6094; and Denman, etal., (2012) Membrane- Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells, PloS One, 7(l):e30264.

[0080] The differentiation process typically comprises two and optionally, three steps: differentiating stem cells into hematopoietic progenitor cells (HPC), differentiating HPC into natural killer cells (NK), and optional expansion of NK cells. The products of differentiation (or expansion) may also be tested for the presence of NK phenotype such as NK-specific gene expression patterns, including expression of NK-specific cell surface markers, e.g., CD45 and CD56. In some embodiments, the step of forming hematopoietic progenitor cells (HPC) comprises forming spheroids by allowing cells to aggregate (optionally assisted by low-speed centrifugation), and incubating the spheroids in presence of one or more cytokines selected from BMP4, VEGF, SCF, IL3, IL6, and TPO. In some embodiments, the combination of BMP4, VEGF, and SCF is used. In some embodiments, the combination of BMP4, VEGF, SCF, IL3, IL6, and TPO is used. In some embodiments, HPC are formed by incubation in the presence of feeder cells such as bone marrow stromal cells. In some embodiments, the step of forming NK cells comprises incubating spheroids in the presence of one or more cytokines selected from IL-3, IL-15, IL-7, SCT, and FLT3L. In some embodiments, the combination of IL-3, IL-15, IL-7, SCT, and FLT3L is used. In some embodiments, feeder cells (stromal cells) are used. In some embodiments, fetal liver stromal cells are used as feeders. In some embodiments, the HPC fraction is enriched for the CD34+cells prior to initiating the NK-differentiation step. In some embodiments, at the completion of the differentiation stage, the cell fraction is tested for surface expression of NK-specific markers such as CD45 and CD56.

[0081] In some embodiments, the donor nucleic acid is introduced into a cell via chemical or electrochemical means (such as lipofection or electroporation). The donor nucleic acid may be introduced as naked double-stranded DNA (dsDNA), e.g., linear dsDNA.

[0082] In some embodiments, the donor nucleic acid is introduced into the cell contained in a vector. The vector sequence prior to insertion of any insert sequence is referred to as “vectorbackbone.” In some embodiments, the vector is a plasmid introduced into the cell by electroporation as stated above. The plasmid is selected from a prokaryotic plasmid, a eukaryotic plasmid, and a shuttle plasmid.

[0083] In some embodiments, the donor nucleic acid is introduced into the cell via encapsulation in a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, liposome, cubosome, or derivation thereof, (see Madigan et al., (2023) Drug delivery systems for CRISPR-based genome editors, Nat. Rev. Drug Discov. 22(11):875). Nanoparticles can comprise a cationic lipid, a helper lipid, a PEG-lipid, or cholesterol. In some embodiments the nanoparticle is decorated with a surface molecule (e.g., a peptide) that targets the nanoparticle to a specific cell or tissue type based on interaction (e.g., specific binding) of the surface molecule present on the nanoparticle with a recognition molecule present on the cell surface. One example of this is surface molecule folate on the nanoparticle binding to the folate receptor on the cell surface.

[0084] In some embodiments, the vector used to deliver the donor nucleic acid is a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector). Suitable vectors are non-replicating in the target cells. In some embodiments, the vector is selected from or designed based on SV40, EBV, HSV, AAV or BPV.

[0085] Adenoviruses and adeno-associated viruses (AAV) are the mostly commonly used delivery vectors in genome editing with AAV (including dual-AAV) most commonly used in CRISPR genome editing. Both single-stranded and double-stranded AAV delivery systems have been developed (reviewed in Xu etal., (2019) Viral delivery systems for CRISPR, Viruses, 11 :28).

[0086] In some embodiments, the invention comprises the addition of nuclease cleavage sites flanking the homology arms (which in turn flank the donor nucleic acid) in the delivery construct. FIGURE 2 illustrates the design of an AAV construct according to the invention. In this example, the endonuclease cleavage sites are placed adjacently to the inverted terminal repeat (ITR).

[0087] FIGURE 3 illustrates the design of a plasmid construct according to the invention. In this example, the endonuclease cleavage sites (shown inside the plasmid circle) are placed at the junction of the donor nucleic acid (e.g., the homology arms flanking the nucleic acid to be inserted) and the plasmid backbone.

[0088] As illustrated in FIGURE 10 (prior art), the existing approach to performing a single gene knock-in (KI) involves a single gene delivery construct (AAV-A) delivering a singledonor nucleic acid A (heavily shaded box) to a single locus (Target A) on a chromosome (chr:A) which is cleaved by a single CRISPR / Cas nucleoprotein complex (CasA). Homology arms (lightly shaded boxes) present in the AAV construct mediate homology-directed repair (HDR) resulting in insertion (knock-in) of the donor nucleic acid A sequence into the Target A.

[0089] In some embodiments, the invention comprises a nucleic acid which is a Sequential Template AAV Cas Knock-in (STACK) construct or “construct”, the construct comprising a viral vector nucleic acid and more than one donor nucleic acid (e.g., two, three, or more donor nucleic acids). The construct further comprises one or more CRISPR / Cas endonuclease cleavage sites (e.g, one, two, three, or more CRISPR / Cas endonuclease cleavage sites). In the construct, the location of each CRISPR / Cas endonuclease cleavage sites is selected from between the viral vector sequences (e.g., inverted terminal repeats, ITRs) and a donor nucleic acid and between two donor nucleic acids from the two or more donor nucleic acids. At least one cleavage site in the construct is identical to at least one cleavage site in the target genome targeted by the construct.

[0090] The construct further comprises homology arms capable of hybridizing to nucleic acid sequences adjacent to the cleavage site in the target genome (e.g., flanking the cleavage site in the target genome). In some embodiments, the homology arms for the STACK constructs are designed to comprise the sequence of at least a portion of the Cas 12a target site. In the example shown in FIGURE 9, each homology arm comprises a portion e.g., a half) of the Casl2a target site sequence. The 20-base pair target (spacer) region is divided into two sections comprising the left 10 base pairs and a right 10 base pairs. The left 10 base pairs of the spacer are the region of the homology arm that immediately flanks the donor nucleic acid at the 5’ end, and the remaining left homology arm can be extended to any length comprising the genomic sequence near the site of insertion. The right 10 base pairs of the spacer are the region of the homology arm that immediately flanks the donor nucleic acid at the 3’ end, and the remaining left homology arm can be extended to any length comprising the genomic sequence near the site of insertion. In some embodiments, each homology arm is 100-500 bp long, for example, 100, 200, 300, 400, 500, 120, 200, or 250 bp long.

[0091] One of skill in nucleic acid chemistry and gene editing would recognize that identity or perfect complementarity are not necessary for forming a stable hybrid between two nucleic acids. Instead, the ability of two nucleic acids to form a stable hybrid depends on reaction conditions such as temperature and ionic strength of the reaction mixture, and the length of thehybridizing nucleic acids, so that less than perfectly complementary nucleic acids can sometimes form stable hybrids. For example, in the case of CRISPR / Cas endonucleases, perfect complementarity is not required between a NATNA spacer and a protospacer in the genome, or a NATNA spacer and a protospacer in the construct. Similarly, perfect complementarity is not required between a homology arm and a nucleic acid sequence in the genome adjacent the insertion site. By extension, identity or perfect complementarity are not required between the cleavage site (e.g., protospacer) in the genome and a cleavage site (e.g., protospacer) in the construct for the two cleavage sites to be capable of forming a stable hybrid with the same NATNA and capable of being cleaved by the same CRISPR / Cas endonuclease.

[0092] In some embodiments, the invention comprises a STACK construct comprising more than one donor nucleic acid. As illustrated in the example shown in FIGURE 11, unlike the traditional virus-based constructs (e.g., FIGURE 10), a single construct (AAV-A+B) now comprises two donor nucleic acids A and B. Further, unlike the traditional virus-based constructs (e.g., FIGURE 10), the embodiments of the invention include constructs further comprising one or more CRISPR / Cas endonuclease cleavage sites. In the example shown in FIGURE 11, at least one cleavage site is required to separate donor nucleic acid A from donor nucleic acid B. The construct in FIGURE 11 includes a CRISPR / Cas cleavage site “Target B” capable of being cleaved by the CRISPR / Cas nucleoprotein complex “CasB.” CasB also cleaves a target locus (Target B) on a chromosome (chr:B), while CasA cleaves another target locus (Target A) on another chromosome (chr:A), or on a different location on the same chromosome as cleaved by CasB (chr:B) (not shown). The construct further comprises homology arms capable of hybridizing to sequences on chromosome A and chromosome B respectively. The homology arms mediate homology-directed repair (HDR) resulting in insertion of the donor nucleic acids A and B into targets A and B respectively on chromosomes A and B respectively. In this embodiment, the STACK approach results in two site-specific insertion events accomplished with a single construct.

[0093] In some embodiments, as illustrated in the example shown in FIGURE 12, a single STACK construct (AAV-A+B) comprises two donor nucleic acids A and B and further comprises two CRISPR / Cas endonuclease cleavage sites: “Target A” capable of being cleaved by the CRISPR / Cas nucleoprotein complex “CasA,” and “Target B” capable of being cleaved by the CRISPR / Cas nucleoprotein complex “CasB.” CasB also cleaves a target locus “Target B” on one chromosome (chr:B), while CasA also cleaves another target locus “Target A” on anotherchromosome (chr:A), (or on a different location on the same chromosome as cleaved by CasB (chr:B), not shown). The construct further comprises homology arms capable of hybridizing to sequences on chromosome A and chromosome B respectively. The homology arms mediate homology-directed repair (HDR) resulting in insertion of the donor nucleic acids A and B into targets A and B respectively on chromosomes A and B respectively. Furthermore, the presence of an endonuclease cleavage site between the viral sequence (e.g., inverted terminal repeat, ITR) and the donor nucleic acid reduces artifacts (e.g., concatemers and partial insertions) resulting from interactions between two viral sequences in the cell.

[0094] In some embodiments, as illustrated in the example shown in FIGURE 13, the STACK construct AAV A+B also comprises not two but three CRISPR / Cas endonuclease cleavage sites, in this example three “Target B” sites capable of being cleaved by the CRISPR / Cas nucleoprotein complex “CasB.” CasB also cleaves a target locus (Target B) on a chromosome (chr:B), while another nucleoprotein complex “CasA” cleaves another target locus (Target A) on another chromosome (chr:A), or a different location on the same chromosome as cleaved by CasB (chr:B) (not shown). The construct further comprises homology arms capable of hybridizing to sequences on chromosome A and chromosome B respectively. The homology arms mediate homology-directed repair (HDR) resulting in insertion of the donor nucleic acids A and B into targets A and B respectively on chromosomes A and B respectively. Furthermore, the presence of endonuclease cleavage sites between the donor nucleic acid and both viral ends (e.g., ITRs) further reduces artifacts (e.g., concatemers and partial insertions) resulting from interactions between two viral sequences in the cell.

[0095] In some embodiments, as illustrated in the example shown in FIGURE 14, more than one (e.g., two) constructs are present, each STACK construct comprising more than one (e.g., two) donor nucleic acids: AAV A+B and AAV B’+C. Each construct further comprises at least two CRISPR / Cas endonuclease cleavage sites, in this example, two “Target B” sites capable of being cleaved by the CRISPR / Cas nucleoprotein complex “CasB.” CasB also cleaves a target locus (Target B) on a chromosome (chr:B), while another nucleoprotein complex “CasA” cleaves a second target locus (Target A) on another chromosome (chr:A), and yet another nucleoprotein complex “CasC” cleaves a third target locus (Target C) on yet another chromosome (chr:C). The construct further comprises homology arms capable of hybridizing to sequences on chromosome A, chromosome B and chromosome C respectively. The homology arms mediate homology-directed repair (HDR) resulting in insertion of the donor nucleic acids A, B, B’, and C into targetsA, B and C respectively. In this example, donor nucleic acids B and B’ have homology arms to the same insertion site in chromosome B. In some embodiments, B and B’ are alternative alleles of the same nucleic acid sequence that can be inserted into the same locus. The design shown in FIGURE 14 enables forming a population of cells with various genotypes at the locus of TargetB, e.g., BB, B’B and B’B’.

[0096] In some embodiments, at least one of the two or more donor nucleic acids in the STACK construct is an expression cassette for a transgene knock-in (KI). In some embodiments, at least one of the two or more donor nucleic acids in the STACK construct is a template for gene knockout (KO). Traditionally, gene knockout using sequence-guided endonucleases (e.g., ZFN, TALEN or CRISPR / Cas endonucleases) relies on non-homologous end joining (NHEJ) and other imperfect repair pathways that hopefully will alter the coding sequence of a gene thereby abolishing transcription, translation or function of the protein encoded by the gene cleaved by the endonuclease. For example, CRISPR / Cas endonucleases generate a mixture of DNA ends in the same reaction: blunt and recessed, including recessed ends with various length of overhang. Cas9 makes blunt ends and 1, 2, and 3-nucleotide overhangs (see e.g., Longo G., et al., (2024) Linking CRISPR-Cas9 double-strand break profdes to gene editing precision with BreakTag, Nat. Biotechnol. May 13, 2024) and Casl2a makes 4 and 5-nucleotide overhangs (see e.g., Zetsche B., et al., (2015) Cpfl is a single RNA-guided endonuclease of a CRISPR-Cas system, Cell 163:759). For this reason, at least some cleavage events are expected to lead to in-frame repair that fails to knock out the target gene.

[0097] The instant invention combines cleavage with a sequence-guided endonuclease (e.g., ZFN, TALEN or CRISPR / Cas endonuclease) with delivery of a donor nucleic acid that includes a stop codon for all three translation frames (the “3x stop” or the “stop cassette” SEQ ID NO: 19). The use of such donor nucleic acid ensures much more reliable gene silencing. In some embodiments, the stop cassette in the STACK construct is flanked by homology arms. Without being bound by a particular theory, the inventors propose that the HDR template will bias repair towards HDR and insertion of the stop cassette. Regardless of the frame of insertion, the stop cassette would place a stop codon in-frame for the translation of mRNA and ensure a nonsense mutation in the target gene. At the same time, only a minority of the repair will proceed via NHEJ, and of those events, only a subset will result in perfect repair and failure to disrupt the target gene.

[0098] In some embodiments, the donor nucleic acid in the construct comprises an HDR template with an expression cassette. In some embodiments, the donor nucleic acid in the construct comprises an HDR template with a stop cassette. In some embodiments, the same construct comprises one HDR template with an expression cassette and one HDR template with a stop cassette.

[0099] In some embodiments, the HDR template comprises a stop cassette and the target gene for the knockout is an immune checkpoint gene or a regulatory gene selected from the group consisting of PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, GISH, CBLB, CIITA, DNMT3A, DNMT3L, FAS, TET1, TET2, TET3, CD155, RASA2, SOCSl, SOCS3, and 2B4. In some embodiments, the HDR template comprises a stop cassette and the target sequence for gene knockout is the TRAC gene or the beta-2 microglobulin (B2M) gene. In some embodiments, the target sequence is in the TRAC gene and comprises SEQ ID NO: 10. In some embodiments, the target sequence is in the PDCD1 gene and comprises SEQ ID NO: 22. In some embodiments, the target sequence is in the CISH gene and comprises SEQ ID NO: 20.

[0100] In some embodiments, the HDR template comprises an expression cassette including a coding sequence for CD47, a chimeric antigen receptor (CAR), a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a portion of an MHC Class I molecule and a fusion including an MHC Class I molecule, e.g., a fusion of HLA-E with HLA-G or HLA-F. In some embodiments, the HDR template comprises an expression cassette including a coding sequence for a synthetic gene fusion comprising a fusion of B2M and HLA-E (SEQ ID NO: 31). In some embodiments, the HDR template comprises an expression cassette including a coding sequence for a ROR1- targeting CAR (SEQ ID NO: 28). In some embodiments, the HDR template comprises an expression cassette including a coding sequence for soluble IL 15 (SEQ ID NO: 29).

[0101] In some embodiment, the STACK construct comprises an HDR template capable of hybridizing to the TRAC locus and including an expression cassette including a coding sequence for an anti-RORl CAR, and further comprises an HDR template capable of hybridizing to the PDCD1 locus and including a stop cassette. The sequences of the constructs comprising the stop cassette with the 120bp, 200bp, 250bp, or 400bp PDCD1 homology arms, and the anti-RORl CAR with TRAC homology arms are provided in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0102] One of skill in the art would envision this design applied to any serotype or variant of AAV as well as any plasmid delivery system where a donor nucleic acid is inserted. More generally any form of donor nucleic acid including single-stranded, double-stranded, linear, and circular donor DNA construct may also be improved by the instant method.

[0103] In some embodiments, the donor DNA comprises a protein expression cassette. In some embodiments, the protein expression cassette comprises sequences that are codon-optimized for expression in mammalian cells. In some embodiments, the expression cassette also incorporates regulatory sequences including transcriptional activator binding sequences, transcriptional repressor binding sequences, enhancers, introns, and the like. In some embodiments, the viral vector supplies a constitutive promoter or an inducible promoter. In some embodiments, the expression cassette includes a promoter selected from EFl a, PGK1, MND, Ubc, CAG, CaMKIIa, and [3- Actin promoter. In some embodiments, the promoter is selected from the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RS V-LTR) promoter, mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, the P-interferon promoter, the hsp70 promoter and EF- 1 cc promoter. In some embodiments, the promoter is an MND promoter.

[0104] In some embodiments, the viral vector supplies a transcription terminator. In some embodiments, the expression cassette comprises a transcription terminator. In some embodiments, the viral vector supplies a polyadenylation site. In some embodiments, the expression cassette comprises a polyadenylation site. In some embodiments, the polyadenylation site is the SV-40 polyadenylation sites.

[0105] In some embodiments, to facilitate homologous recombination, the donor DNA comprises the expression cassette described above joined to homology arms. Homology arms are sequences capable of hybridizing to sequences located 5’ (upstream) and 3’ (downstream) of the insertion site in the genome. In some embodiments, the homology arms are about 500 bp long. In some embodiments, the donor DNA is cloned into a viral vector plasmid. The plasmid is used to package the sequences into a virus capable of delivering the payload of donor DNA into the target cell.

[0106] In some embodiments, the expression cassette described herein is inserted into a double-strand break in the genome of the cell. In some embodiments, the introduction of theengineered protein coincides with inactivation of another gene by the insertion of the engineered fusion protein (gene knock-out and simultaneous gene knock-in). In some embodiments, the insertion site and an inactivated gene is selected from TRAC, CBLB, / U / W (beta-2 microglobulin), PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, CIITA, DNMT3A, DNMT3L, FAS, TETl, TET2, TET3, CD 155, RASA2, SOCS1, SOCS3, and 2B4.

[0107] In some embodiments, the inserted expression cassette comprises a chimeric antigen receptor (CAR)-coding sequence. A CAR is a protein expressed by engineered immune cells such as CAR-T cells or CAR-natural killer (CAR-NK) cells or other immune cell types engineered to target diseased cells or other pathogenic entities in a subject or a human patient. A typical CAR comprises an extracellular domain comprising an antigen binding region, a transmembrane domain, and one or more intracellular activation (co-stimulatory) domains. In some embodiments, the CAR also comprises a hinge domain. In some embodiments, the CAR also comprises a leader peptide directing the CAR to the cell membrane.

[0108] In some embodiments, the extracellular domain of the CAR comprising the antigen binding region targets a tumor antigen. In some embodiments, the tumor antigen is selected from CD19, CD-371, CD-269 (BCMA), CA-125, CD20, CXCR5, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, CD99, CLL-1, CD47, CD33, CS1, ROR1, c-Met, TROP2, EphA2, GD2, GPC3, epithelial tumor antigen, melanoma-associated antigen, or a mutated protein selected from TP53, Ras and BRAF.

[0109] In some embodiments, the antigen binding region comprises a single-chain variable fragment (scFv). An scFv comprises a variable region of an antibody light chain (VL) linked to a variable region of an antibody heavy chain (VH). In some embodiments, the VL is linked to the VH via a peptide linker that comprises from about 5 to about 40 amino acids. The linker can be a naturally occurring sequence or an engineered sequence. For example, in some embodiments, the linker is derived from a human protein, e.g., an immunoglobulin selected from IgG, IgA, IgD, IgE, or IgM. In some embodiments, the linker is a glycine and serine rich linker having the sequence (GxSy)n. Additional linker examples and sequences are disclosed in the U.S. Patent No. 5,525,491 Serine-rich peptide linkers, U.S. Patent No. 5,482,858 Polypeptide linkers for production of biosynthetic proteins, and a publication W02014087010 Improved polypeptides directed against IgE. In some embodiments, the scFv, the CAR domains or the entire CAR is fully human or ishumanized to reduce immunogenicity in human patients. In some embodiments, the CAR sequence is optimized for codon usage in human cells.

[0110] In some embodiments, the transmembrane domain of the CAR is derived from a membrane-bound or transmembrane protein. For example, the transmembrane domain of the CAR may be the transmembrane domain of a T cell receptor alpha-chain or beta-chain, a CD3-zeta chain, CD28, CD3-epsilon chain, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, DNAM1, NKp44, NKp46, NKG2D, 2B4, or a GITR.

[0111] The intracellular signaling domain of a CAR is responsible for activation of one or more effector functions of the immune cell expressing the CAR. In some embodiments, the intracellular signaling domain of the CAR comprises one or more subdomains each comprising a part of or the entire sequence of the TCR zeta chain, CD3 zeta chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, ICOS / CD278, IL-2Rbeta / CD122, IL-2Ralpha / CD132, DAP10, DAP12, DNAM1, TLR1, TLR2, TLR4, TLR5, TLR6, MyD88, CD40 or a combination thereof.

[0112] The present invention involves manipulating nucleic acids, including genomic DNA and plasmid DNA that were isolated or extracted from a sample. Methods of nucleic acid extraction are well known in the art. See J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, N.Y.). A variety of reagent and kits are commercially available for extracting nucleic acids (DNA or RNA) from biological samples, including products from BD Biosciences (San Jose, Cal.), Clontech (TaKaRa Bio ); Epicentre Technologies (Madison, Wise.); Gentra Systems, (Minneapolis, Minn.); Qiagen (Valencia, CaL); Ambion (Austin, Tex.); BioRad Laboratories (Hercules, Cal.); KAPA Biosystems (Roche Sequencing Solutions, Pleasanton, Cal.) and more.

[0113] In some embodiments, the invention involves intermediate purification or separation steps for nucleic acids, e.g., to remove unused reactants from the DNA. The purification or separation may be performed by a size selection method selected from gel electrophoresis, affinity chromatography and size exclusion chromatography. In some embodiments, size selection can be performed using Solid Phase Reversible Immobilization (SPRI) technology from Beckman Coulter (Brea, Cal.).

[0114] In some embodiments, the donor DNA nucleic acid is generated using chemical synthesis such as phosphoramidite chemistry on solid support or any other nucleic acid synthesis method available to the user. The chemically synthesized donor DNA nucleic acid could be singlestranded, linear double-stranded, closed double-stranded, or partially double-stranded and singlestranded. Chemically synthesized donor DNA nucleic acid may comprise native or non-native (e.g., chemically modified) nucleic acid bases or bases other than adenine, guanine, cytosine and thymine. In some embodiments, chemically modified nucleic acid bases provide protection from nuclease degradation, reduce activation of anti-viral response pathways, or activation of DNA damage response pathways in the host cell.

[0115] In some embodiments, the donor DNA is introduced in the genome at the site of cleavage by a sequence-specific endonuclease. In some embodiments, the sequence-specific endonuclease is selected from a rare-cutting restriction enzyme, a TALEN, a Zinc-finger nuclease (ZFN), a Fanzor (Saito et al. (2023) Fanzor is a eukaryotic programmable RNA-guided endonuclease, Nature 620:660) and a CRISPR endonuclease.

[0116] In some embodiments, the sequence-specific endonuclease is a CRISPR endonuclease. In some embodiments, the CRISPR endonuclease is part of a nucleoprotein complex comprising the CRISPR endonuclease and CRISPR guide RNA (nucleic acid targeting nucleic acid or NATNA). In some embodiments, the NATNA comprises one or more DNA nucleotides and is a CRISPR hybrid RNA-DNA or chRDNA. In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. In some embodiments, the NATNA is selected from the embodiments described in the International Application Pub. No. WO2022086846 DNA-containing polynucleotides and guides for CRSIPR Type V systems, and methods of making and using the same.

[0117] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus is found in many prokaryotic genomes and provides resistance to invasion of foreign nucleic acids. Structure, nomenclature, and classification of CRISPR loci are reviewed in Makarova et al, Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 2011 June; 9(6): 467-477. In some embodiments, the endonuclease is a catalytically inactive CRISPR endonuclease (e.g., catalytically inactive Cas9 or Casl2a) conjugated to thecleavage domain of the restriction endonuclease Fok I. (see e.g, Guilinger, J. P., et al., (2014). Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification, Nature biotechnology, 32(6), 577-582.

[0118] Briefly, a typical CRISPR locus includes a number of short repeats regularly interspaced with spacers. The CRISPR locus also includes coding sequences for CRISPR- associated (Cas) genes. A spacer-repeat sequence unit encodes a CRISPR RNA (crRNA). In vivo, a mature crRNAs are processed from a polycistronic transcript referred to as pre-crRNA or pre- crRNA array. The repeats in the pre-crRNA array are recognized by Cas-encoded proteins that bind to and cleave the repeats liberating mature crRNAs. CRISPR systems perform cleavage of a target nucleic acid wherein Cas proteins and crRNA form a CRISPR ribonucleoproteins (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid invading a bacterial cell) and the Cas nuclease proteins cleave the target nucleic acid.

[0119] Type I CRISPR systems include means for processing the pre-crRNA array that include a multi-protein complex called CASCADE (CRISPR-associated complex for antiviral defense) comprised of subunits CasA, B, C, D and E. The Cascade-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. The bound nucleoprotein complex recruits the Cas3 helicase / nuclease to facilitate cleavage of target nucleic acid.

[0120] Type II CRISPR systems include a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a crRNA repeat in the pre-crRNA array and recruits endogenous RNaselll to cleave the pre-crRNA array. The tracrRNA / crRNA complex can associate with a nuclease, e.g., Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease, for target nucleic acid cleavage.

[0121] Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave the pre-crRNA array with the help of one or more CRISPR polymerase- like proteins.

[0122] Type V CRISPR systems comprise a different set of Cas-like genes, including Casl2, Csfl, Csf2, Csf3 and Csf4 which are distant homologues of Cas genes in Type I-III CRISPR systems.

[0123] CRISPR endonucleases require a nucleic acid targeting nucleic acid (NATNA) also known as guide RNAs. The endonuclease is capable of forming a ribonucleoprotein complex (RNP) with one or more guide RNAs. In some embodiments, the endonuclease is a Type II CRISPR endonuclease and NATNA comprises tracrRNA and crRNA.

[0124] In some embodiments, NATNA is selected from the embodiments described in U.S.Patent No. 9,260,752. Briefly, a NATNA can comprise, in the order of 5' to 3', a spacer extension, a spacer, a minimum CRISPR repeat, a single guide connector, a minimum tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some instances, a nucleic acid-targeting nucleic acid can comprise, a tracrRNA extension, a 3' tracrRNA sequence, a minimum tracrRNA, a single guide connector, a minimum CRISPR repeat, a spacer, and a spacer extension in any order.

[0125] In some embodiments, the guide nucleic acid-targeting nucleic acid can comprise a single guide NATNA. The NATNA comprises a spacer sequence which can be engineered to hybridize to the target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence. Optionally, NATNA can have a spacer extension and a tracrRNA extension. These elements can include elements that can contribute to stability of NATNA. The CRISPR repeat and the tracrRNA sequence can interact, to form a base-paired, double-stranded structure. The structure can facilitate binding of the endonuclease to the NATNA.

[0126] In some embodiments, the single guide NATNA comprises a spacer sequence located 5' of a first duplex which comprises a region of hybridization between a minimum CRISPR repeat and minimum tracrRNA sequence. The first duplex can be interrupted by a bulge. The bulge facilitates recruitment of the endonuclease to the NATNA. The bulge can be followed by a first stem comprising a linker connecting the minimum CRISPR repeat and the minimum tracrRNA sequence. The last paired nucleotide at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to a mid-tracrRNA. The mid-tracrRNA can comprise one or more additional hairpins.

[0127] In some embodiments, the NATNA can comprise a double guide nucleic acid structure. The double guide NATNA comprises a spacer extension, a spacer, a minimum CRISPR repeat, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The double guide NATNA does not include the single guide connector. Instead, the minimum CRISPR repeat sequence comprises a 3' CRISPR repeat sequence and the minimum tracrRNAsequence comprises a 5' tracrRNA sequence and the double guide NATNAs can hybridize via the minimum CRISPR repeat and the minimum tracrRNA sequence.

[0128] In some embodiments, NATNA is an engineered guide RNA comprising one or more DNA residues (CRISPR hybrid R / DNA or chRDNA). In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617 International Application Pub. No. WO2022086846 DNA-containing polynucleotides and guides for CRSIPR Type V systems, and methods of making and using the same. Briefly, some chRDNA for use with a Type II CRISPR system may be composed of two strands forming a secondary structure that includes an activating region composed of an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. Other chRDNA may be a single guide D(R)NA for use with a Type II CRISPR system comprising a targeting region, and an activating region composed of and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. For example, the targeting region may comprise DNA or a mixture of DNA and RNA, and an activating region may comprise RNA or a mixture of DNA and RNA.

[0129] The instant invention comprises introducing endonuclease cleavage sites into the donor nucleic acid delivery constructs at positions flanking the donor nucleic acid (see e.g., FIGURE 2 and FIGURE 3). In particular, in an AAV construct, the sites can be engineered according to the examples shown in FIGURE 4, i.e., near one or both ITRs. The cleavage site corresponds to the chosen endonuclease. In some embodiments, the endonuclease used for concatemer reduction is the same as the endonuclease used to insert the donor DNA into the genome (FIGURE 5). The following are some selected examples of endonucleases that can be used in the method of the invention.

[0130] In some embodiments, the endonuclease is a CRISPR endonuclease, and the cleavage site comprises a target-binding sequence for the nucleic acid targeting nucleic acid (NATNA) known as “protospacer” and a protospacer-adjacent motif (PAM). In some embodiments, the PAM is located to the 5’ of the protospacer. In some embodiments, the PAM is located to the 3’ of the protospacer.

[0131] In some embodiments, the CRISPR endonuclease is a Type V CRISPR endonuclease (e.g., Casl2a), and the PAM consists of a sequence selected from 5’-TTN-3’, 5’- TTTN-3’ and 5’-TTTV-3’. (FIGURE 6)

[0132] In some embodiments, the CRISPR endonuclease is a Type II CRISPR endonuclease e.g., Cas9), and the PAM consists of a sequence selected from 5'-NGG-3', 5'- NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3'. (FIGURE 7)

[0133] In some embodiments, the CRISPR endonuclease is a Type I (CRISPR CASCADE) endonuclease, and the PAM consists of a sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’- ATG-3’, 5’-GAG-3’, 5’-CAG-3’, 5’-GTG-3’, 5’-TAA-3’, 5’-TGG-3’, 5’-AAA-3’, 5’-AAC-3’, 5’- AAT-3’, 5 ’-ATA-3’, 5 ’-TAG-3’, and 5’-TTG-3’.

[0134] In some embodiments, the PAM is located on the vector side of the protospacer (P AM-out orientation) as shown in FIGURE 6 and FIGURE 7. In some embodiments, this orientation assures that the CRISPR endonuclease remains bound to the vector portion (or portions) of the construct, and the insert portion of the construct is not bound to the endonuclease and can be for example, captured and removed.

[0135] In some embodiments, the PAM is located on the insert side of the protospacer (PAM-in orientation) (diagram not shown). In some embodiments, this orientation assures that the CRISPR endonuclease remains bound to the insert portion of the construct, and the vector portion (or portions) of the construct is not bound to the endonuclease and can be for example, removed.

[0136] In some embodiments, the endonuclease is a Fanzor, a eukaryotic CRISPR-like endonuclease (Saito et al. (2023) Fanzor is a eukaryotic programmable RNA-guided endonuclease, Nature 620:660). In some embodiments, the Fanzor cleavage site comprises a target-adjacent motif (TAM) and a binding site for the guide RNA (omega-RNA). In some embodiments, TAM is located 5’-of the binding site for the guide RNA. In some embodiments, the TAM is selected from 5’-CATA-3’, 5’-TTAAN-3’, 5’-CCG-3’, and 5 ’-TAG-3.’

[0137] In some embodiments the endonuclease is a zinc finger nuclease (ZFN), or a ZFN- Fok I fusion. In such embodiments, the cleavage site is about 22-52 bases long and comprises a pair of ZFN recognition sequences, each 9-18 nucleotides long, separated by a spacer, which is 4- 18 nucleotides long. (See e.g.., Kim Y.G., et al., (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl Acad Sci USA. 93(3): 1156-1160.

[0138] In some embodiments, the endonuclease is a transcription activator-like effector nuclease (TALEN), or a TALEN-Fok I fusion. In such embodiments, the cleavage site is about 48- 85 nucleotides long and comprises a pair of TALEN recognition sequences, each 18-30 nucleotides long, separated by a spacer, which is 12-25 nucleotides long. (See e.g., Christian M. et al., (2010) Targeting DNA double-strand breaks with TAL effector nucleases, Genetics. 186 (2): 757-61.)

[0139] In some embodiments, the endonuclease is a homing endonuclease (also known as a meganuclease), and the cleavage site comprises a recognition sequence of 14-40 bp.

[0140] In some embodiments, the endonuclease used in the concatemer reduction method disclosed herein is the same endonuclease that is used for inserting the donor DNA into the genome. In some embodiments, the endonuclease used in the concatemer reduction method disclosed herein is a different endonuclease (including a different type of endonuclease) that is used for inserting the donor DNA into the genome.

[0141] In some embodiments, the invention is a composition comprising a nucleic acid construct for delivery of a donor nucleic acid into a target cell with reduced formation of concatemers of the donor nucleic acid in the genome of the cell, the construct comprising: a donor nucleic acid sequence and at least one cleavage site for a sequence-guided endonuclease.

[0142] The donor nucleic acid present in the construct is the nucleic acid to be introduced into the cell. The donor nucleic acid may comprise an expression cassette, i.e., a protein coding sequence or a sequence coding for a functional RNA, e.g., micro-RNA, RNAi, shRNA, and guide RNA for a CRISPR endonuclease. The expression cassette may further comprise at least one of a promoter, a transcription terminator, and a polyadenylation signal. The promoter may be selected from the EFla promoter, the PGK1 promoter, the MND promoter, the Ubc promoter, the CAG promoter, the CaMKIIa promoter, the [3-Actin promoter, the P-interferon promoter, the hsp70 promoter, the MMTV-LTR promoter, the RSV-LTR promoter the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.

[0143] In some embodiments, the donor nucleic acid contains a sequence used to replace a sequence in the cellular genome, e.g., to replace a mutant sequence with a non-mutant sequence (to restore a function) or to replace a non-mutant sequence with a mutant sequence (to block an undesired function).

[0144] The donor nucleic acid may also comprise one or two homology arms having a sequence capable of hybridizing to a sequence adjacent to a desired integration site and capable of promoting homology driven recombination (HDR) that would facilitate insertion of at least a portion of the donor DNA (e.g., the expression cassette) into the integration site. In some embodiments, the homology arms are between 10 and 100 base pairs long. In some embodiments, the homology arms are between 100 and 1000 base pairs long. In some embodiments, the homology arms are about 500-550 base pairs long. In some embodiments, the homology arms are single or double stranded stretches of nucleic acids.

[0145] The endonuclease cleavage sites present in the construct correspond to the endonuclease used in the step of concatemer reduction described herein. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18 nucleotides long ZFN recognition sequences separated by a 4-18 nucleotides long spacer. In some embodiments, the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALEN recognition sequences, each 18-30 nucleotides long, separated by a 12-25 nucleotide long spacer. In some embodiments, the endonuclease is a CRISPR endonuclease, and the cleavage site comprises a NATNA recognition site (protospacer) and a protospacer adjacent motif (PAM). In some embodiments, the endonuclease is a Fanzor endonuclease, and the cleavage site comprises an omega-RNA recognition site and a target adjacent motif (TAM).

[0146] In some embodiments, the composition further comprises the endonuclease whose cleavage site is present in the construct. In some embodiments, the composition further comprises the NATNA required by the endonuclease for cleavage. In some embodiments, the appropriate NATNA is selected from CRISPR guide RNA (gRNA), CRISPR hybrid RNA-DNA (chRDNA) and Fanzor omega-RNA.

[0147] In some embodiments, the composition comprises the nucleic acid construct comprising the donor nucleic acid designed as described herein and containing cleavage sites for sequence- guided endonuclease and further comprises the sequence-guided endonuclease capable of cleaving the cellular genome at the desired integration site for the donor nucleic acid and further capable of cleaving the cleavage sites in the nucleic acid construct (before or after integration into the cellular genome) so that with the introducing the composition into a cell, the occurrence of concatemers or imperfect integrations of the donor nucleic acid in the cellular genome is reduced.

[0148] In some embodiments, the invention is a composition comprising a nucleic acid which is a donor nucleic acid delivery construct (“a STACK construct”), the construct comprising a viral vector nucleic acid and more than one donor nucleic acid (e.g., two, three, or more donor nucleic acids) and further comprising one or more sequence-guided endonuclease cleavage sites. In some embodiments, the sequence-guided endonuclease cleavage sites are CRISPR / Cas endonuclease cleavage sites (e. ., one, two, three, or more CRISPR / Cas endonuclease cleavage sites). In the construct, the location of each sequence-guided cleavage site is selected from between the viral vector sequences (e.g., inverted terminal repeats, ITRs) and a donor nucleic acid and between two donor nucleic acids from the two or more donor nucleic acids. At least one cleavage site in the construct is identical to at least one cleavage site in the target genome targeted by the construct so that the sequence-guided endonuclease is capable of cleaving the construct and the target sequence in the genome. In some embodiments, the construct in the composition further comprises homology arms flanking the donor nucleic acid, the homology arms capable of hybridizing to nucleic acid sequences adjacent to the cleavage site in the target genome (e.g., flanking cleavage site in the target genome). In some embodiments, the donor nucleic acid in the construct comprises an expression cassette. In some embodiments, the donor nucleic acid in the construct comprises a stop cassette. In some embodiments, the same construct comprises at least one expression cassette and at least one stop cassette. In some embodiments, the HDR template is capable of hybridizing to a sequence in a locus selected from PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, GISH, CBLB, 2B4, TRAC, GUTA, DNMT3A, DNMT3L, FAS, TET1, TET2, TET3, CD 155, RASA2, SOCS1, SOCS3 and B2M. In some embodiments, the HDR template comprises an expression cassette encoding a gene selected from CIITA, a chimeric antigen receptor (CAR), a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a portion of an MHC Class I molecule and a fusion including an MHC Class I molecule, e.g., a fusion of HLA- E with HLA-G or HLA-F. In some embodiments, the construct comprises a sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and SEQ ID NO: 30.

[0149] In some embodiments, the sequence-guided endonuclease is a CRISPR / Cas endonuclease. In some embodiments, the composition further comprises one or more components of a CRISPR / Cas nucleoprotein complex (NPC) or a precursor of such components (e.g., mRNA) or a fully assembled NPC.EXAMPLES

[0150] Example 1. Preparation of Cytotoxic T Cells (CD4+ and CD8+) from PBMCs and Culture of Primary Cells

[0151] This Example illustrates the preparation of CD4+and CD8+T cells from donor peripheral blood mononuclear cells (PBMCs).

[0152] CD4+and CD8+T cells were prepared from donor PBMCs essentially as follows. T cells were isolated from peripheral blood mononuclear cells (PBMCs) using RoboSep-S and EasySep™ Human T cell Isolation Kit (both from STEMCELL Technologies, Cambridge, Mass.) and activated for 3 days in the presence of anti-CD3 / CD28 beads (Dynabeads™; Gibco 11132D) in ImmunoCult-XF complete medium (ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies), CTS Immune Cell SR (Gibco A2596102), Antibiotics-Antimycotics (100X, Corning 30-004-C1)) supplemented with recombinant human (rh) IL-2 (100 units / mL). After 3 days, beads were removed via magnetic separation and cells were expanded for 1 day in ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL).

[0153] Example 2. Reducing concatemer formation in engineered T cells at the B2M locus

[0154] In this experiment, three plasmids contained the same donor DNA but differed by the presence or number of flanking endonuclease cleavage sites.

[0155] A. Design of B2M-HLA-E insertion sequence

[0156] The target insertion site (CRISPR Casl2a endonuclease cleavage site) in the human genome was located in the beta-2 microglobulin (B2M) locus (SEQ ID NO: 5). The same Casl2a endonuclease cleavage site was inserted into plasmids carrying protein expression cassettes as donor DNA. The control plasmid pCB6837 (SEQ ID NO: 7) contained an insert comprising a protein expression cassette including the coding sequence for the following fusion peptide: a P2A skipping peptide sequence, a B2M signal peptide sequence, an HLA-G peptide sequence, a B2M protein sequence, an HLA-E protein sequence. The coding sequence was followed by a BGH terminator sequence. The expression cassette was flanked by a 5’ and 3’ homology arms for the B2M locus. The left and right AAV2 ITRs were located upstream and downstream of the B2M homology arms. The plasmid pCB7554 (SEQ ID NO: 8) contained the same insert as the control plasmid and also contained a single B2M CRISPR Casl2a endonuclease cleavage site inserted between the 3’ homology arm and the right ITR. The plasmid pCB7555 (SEQ ID NO: 9) containedthe same insert as the control plasmid and also contained B2M CRISPR Casl2a endonuclease cleavage sites inserted both between the 3’ homology arm and the right ITR, and between the 5’ homology arm and the left ITR. Plasmids were provided to a commercial manufacturer for packaging into separate AAV6 viruses.

[0157] B. Cloning of a Casl2 protein

[0158] The Acidaminococcus spp. (strain BV3L6) catalytically active Casl2a protein sequence (SEQ ID NO: 1) was codon optimized for expression in E. coli cells. At the C-terminus, a glycineserine linker and one nuclear localization sequence (NLS) (SEQ ID NO: 4) was added. Oligonucleotide sequences coding for the Casl2a-NLS protein (referred to as the AsCasl2a and Cast 2a protein in the following examples) were provided to commercial manufacturers for synthesis. DNA sequences were then cloned into suitable bacterial expression vectors using standard cloning methods.

[0159] C. Expression and purification of a Casl2a protein

[0160] The AsCasl2a protein was expressed in E. coli using an expression vector and purified using affinity chromatography, ion exchange, and size exclusion chromatography, essentially as described in Swarts et al. (2017) Dependent DNA targeting by CRISPR Casl2a, Molecular Cell 66:221-233.

[0161] D. Production of Casl2a guide components

[0162] Cast 2a guides were produced by linking a targeting region of a B2M target sequence (SEQ ID NO: 6) to a particular Cast 2a guide activating region (SEQ ID NO: 2).

[0163] Casl2a guide sequences (such as crRNAs and CRISPR hybrid RNA-DNA or chRDNA) were provided to a commercial manufacturer for synthesis.

[0164] E. Assembly of a Casl2a guide / nucleoprotein complex

[0165] Acidaminococcus spp. Cast 2a (AsCasl2a) tagged with a C-terminal nuclear localization sequence (NLS) was recombinantly expressed in E. coli and purified using chromatographic methods. Nucleoprotein complexes were formed at a concentration of 80 pmol Casl2a protein:240 pmol guide, unless otherwise stated. Prior to assembly with Casl2a protein, each of the guide components (e.g., crRNA or chRDNA) was adjusted to the desired total concentration (240 pmol) in a final volume of 1 pl, incubated for 2 minutes at 95°C, removed from a thermocycler, and allowed to equilibrate to room temperature. The Casl2a protein was diluted to an appropriate concentration in binding buffer (60mM TRIS-acetate, 150 mM potassiumacetate, 30 mM magnesium acetate, at pH 7.9) to a final volume of 1.5 pl and mixed with the 1 pl of the guide components, followed by incubation at 37°C for 10 minutes.

[0166] F. Isolation and Activation of Human T cells

[0167] The Casl2a guide / nucleoprotein complexes were transfected into primary activated T cells (CD4+and CD8+) using the Nucleofector™ 96-well Shuttle System (Lonza, Allendale, NJ). The Casl2a guide / nucleoprotein complex were dispensed in a 2.5 pl final volume into individual wells of a 96-well plate. The suspended T cells were pelleted by centrifugation for 10 minutes at 200 x g, washed with calcium and magnesium-free phosphate buffered saline (PBS), and resuspended in 10 ml of calcium and magnesium-free PBS. The cells were counted using the Countess® II Automated Cell Counter (Life Technologies; Grand Island, NY).

[0168] 2.2 x 107cells were transferred to a 15 ml conical tube and pelleted. The PBS was aspirated, and the cells resuspended in Nucleofector™ P3 solution (Lonza) to a density of 2xl05- 106cells / ml per sample. 20 pl of the cell suspension was then added to each well containing 2.5 pl of the Cast 2a guide / nucleoprotein complexes, and the entire volume from each well was transferred to a well of a 96-well Nucleocuvette™ Plate (Lonza). The plate was loaded onto the Nucleofector™ 96-well Shuttle (Lonza) and cells nucleofected using the CA137 Nucleofector™ program (Lonza). Post-nucleofection, 77.5 pl of ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL) was added to each well, and the entire volume of transfected cell suspension was transferred to a 96-well cell culture plate containing 100 pl pre-warmed ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL). The plate was transferred to a tissue culture incubator and maintained at 37°C in 5% CO2 before next step.

[0169] G. Primary T cell transduction with rAAV

[0170] The day before the transduction AAV6 virus was removed from -80 °C storage and thawed overnight at 4°C.

[0171] The T cells transfected with B2M (SEQ ID NO: 6)-targeting Casl2a chRDNA guide / nucleoprotein complexes, were transduced with AAV6 between 1 minute and 4 hours after nucleofection, cells were infected with the AAV6 virus packaged with B2M-HLA-E constructs have none, one or two Casl2a cleavage sites (SEQ ID NO: 7 - 9) at an MOI of 2 x 105. T cells were cultured in ImmunoCult-XF complete medium (STEMCELL Technologies) supplemented with IL-2 (100 units / mL) for 24 hours after the transductions. The next day, the transduced T cells were transferred to 50 mL conical tubes and centrifuged at 300 x g for approximately 7-10 minutesto pellet the cells. The supernatant was discarded, the pellet was gently resuspended, and the T cells pooled in an appropriate volume of ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL).

[0172] The transduced T cells were enumerated and diluted to a density of 1 x 106cells / mL in ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL) every 48-72 hours, as needed.

[0173] After eight days of expansion, 10 million cells were harvested centrifuged, supernatant aspirated and washed with lOmL of PBS. Genomic DNA was extracted using the Puregene kit (Qiagen, Valencia, Cal.) following the manufacturer’s protocol. Extracted DNA was further purified using AMPure® PD beads per manufacturer protocol before targeted digestion with the Cas9 Sequencing Kit (Oxford Nanopore Technologies, Oxford, United Kingdom) using Cas9 guides which flanked the B2M target region. Digested samples were sequenced using the GridlON Mkl (Oxford Nanopore Technologies). Results are shown in Table 1, and in Figure 8A.

[0174] Table 1. Reduction in Imperfect Insertion in human T cells* Informative reads

[0175] The imperfect insertion events observed across the three samples were partitioned into concatemers and other imperfect insertion events. Results are shown in Table 2, and Figure 8B.

[0176] Table 2. Reduction in Concatemer Insertion in human T cells

[0177] Example 3. Reducing concatemer formation in engineered T cells at the TRAC locus (prophetic)

[0178] In this experiment, three plasmids contained an anti-RORl CAR donor DNA sequence with or without the of flanking endonuclease cleavage sites are traduced into human primary T cells editing with a Casl2a nucleoprotein complex.

[0179] A. Design of anti-RORl CAR insertion sequence

[0180] The target insertion site (CRISPR Casl2a endonuclease cleavage site) in the human genome is located in the T cell alpha constant region TRAC) locus (SEQ ID NO: 10). The same Casl2a endonuclease cleavage sites is inserted into plasmids as follows. The unmodified plasmid pCB7306 (SEQ ID NO: 12) contains a fusion of EFl alpha promoter, a CD8 alpha signal peptide coding sequence, an anti-RORl single chain variable fragment (scFv) coding sequence, a CD8 hinge region coding sequence, a CD8 transmembrane region coding sequence, a 4 IBB signaling domain coding sequence, a CD3 zeta signaling domain coding sequence, and a BGH terminator sequence flanked by a 5’ and 3’ homology arms for the TRAC locus, and with a left and right AAV2 ITR upstream and downstream of the TRAC homology arms. The plasmid pCB7870 (SEQ ID NO: 13) contains the same insert and also contains a single TRAC cleavage site inserted between the 3’ homology arm and the right ITR. The plasmid pCB7871 (SEQ ID NO: 14) contains the same insert and also contains a TRAC cleavage site inserted both between the 3’ homology arm and the right ITR, and between the 5’ homology arm and the left ITR.

[0181] Plasmids are provided to a commercial manufacturer for packaging into separate AAV6 viruses.

[0182] B. Casl2a editing components

[0183] The Casl2a protein is produced as described in Example 2. The Casl2a guides are produced by linking a targeting region of a TRAC target sequence (SEQ ID NO: 10) to a particular Casl2a guide activating region (SEQ ID NO: 2). The Casl2a guide sequences (SEQ ID NO: 11) are provided to a commercial manufacturer for synthesis.

[0184] C. Transfection and transduction of human primary T cells

[0185] Casl2a editing reagents and AAV6 are prepare as described in Example 2.

[0186] Human T cells are isolated and activated as described in Example 1 and edited using the Casl2a protein and TRAC targeting guides as described in Example 2. Cells are transduced with anti-RORl CAR AAV6 with and without ITR flanked targeting regions as described in Example 2. Edited cells are enumerated and subjected to long-read sequencing as described in Example 2.

[0187] Example 4. A STACK construct for anti-RORl CAR insertion and PDCD1 gene knockout.

[0188] This example describes the design of a STACK construct comprising an anti-RORl CAR to be inserted into the TRAC locus and a stop cassette for insertion into the PDCD1 locus for gene knock-out.

[0189] A Casl2a target site in the TRAC locus was selected as the first target site (SEQ ID NO: 10). A first donor nucleic acid (AAV-A donor nucleic acid) was designed to include a left (upstream) 500bp homology arm an expression cassette for an anti-RORl CAR (SEQ ID NO: 28) comprising an EF 1 alpha promoter, a CAR coding sequence, and a BGH polyadenylation sequence, and a right (downstream) 500 bp homology arm, where the homology arms are capable of hybridizing to the first target site in the TRAC locus according to the scheme shown in FIGURE 9.

[0190] A Casl2a target site in the PDCD1 locus was selected as a second target site (SEQ ID NO: 22). A second donor nucleic acid (AAV-B donor nucleic acid) was designed with a stop cassette (SEQ ID NO: 19). Four sets of homology arms were designs of varying length of 120bp, 200bp, 250bp, or 400bp in length where the homology arms are capable of hybridizing to the second target site in the PDCD1 locus according to the scheme shown in FIGURE 9.

[0191] For the design of the STACK construct, the PDCD1 Casl2a cleavage site (SEQ IDNO: 22) was added downstream (in the 3’ direction) of an AAV6 left ITR (SEQ ID NO: 26), followed by the stop cassette (SEQ ID NO: 19) flanked by variable length homology arms for the PDCD1 locus, followed by the TRAC Cast 2a cleavage site (SEQ ID NO: 10), followed by the anti-RORl CAR expression cassette (SEQ ID NO: 28), and then the AAV6 right ITR (SEQ ID NO: 27). The sequence of the STACK constructs comprising the 120bp, 200bp, 250bp, or 400bpPDCD1 homology arms, stop cassette, and anti-RORl CAR flanked by ITRs of AAV6 are provided in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0192] Example 6. Cloning of AA V donor cassette, AA V production and AA V transduction of primary cells

[0193] This Example describes the design and cloning of donor nucleic acids into an AAV vector to form a STACK construct, production of AAV, and codelivery of Casl2a RNP and AAV transduction of primary cells with AAV for site-specific integration of a CAR expression cassette into primary cells.

[0194] A. In silico design of AAV donor element cassettes and rAAV production

[0195] The sequences of the STACK constructs described in Example 4 were provided to a commercial manufacturer for synthesis into suitable recombinant AAV (rAAV) plasmids. The resulting rAAV plasmids containing STACK constructs were provided to a commercial manufacturer for packaging into an AAV6 virus.

[0196] B. Primary T cell transduction with rAAV

[0197] Primary activated T cells are obtained from PBMCs as described in Example 1. Casl2a-guide nucleoprotein complexes (NPCs) are prepared (Example 2 D-E) containing a chRDNA targeting TRAC (SEQ ID NO: 11) and PDCD1 (SEQ ID NO: 23) and the T cells are transfected with the NPCs (Example 2F), and AAV6 virus packaged with a STACK donor sequence (SEQ ID NO: 15-18) are transduced with the AAV6 virus (Example 2G).

[0198] To confirm the integration of the AAV-A donor nucleic (anti-ROR l CAR, SEQ ID NO: 28) acid at the TRAC locus and the AAV-B donor nucleic acid (the stop cassette, SEQ ID NO: 19) at the PDCD1 locus, genomic DNA from transfected and transduced cells is isolated and a combination of long-read next-generation sequencing is performed using the SEQUEL lie SYSTEM Long-read sequencing (Pacific Biosciences, Menlo Park, Cal) sequencer for the insertion of the anit-RORl CAR at the TRAC locus and short read next generation sequencing is performed using the MiSeq Sequencing System (Illumina, San Diego, Cal) for the insertion of the stop cassette at the PDCD1 locus.

[0199] Example 7. In vitro cytotoxicity of CAR-T cells transfected with the STACK construct

[0200] The cytotoxicity of CAR-T cells of Example 6 is evaluated in vitro against a R0R1 expressing target cell line. Briefly, target cells are labeled with CellTrace™ Violet (CTV;ThermoFisher Scientific, C34557) to distinguish them from effector CAR-T cells, and cells are co-cultured at effectontarget (E:T) ratios of 0:1, 1 :20, 1 :10, 1 :5, 1 :3, 1 : 1, 3:1, and 10: 1 (3 cocultures per E:T ratio). Cytotoxicity is measured by gating on CTV cell population (target cells) and live cells as measured by propidium iodide (PI) after 48 hours in co-culture. Data is analyzed by flow cytometry (Intellicyt iQue Screener Plus). Specific lysis of target cells is calculated for each well using the following formula:Specific lysis = 1 - (number of live target cells in the test sample / number of live target cells in the control sample).

[0201] Example 8. A STACK construct for anti-RORl CAR and IL15 gene insertions

[0202] This example describes the design of a STACK construct comprising an anti-RORlCAR to be inserted into the TRAC locus and an interleukin 15 (IL15) expression cassette for insertion into the C1SH locus.

[0203] A Casl2a target site in the TRAC locus was selected as the first target site (TargetA, SEQ ID NO: 10). A first donor nucleic acid (AAV-A donor nucleic acid) was designed to include a left (upstream) 250 bp TRAC homology arm, the anti-RORl CAR expression cassette (SEQ ID NO: 28), and a right (downstream) 250 bp TRAC homology arm.

[0204] A Casl2a target site in the CISH locus was selected as a second target site (TargetB, SEQ ID NO: 20). A second donor nucleic acid (AAV-B donor nucleic acid) was designed to include a right (upstream) 250 bp CISH homology arm, and IL15 expression cassette (SEQ ID NO: 29) including an EFl alpha promoter, an IL-2 signal peptide, an IL 15 coding sequence, and SV40 early polyadenylation sequence, and a left (downstream) 250 bp CISH homology arm.

[0205] For the design of the STACK construct, AAV6 left ITR (SEQ ID NO: 26) and rightITR (SEQ ID NO: 27) were added to the construct resulting in the STACK construct comprising in the 5’-3’ direction: AAV6 left ITR, TRAC Casl2a target site, the anti-RORl CAR expression cassette, CISH Casl2a target site, the IL-15 expression cassette, AAV6 right ITR. The final construct SEQ ID NO: 30 is synthesized and packaged in AAV by a commercial manufacturer.

[0206] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

[0207] SEQUENCE TABLE

Claims

CLAIMSWhat is claimed is:

1. A nucleic acid construct for delivery of a donor nucleic acid into a genome of a target cell, the construct comprising: a) at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; b) at least one cleavage site for a sequence-guided endonuclease, the cleavage site located outside of the homology arms relative to the donor nucleic acid; and c) a vector backbone.

2. The nucleic acid construct of claim 1, wherein the at least one cleavage site for the sequence-guided endonuclease comprises a sequence substantially identical to the target sequence in the genome of the cell, and wherein the sequence-guided endonuclease capable of cleaving the construct is also capable of cleaving the target site in the genome.

3. The nucleic acid construct of claim 2, wherein the at least one donor nucleic acid comprises in the 5’-3’ direction: a) a first donor nucleic acid flanked by a first set of first and second homology arms capable of hybridizing to a first target sequence in the genome of the cell, and b) a second donor nucleic acid flanked by a second set of first and second homology arms capable of hybridizing to a second target sequence in the genome of the cell, wherein one cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid.

4. The nucleic acid construct of claim 3, comprising two or more cleavage sites for a sequence-guided endonuclease, wherein at least two cleavage sites are for different sequence-guided endonucleases.

5. The nucleic acid construct of claim 4, comprising three cleavage sites for a sequence- guided endonuclease, wherein the first cleavage site is located between the vector backbone and the first homology arm of the first donor nucleic acid; the second cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid; and the third cleavage site is locatedbetween the second homology arm of the second donor nucleic acid and the vector backbone.

6. The nucleic acid construct of claim 1, wherein the endonuclease is a CRISPR endonuclease, and the cleavage site comprises a protospacer and a protospacer adjacent motif (PAM).

7. The nucleic acid construct of claim 6, wherein the protospacer is substantially identical to a sequence in the target sequence in the genome of the cell.

8. The nucleic acid construct of claim 7, wherein the protospacer is substantially identical to a sequence in a gene selected from the group consisting of PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, CISH, CBLB, 2B4, TRAC, GUTA, DNMT3A, DNMT3L, FAS, TET1, TET2, TET3, CD155, RASA2, SOCSl, SOCS3 and B2M.

9. The nucleic acid construct of claim 8, wherein the protospacer is substantially identical to a sequence in the PDCD1 gene and comprises or consists essentially of SEQ ID NO: 22.

10. The nucleic acid construct of claim 8, wherein the protospacer is substantially identical to a sequence in the TRAC gene and comprises or consists essentially of SEQ ID NO: 10.

11. The nucleic acid construct of claim 8, wherein the protospacer is substantially identical to a sequence in the B2M gene and comprises or consists essentially of SEQ ID NO: 5.

12. The nucleic acid construct of claim 8, wherein the protospacer is substantially identical to a sequence in the CISH gene and comprises or consists essentially of SEQ ID NO: 20.

13. The nucleic acid construct of claim 6, wherein the CRISPR endonuclease is a Type V CRISPR endonuclease, and the PAM consists of a sequence selected from 5’-TTN-3’, 5’- TTTN-3’ and 5’-TTTV-3’.

14. The nucleic acid construct of claim 6, wherein the CRISPR endonuclease is a Type II CRISPR endonuclease, and the PAM consists of a sequence selected from 5'-NGG-3', 5'- NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'- NNNACA-3’.

15. The nucleic acid construct of claim 6, wherein the CRISPR endonuclease is a Type I (CRISPR CASCADE) endonuclease, and the PAM consists of a sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5’-GAG-3’, 5’-CAG-3’, 5’-GTG-3’, 5’-TAA-3’, 5’- TGG-3’, 5’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5’-ATA-3’, 5’-TAG-3’, and 5’-TTG-3’.

16. The nucleic acid construct of claim 1, wherein the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALEN recognition sequences each comprising 18-30 nucleotides separated by a spacer comprising 12-25 nucleotides.

17. The nucleic acid construct of claim 1, wherein the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18 nucleotides long ZFN recognition sequences each comprising 9-18 nucleotides separated by a spacer comprising 4-18 nucleotides.

18. The nucleic acid construct of claim 1, wherein the vector is a plasmid vector, or a viral vector selected from adenovirus (AdV), adeno-associated virus (AAV) and a lentivirus.

19. The nucleic acid construct of claim 1, wherein the donor nucleic acid comprises an expression cassette.

20. The nucleic acid construct of claim 19, wherein the expression cassette comprises a protein coding sequence encoding a protein selected from the group consisting of CD47, a chimeric antigen receptor (CAR), a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a portion of an MHC Class I molecule and a fusion including an MHC Class I molecule.

21. The nucleic acid construct of claim 20, comprising SEQ ID NO: 31.

22. The nucleic acid construct of claim 20, comprising SEQ ID NO: 29.

23. The nucleic acid construct of claim 20, comprising SEQ ID NO: 28.

24. The nucleic acid construct of claim 19, wherein the expression cassette comprises a promoter selected from the EFla promoter, the PGK1 promoter, the MND promoter, the Ubc promoter, the CAG promoter, the CaMKIIa promoter, the 0-Actin promoter, the P- interferon promoter, the hsp70 promoter, the MMTV-LTR promoter, the RSV-LTR promoter the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.

25. The nucleic acid construct of claim 19, wherein the expression cassette encodes a functional RNA.

26. The nucleic acid construct of claim 25, wherein the functional RNA is selected from the group consisting of micro-RNA, small inhibitory RNA (siRNA), small hairpin RNA (shRNA), and guide RNA for a CRISPR endonuclease (NATNA).

27. The nucleic acid construct of claim 1, wherein the donor nucleic acid comprises one, two or three stop codons wherein the two or three stop codons are in different translation frames.

28. The nucleic acid construct of claim 27, comprising SEQ ID NO: 19.

29. The nucleic acid construct of claim 1, wherein the donor nucleic acid comprises a splice site selected from the group consisting of a donor splice site, an acceptor splice site and a donor-acceptor splice site.

30. The nucleic acid construct of claim 1, comprising a sequence selected from the group consisting of SEQ ID NO: 15-18 and 30.

31. A composition comprising the nucleic acid construct of claim 1.

32. The composition of claim 31, further comprising one or more sequence-guided endonucleases capable of cleaving the one or more sequence-guided endonuclease recognition sites in the nucleic acid construct.

33. The composition of claim 32, wherein the sequence-guided endonuclease is a CRISPR endonuclease, and the composition further comprises a nucleic acid targeting nucleic acid (NATNA) capable of binding the protospacer and promoting cleavage by the CRISPR endonuclease.

34. A cell comprising the nucleic acid construct of claim 1.

35. The cell of claim 34, wherein the cell is a bacterial cell, a plant cell, a human cell, or a non-human animal cell.

36. The cell of claim 35, wherein the human cell is in vitro.

37. The cell of claim 35, wherein the human cell is not a human germline cell.

38. The cell of claim 34, wherein the human cell is not a human totipotent cell.

39. A method for integrating a donor nucleic acid sequence into a cellular genome in a population of cells resulting in a reduced number of cells comprising concatemers of the donor nucleic acid in the cellular genome, the method comprising introducing into the cells of a population of cells the nucleic acid construct of claim 1, and further introducinginto the cells the sequence-guided endonuclease capable of cleaving the sequence-guided endonuclease recognition sites in the nucleic acid construct.

40. The method of claim 39, further comprising integration of at least a portion of the donor nucleic acid into the target cell’s chromosome.

41. The method of claim 39, wherein only one copy of at least a portion of the donor nucleic acid integrates into the genomes of cells of a population of cells.

42. The method of claim 39, wherein a number of cells comprising concatemers of the donor nucleic acid integrated into the genome is reduced relatively to the number of cells comprising concatemers of the donor nucleic acid integrated into the genome where the donor nucleic acid is introduced via a nucleic acid construct lacking any cleavage sites for the sequence-guided endonuclease.

43. The method of claim 39, further comprising integration of one or more additional donor nucleic acids into the cellular genome.

44. The method of claim 39, wherein the at least one donor nucleic acid in the nucleic acid construct comprises in the 5’-3’ direction: a) a first donor nucleic acid flanked by a first set of first and second homology arms capable of hybridizing to a first target sequence in the genome of the cell, and b) a second donor nucleic acid flanked by a second set of first and second homology arms capable of hybridizing to a second target sequence in the genome of the cell, wherein one cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid.

45. The method of claim 39, wherein the nucleic acid construct comprises two or more cleavage sites for a sequence-guided endonuclease, wherein at least two cleavage sites are for different sequence-guided endonucleases.

46. The method of claim 39, wherein the nucleic acid construct comprises three cleavage sites for a sequence-guided endonuclease, wherein the first cleavage site is located between the vector backbone and the first homology arm of the first donor nucleic acid; the second cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid; and the third cleavage site is located between the second homology arm of the second donor nucleic acid and the vector backbone.

47. The method of claim 39, wherein the sequence-guided endonuclease is a CRISPR endonuclease, and the cleavage site comprises a protospacer and a protospacer adjacent motif (PAM).

48. The method of claim 47, wherein the protospacer is substantially identical to a sequence in the target sequence in the genome of the cell.

49. The method of claim 47, wherein the protospacer is substantially identical to a sequence in a gene selected from the group consisting of PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, GISH, CBLB, 2B4, TRAC, CIITA, DNMT3A, DNMT3L, FAS, TET1, TET2, TET3, CD155, RASA2, SOCS1, SOCS3 and B2M.

50. The method of claim 45, wherein the protospacer is substantially identical to a sequence in the PDCD1 gene and comprises or consists essentially of SEQ ID NO: 22.

51. The method of claim 45, wherein the protospacer is substantially identical to a sequence in the TRAC gene and comprises or consists essentially of SEQ ID NO: 10.

52. The method of claim 45, wherein the protospacer is substantially identical to a sequence in the B2M gene and comprises or consists essentially of SEQ ID NO: 5.

53. The method of claim 45, wherein the protospacer is substantially identical to a sequence in the CISH gene and comprises or consists essentially of SEQ ID NO: 20.

54. The method of claim 47, wherein the CRISPR endonuclease is a Type V CRISPR endonuclease, and the PAM consists of a sequence selected from 5’-TTN-3’, 5’-TTTN-3’ and 5’-TTTV-3’.

55. The method of claim 47, wherein the CRISPR endonuclease is a Type II CRISPR endonuclease, and the PAM consists of a sequence selected from 5'-NGG-3', 5'- NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'- NNNACA-3’.

56. The method of claim 47, wherein the CRISPR endonuclease is a Type I (CRISPR CASCADE) endonuclease, and the PAM consists of a sequence selected from 5’-AAG- 3’, 5’-AGG-3’, 5’-ATG-3’, 5’-GAG-3’, 5’-CAG-3’, 5’-GTG-3’, 5’-TAA-3’, 5’-TGG-3’, 5 ’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5 ’-ATA-3’, 5 ’-TAG-3’, and 5’-TTG-3’.

57. The method of claim 39, wherein the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALENrecognition sequences each comprising 18-30 nucleotides separated by a spacer comprising 12-25 nucleotides.

58. The method of claim 39, wherein the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18 nucleotides long ZFN recognition sequences each comprising 9-18 nucleotides separated by a spacer comprising 4-18 nucleotides.

59. The method of claim 39, wherein the vector is a plasmid vector, or a viral vector selected from adenovirus (AdV), adeno-associated virus (AAV) and a lentivirus.

60. The method of claim 39, wherein the donor nucleic acid comprises an expression cassette.

61. The method of claim 60, wherein the expression cassette comprises a protein coding sequence encoding a protein selected from the group consisting of CD47, a chimeric antigen receptor (CAR), a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a portion of an MHC Class I molecule and a fusion including an MHC Class I molecule.

62. The method of claim 61, wherein the expression cassette comprises SEQ ID NO: 31.

63. The method of claim 61, wherein the expression cassette comprises SEQ ID NO: 29.

64. The method of claim 61, wherein the expression cassette comprises SEQ ID NO: 28.

65. The method of claim 60, wherein the expression cassette comprises a promoter selected from the EF 1 a promoter, the PGK1 promoter, the MND promoter, the Ubc promoter, the CAG promoter, the CaMKIIa promoter, the P- Actin promoter, the -interferon promoter, the hsp70 promoter, the MMTV-LTR promoter, the RSV-LTR promoter the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.

66. The method of claim 60, wherein the expression cassette encodes a functional RNA.

67. The method of claim 66, wherein the functional RNA is selected from the group consisting of micro-RNA, small inhibitory RNA (siRNA), small hairpin RNA (shRNA), and guide RNA for a CRISPR endonuclease (NATNA).

68. The method of claim 39, wherein the donor nucleic acid comprises one, two or three stop codons wherein the two or three stop codons are in different translation frames.

69. The method of claim 68, wherein the donor nucleic acid comprises of SEQ ID NO: 19.

70. The method of claim 39, wherein the donor nucleic acid comprises a splice site selected from the group consisting of a donor splice site, an acceptor splice site and a donoracceptor splice site.

71. The method of claim 39, wherein the nucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NO: 15-18 and 30.

72. The method of claim 60, further comprising detecting sustained expression of a protein or RNA encoded by the expression cassette.

73. The method of claim 39, wherein the population of cells is a population of bacterial cells, plant cells, human cells, or non-human animal cells.

74. The method of claim 73, wherein the human cells are present in vitro.

75. The method of claim 73, wherein the human cells are present in vivo but is not a human germline cell or a human totipotent cell and the method does not alter genetic identity of a human being.

76. The method of claim 73, wherein the human cells are immune cell selected from T cells, T cell precursors, B cells, B cell precursors, macrophages, and macrophage precursors.

77. A method of manufacturing the nucleic acid construct of claim 1, the method comprising linking together a) at least one donor nucleic acid to be introduced into a genome of a cell flanked by homology arms capable of hybridizing to a target sequence in the genome of the cell; b) at least one cleavage site for a sequence-guided endonuclease, the cleavage site located outside of the homology arms relative to the donor nucleic acid; and c) a vector backbone.

78. The method of claim 77, wherein the method comprising linking in the 5’-3’ direction: a) a first donor nucleic acid flanked by a first set of first and second homology arms capable of hybridizing to a first target sequence in the genome of the cell, and b) a second donor nucleic acid flanked by a second set of first and second homology arms capable of hybridizing to a second target sequence in the genome of the cell, wherein one cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid.

79. The method of claim 77, wherein the nucleic acid construct comprises two or more cleavage sites for a sequence-guided endonuclease, wherein at least two cleavage sites are for different sequence-guided endonucleases.

80. The method of claim 77, wherein the nucleic acid construct comprises three cleavage sites for a sequence-guided endonuclease, wherein the first cleavage site is located between the vector backbone and the first homology arm of the first donor nucleic acid; the second cleavage site is located between the second homology arm of the first donor nucleic acid and the first homology arm of the second donor nucleic acid; and the third cleavage site is located between the second homology arm of the second donor nucleic acid and the vector backbone.

81. The method of claim 77, wherein the sequence-guided endonuclease is a CRISPR endonuclease, and the cleavage site comprises a protospacer and a protospacer adjacent motif (PAM).

82. The method of claim 81, wherein the protospacer is substantially identical to a sequence in the target sequence in the genome of the cell.

83. The method of claim 81, wherein the protospacer is substantially identical to a sequence in a gene selected from the group consisting of PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, CISH, CBLB, 2B4, TRAC, CIITA, DNMT3A, DNMT3L, FAS, TETl, TET2, TET3, CD 155, RASA2, SOCS1, SOCS3 and B2M.

84. The method of claim 83, wherein the protospacer is substantially identical to a sequence in the PDCD1 gene and comprises or consists essentially of SEQ ID NO: 22.

85. The method of claim 83, wherein the protospacer is substantially identical to a sequence in the TRAC gene and comprises or consists essentially of SEQ ID NO: 10.

86. The method of claim 83, wherein the protospacer is substantially identical to a sequence in the B2M gene and comprises or consists essentially of SEQ ID NO: 5.

87. The method of claim 83, wherein the protospacer is substantially identical to a sequence in the CISH gene and comprises or consists essentially of SEQ ID NO: 20.

88. The method of claim 81, wherein the CRISPR endonuclease is a Type V CRISPR endonuclease, and the PAM consists of a sequence selected from 5’-TTN-3’, 5’-TTTN-3’ and 5’-TTTV-3’.

89. The method of claim 81, wherein the CRISPR endonuclease is a Type II CRISPR endonuclease, and the PAM consists of a sequence selected from 5'-NGG-3', 5'- NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'- NNNACA-3’.

90. The method of claim 81, wherein the CRISPR endonuclease is a Type I (CRISPR CASCADE) endonuclease, and the PAM consists of a sequence selected from 5’-AAG- 3’, 5’-AGG-3’, 5’-ATG-3’, 5’-GAG-3’, 5’-CAG-3’, 5’-GTG-3’, 5’-TAA-3’, 5’-TGG-3’, 5 ’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5 ’-ATA-3’, 5 ’-TAG-3’, and 5’-TTG-3’.

91. The method of claim 77, wherein the endonuclease is a transcription activator-like effector nuclease (TALEN), and the cleavage site comprises a pair of TALEN recognition sequences each comprising 18-30 nucleotides separated by a spacer comprising 12-25 nucleotides.

92. The method of claim 77, wherein the endonuclease is a zinc finger nuclease (ZFN), and the cleavage site comprises a pair of 9-18 nucleotides long ZFN recognition sequences each comprising 9-18 nucleotides separated by a spacer comprising 4-18 nucleotides.

93. The method of claim 77, wherein the vector is a plasmid vector, or a viral vector selected from adenovirus (AdV), adeno-associated virus (AAV) and a lentivirus.

94. The method of claim 77, wherein the donor nucleic acid comprises an expression cassette.

95. The method of claim 94, wherein the expression cassette comprises a protein coding sequence encoding a protein selected from the group consisting of CD47, a chimeric antigen receptor (CAR), a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, a chemokine, a chemokine receptor, a chemokine-chemokine receptor fusion, a portion of an MHC Class I molecule and a fusion including an MHC Class I molecule.

96. The method of claim 95, wherein the expression cassette comprises SEQ ID NO: 31.

97. The method of claim 95, wherein the expression cassette comprises SEQ ID NO: 28.

98. The method of claim 95, wherein the expression cassette comprises SEQ ID NO: 29.

99. The method of claim 94, wherein the expression cassette comprises a promoter selected from the EF 1 a promoter, the PGK1 promoter, the MND promoter, the Ubc promoter, the CAG promoter, the CaMKIIa promoter, the 0- Actin promoter, the 0-interferon promoter, the hsp70 promoter, the MMTV-LTR promoter, the RSV-LTR promoter the SV40 earlyand late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.

100. The method of claim 94, wherein the expression cassette encodes a functional RNA101. The method of claim 100, wherein the functional RNA is selected from the group consisting of micro-RNA, small inhibitory RNA (siRNA), small hairpin RNA (shRNA), and guide RNA for a CRISPR endonuclease (NATNA).

102. The method of claim 77, wherein the donor nucleic acid comprises one, two or three stop codons wherein the two or three stop codons are in different translation frames.

103. The method of claim 102, wherein the donor nucleic acid comprises SEQ ID NO: 19.

104. The method of claim 77, wherein the donor nucleic acid comprises a splice site selected from the group consisting of a donor splice site, an acceptor splice site and a donor-acceptor splice site.

105. The method of claim 77, wherein the nucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NO: 15-18 and 30.

106. A method of making a cell comprising a donor nucleic acid integrated into the genome of the cell, the method comprising introducing into the cell a nucleic acid construct of claim 1.

107. The method of claim 106, wherein the cell is selected from the group consisting of a bacterial cell, a plant cell, a human cell, or a non-human animal cell.

108. The method of claim 107, wherein the human cell is present in vitro.

109. The method of claim 107, wherein the human cell is present in vivo but is not a human germline cell or a human totipotent cell and the method does not alter genetic identity of a human being.

110. The method of claim 107, wherein the human cell is an immune cell selected from a T cell, a T cell precursor, a B cell, a B cell precursor, a macrophage, and a macrophage precursor.

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