Compositions and methods for improved gene editing

Abstract

The present disclosure provides methods for introducing site-specific mutations into target cells and methods for determining the efficacy of enzymes capable of introducing site-specific mutations. The present disclosure also provides methods for providing biallelic sequence integration, methods for integrating a sequence of interest into a locus in the genome of a cell, and methods for introducing stable episomal vectors into cells. The present disclosure further provides methods for generating human cells resistant to diphtheria toxin.

Description

[Technical field]

[0001] The present disclosure provides methods for introducing site-specific mutations into target cells and for determining the efficacy of enzymes capable of introducing site-specific mutations. The present disclosure also provides methods for providing biallelic sequence integration, for integrating a sequence of interest into a locus in the genome of a cell, and for introducing a stable episomal vector into a cell. The present disclosure further provides a method for generating human cells resistant to diphtheria toxin. [Background technology]

[0002] Targeted nucleic acid modification by programmable site-specific nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided Cas9 is a very promising approach to studying gene function and has great potential to provide novel therapeutics for genetic diseases. Typically, programmable nucleases generate double-strand breaks (DSBs) in the target sequence. The DSBs can then be repaired by mutation via the non-homologous end joining (NHEJ) pathway, or the DNA around the cleavage site can be replaced with a template introduced simultaneously via the homology-directed repair (HDR) pathway. For an overview of targeted nucleic acid modification, see, for example, Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3.

[0003] The drawbacks of relying on NHEJ and HDR include, for example, low efficiency of HDR and undesirable off-target activity by NHEJ. Low efficiency of HDR makes it particularly difficult to select precise on-target modification (see, for example, Non-Patent Document 1, Non-Patent Document 4, Non-Patent Document 5). Various attempts to force HDR over NHEJ include, for example, generating one or more single-stranded nicks in target DNA instead of DSB (see, for example, Non-Patent Document 6, Non-Patent Document 7). However, there is still a need in the art for improved selection of HDR events, for example, when biallelic integration or gene silencing, which is typically achieved by HDR template, is desired.

[0004] Although HDR is less error-prone than NHEJ, HDR still tends to generate undesired modifications that compete with the targeted modification. Therefore, base editing has recently emerged as a powerful and precise gene editing technique that facilitates the replacement of single base pairs at specific locations in the genome. Compared with HDR-based methods for site-specific modification, base editing provides a more effective way to introduce single nucleotide mutations, overcoming some of the limitations associated with HDR. Base editing involves the site-specific modification of a single DNA base along with the manipulation of natural DNA repair mechanisms to avoid faithful repair of the modified base. Base editors are typically chimeric proteins that include a DNA targeting module and a catalytic domain that can deaminate, for example, a cytidine base to thymine or an adenine base to guanine. For example, the DNA targeting module can be based on catalytically inactive Cas9 (dCas9) or a Cas9 nickase variant (Cas9n) guided by a guide RNA molecule (sgRNA or gRNA). The catalytic domain can be a cytidine deaminase or an adenine deaminase. It is not necessary to generate DSBs to edit DNA bases and limit the generation of insertions and deletions (indels) at target and off-target sites. Therefore, base editing does not depend on the cellular HDR machinery, and is therefore more efficient than HDR and causes less inaccurate modifications due to NHEJ. Modified base editing systems are described, for example, in Non-Patent Document 8, Non-Patent Document 9, Non-Patent Document 10, and Non-Patent Document 11. For an overview of base editing, see, for example, Non-Patent Document 12, Non-Patent Document 13, and Non-Patent Document 14. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Humbert et al.,Crit Rev Biochem Mol Biol(2012)47:264-281 [Non-Patent Document 2] Perez-Pinera et al., Curr Opin Chem Biol (2012) 16:268-277 [Non-Patent Document 3] Pan et al., Mol Biotechnol (2013) 55:54-62 [Non-Patent Document 4] Peng et al., FEBS J (2016) 283:1218-1231 [Non-Patent Document 5] Liu et al., J Biol Chem (2017) 292:5624-5633) [Non-Patent Document 6] Richardson et al., Nature Biotechnol (2016) 34:339-344 [Non-Patent Document 7] Kocher et al., Mol Ther (2017) 25:2585-2598 [Non-Patent Document 8] Gaudelli et al., Nature (2017) 551:464-471 [Non-Patent Document 9] Rees et al., Nature Comm (2017) 8:15790 [Non-Patent Document 10] Billon et al., Mol Cell (2017) 67:1068-1079 [Non-Patent Document 11] Zafra et al., Nat Biotechnol (2018) 36:888-893 [Non-Patent Document 12] Hess et al., Mol Cell (2017) 68:26-43 [Non-Patent Document 13] Eid et al., Biochem J (2018) 475:1955-1964 [Non-Patent Document 14] Komor et al., ACS Chem Biol (2018) 13:383-388 [Summary of the Invention] [Problem to be solved by the invention]

[0006] Because many genetic diseases can be attributed to the change of specific nucleotides at specific positions in genome (e.g., C to T change in specific codons of disease-related genes), base editing can serve as a promising therapeutic approach for treating genetic disorders based on single nucleotide variants.However, despite the improvements over traditional CRISPR / Cas9 editing, base editing efficiency remains low to moderate, and in addition, it suffers from genome-wide mismatches.Therefore, there is still a need in the art for improved base editing systems with higher efficiency.

[0007] Various publications are cited herein, the disclosures of which in their entireties are incorporated herein by reference. [Means for solving the problem]

[0008] In some embodiments, the disclosure provides a method of introducing site-specific mutations in a target polynucleotide in a target cell in a cell population, the method comprising: (a) introducing into the cell population: (i) a base editing enzyme; (ii) a first guide polynucleotide that (1) hybridizes to a gene encoding a cytotoxic agent (CA) receptor and (2) forms a first complex with the base editing enzyme, where the base editing enzyme of the first complex provides a mutation in the gene encoding the CA receptor, where the mutation in the gene encoding the CA receptor forms a CA-resistant cell in the cell population; and (iii) a second guide polynucleotide that (1) hybridizes to the target polynucleotide and (2) forms a second complex with the base editing enzyme, where the base editing enzyme of the second complex provides a mutation in the target polynucleotide; (b) contacting the cell population with a CA; and (c) enriching for target cells comprising the mutation in the target polynucleotide by selecting CA-resistant cells from the cell population.

[0009] In some embodiments, the disclosure provides a method of determining efficacy of a base editing enzyme in a cell population, the method comprising: (a) introducing into the cell population: (i) a base editing enzyme; (ii) a first guide polynucleotide that (1) hybridizes to a gene encoding a cytotoxic agent (CA) receptor and (2) forms a first complex with the base editing enzyme, where the base editing enzyme of the first complex introduces a mutation in the gene encoding the CA receptor, where the mutation in the gene encoding the CA receptor forms CA-resistant cells in the cell population; and (iii) a second guide polynucleotide that (1) hybridizes to a target polynucleotide and (2) forms a second complex with the base editing enzyme, where the base editing enzyme of the second complex introduces a mutation in the target polynucleotide; (b) contacting the cell population with a CA to isolate CA-resistant cells; and (c) determining the ratio of CA-resistant cells to the total cell population to determine the efficacy of the base editing enzyme.

[0010] In some embodiments, the base editing enzyme comprises a DNA targeting domain and a DNA editing domain.

[0011] In some embodiments, the DNA targeting domain comprises Cas9. In some embodiments, Cas9 comprises a mutation in the catalytic domain. In some embodiments, the base editing enzyme comprises a catalytically inactive Cas9 and a DNA editing domain. In some embodiments, the base editing enzyme comprises a Cas9 (nCas9) capable of generating single-stranded DNA breaks, and a DNA editing domain. In some embodiments, nCas9 comprises a mutation relative to wild-type Cas9 at amino acid residue D10 or H840 (numbering is relative to SEQ ID NO: 3). In some embodiments, Cas9 is at least 90% identical to SEQ ID NO: 3 or 4.

[0012] In some embodiments, the DNA editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) deaminase, activation-induced cytidine deaminase (AID), ACF1 / ASE deaminase, ADAT deaminase, or ADAR deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is APOBEC1.

[0013] In some embodiments, the base editing enzyme further comprises a DNA glycosylase inhibitor domain. In some embodiments, the DNA glycosylase is uracil DNA glycosylase inhibitor (UGI). In some embodiments, the base editing enzyme comprises nCas9 and cytidine deaminase. In some embodiments, the base editing enzyme comprises nCas9 and adenosine deaminase. In some embodiments, the base editing enzyme comprises a polypeptide sequence at least 90% identical to SEQ ID NO:6. In some embodiments, the base editing enzyme is BE3.

[0014] In some embodiments, the first and / or second guide polynucleotide is an RNA polynucleotide. In some embodiments, the first and / or second guide polynucleotide further comprises a tracrRNA sequence.

[0015] In some embodiments, the cell population is human cells.

[0016] In some embodiments, the mutation in the gene encoding the CA receptor is a cytidine (C) to thymine (T) point mutation. In some embodiments, the mutation in the gene encoding the CA receptor is an adenine (A) to guanine (G) point mutation.

[0017] In some embodiments, the CA is diphtheria toxin. In some embodiments, the cytotoxic agent (CA) receptor is a receptor for diphtheria toxin. In some embodiments, the CA receptor is heparin-binding EGF-like growth factor (HB-EGF). In some embodiments, the HB-EGF comprises the polypeptide sequence of SEQ ID NO:8.

[0018] In some embodiments, the base editing enzyme of the first complex provides a mutation at one or more of amino acids 107-148 of HB-EGF. In some embodiments, the base editing enzyme of the first complex provides a mutation at one or more of amino acids 138-144 of HB-EGF. In some embodiments, the base editing enzyme of the first complex provides a mutation at amino acid 141 of HB-EGF. In some embodiments, the base editing enzyme of the first complex provides a mutation from GLU141 to LYS141 in the amino acid sequence of HB-EGF.

[0019] In some embodiments, the base editing enzyme of the first complex provides a mutation in a region of HB-EGF that binds to diphtheria toxin. In some embodiments, the base editing enzyme of the first complex provides a mutation in HB-EGF that renders the target cell resistant to diphtheria toxin. In some embodiments, the mutation in the target polynucleotide is a point mutation of cytidine (C) to thymine (T) in the target polynucleotide. In some embodiments, the mutation in the target polynucleotide is a point mutation of adenine (A) to guanine (G) in the target polynucleotide.

[0020] In some embodiments, the base editing enzyme is introduced into the cell population as a polynucleotide encoding the base editing enzyme. In some embodiments, the polynucleotide encoding the base editing enzyme, the (ii) first guide polynucleotide, and the (iii) second guide polynucleotide are on a single vector. In some embodiments, the polynucleotide encoding the base editing enzyme, the (ii) first guide polynucleotide, and the (iii) second guide polynucleotide are on one or more vectors. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adenovirus, a lentivirus, or an adeno-associated virus.

[0021] In some embodiments, the disclosure provides a method of providing biallelic integration of a sequence of interest (SOI) into a toxin sensitivity gene (TSG) locus in the genome of a cell, the method comprising: (a) introducing into a cell population: (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to the TSG locus; and (iii) a donor polynucleotide comprising: (1) a 5' homology arm, a 3' homology arm, and a mutation in the native coding sequence of TSG, where the mutation confers resistance to the toxin; and (2) an SOI; wherein introduction of (i), (ii), and (iii) results in integration of the donor polynucleotide into the TSG locus; (b) contacting the cell population with the toxin; and (c) selecting one or more cells that are resistant to the toxin, wherein the one or more cells that are resistant to the toxin comprise a biallelic integration of the SOI.

[0022] In some embodiments, the donor polynucleotide is integrated by homology directed repair (HDR). In some embodiments, the donor polynucleotide is integrated by non-homologous end joining (NHEJ).

[0023] In some embodiments, the TSG locus comprises an intron and an exon. In some embodiments, the donor polynucleotide further comprises a splicing acceptor sequence. In some embodiments, the nuclease capable of generating a double-stranded break generates a break in an intron. In some embodiments, the mutation in the native coding sequence of TSG is in an exon of the TSG locus.

[0024] In some embodiments, the disclosure provides a method of integrating a sequence of interest (SOI) into a target locus in the genome of a cell, comprising: (a) administering to a cell population: (i) a nuclease capable of generating a double stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to a toxin sensitivity gene (TSG) locus in the genome of the cell, where TSG is an essential gene; and (iii) (1) a functional TSG gene comprising a mutation in the native coding sequence of TSG, where the mutation confers resistance to a toxin; and (2) The present invention provides a method for the preparation of a method for the preparation of a cell population comprising: (a) introducing a donor polynucleotide comprising an SOI and (b) a sequence for genomic integration at a target locus, wherein introduction of (i), (ii), and (iii) results in inactivation of a TSG in the genome of the cell by a nuclease and integration of the donor polynucleotide at the target locus; (b) contacting the cell population with a toxin; and (c) selecting one or more cells that are resistant to the toxin, wherein the one or more cells that are resistant to the toxin comprise an SOI integrated into the target locus.

[0025] In some embodiments, sequences for genomic integration are obtained from a transposon or retroviral vector.

[0026] In some embodiments, the functional TSG of the donor polynucleotide or episomal vector is resistant to inactivation by nucleases. In some embodiments, a mutation in the native coding sequence of the TSG removes a protospacer adjacent motif from the native coding sequence. In some embodiments, the guide polynucleotide cannot hybridize to the functional TSG of the donor polynucleotide or episomal vector.

[0027] In some embodiments, the nuclease capable of generating a double-stranded break is Cas9. In some embodiments, the Cas9 is capable of generating sticky ends. In some embodiments, the Cas9 comprises the polypeptide sequence of SEQ ID NO: 3 or 4.

[0028] In some embodiments, the guide polynucleotide is an RNA polynucleotide. In some embodiments, the guide polynucleotide further comprises a tracrRNA sequence.

[0029] In some embodiments, the donor polynucleotide is a vector. In some embodiments, the mutation in the native coding sequence of TSG is a substitution mutation, an insertion, or a deletion. In some embodiments, the mutation in the native coding sequence of TSG is a mutation in the toxin-binding region of the protein encoded by TSG. In some embodiments, the TSG locus comprises a gene encoding heparin-binding EGF-like growth factor (HB-EGF). In some embodiments, the TSG encodes HB-EGF (SEQ ID NO: 8).

[0030] In some embodiments, the mutation in the native coding sequence for TSG is a mutation at one or more of amino acids 107-148 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence for TSG is a mutation at one or more of amino acids 138-144 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence for TSG is a mutation at amino acid 141 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence for TSG is a mutation from GLU141 to LYS141 of HB-EGF (SEQ ID NO:8).

[0031] In some embodiments, the toxin is diphtheria toxin. In some embodiments, a mutation in the native coding sequence of TSG renders the cell resistant to diphtheria toxin. In some embodiments, the toxin is an antibody-drug conjugate and TSG encodes a receptor for the antibody-drug conjugate.

[0032] In some embodiments, the disclosure provides a method of providing resistance to diphtheria toxin in a human cell, the method comprising introducing into the cell (i) a base editing enzyme and (ii) a guide polynucleotide that targets a heparin-binding EGF-like growth factor (HB-EGF) receptor in the human cell, wherein the base editing enzyme forms a complex with the guide polynucleotide, and the base editing enzyme is targeted to HB-EGF and provides a site-specific mutation in HB-EGF to provide diphtheria toxin resistance in the human cell.

[0033] In some embodiments, the base editing enzyme comprises a DNA targeting domain and a DNA editing domain.

[0034] In some embodiments, the DNA targeting domain comprises Cas9. In some embodiments, Cas9 comprises a mutation in the catalytic domain. In some embodiments, the base editing enzyme comprises a catalytically inactive Cas9 and a DNA editing domain. In some embodiments, the base editing enzyme comprises a Cas9 (nCas9) capable of generating single-stranded DNA breaks, and a DNA editing domain. In some embodiments, nCas9 comprises a mutation relative to wild-type Cas9 at amino acid residue D10 or H840 (numbering is relative to SEQ ID NO: 3). In some embodiments, Cas9 is at least 90% identical to SEQ ID NO: 3 or 4.

[0035] In some embodiments, the DNA editing domain comprises a deaminase. In some embodiments, the deaminase is selected from cytidine deaminase and adenosine deaminase. In some embodiments, the deaminase is cytidine deaminase. In some embodiments, the deaminase is adenosine deaminase. In some embodiments, the deaminase is selected from apolipoprotein B mRNA editing complex (APOBEC) deaminase, activation-induced cytidine deaminase (AID), ACF1 / ASE deaminase, ADAT deaminase, and TadA deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytidine deaminase is APOBEC1. In some embodiments, the base editing enzyme further comprises a DNA glycosylase inhibitor domain. In some embodiments, the DNA glycosylase is uracil DNA glycosylase inhibitor (UGI).

[0036] In some embodiments, the base editing enzyme comprises nCas9 and cytidine deaminase. In some embodiments, the base editing enzyme comprises nCas9 and adenosine deaminase. In some embodiments, the base editing enzyme comprises a polypeptide sequence at least 90% identical to SEQ ID NO:6. In some embodiments, the base editing enzyme is BE3.

[0037] In some embodiments, the guide polynucleotide is an RNA polynucleotide. In some embodiments, the guide polynucleotide further comprises a tracrRNA sequence.

[0038] In some embodiments, the site-specific mutation is in one or more of amino acids 107-148 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is in one or more of amino acids 138-144 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is at amino acid 141 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is a mutation of GLU141 to LYS141 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is in a region of HB-EGF that binds to diphtheria toxin.

[0039] In some embodiments, the disclosure provides a method for integrating and enriching a sequence of interest (SOI) into a target locus in the genome of a cell, comprising: (a) providing to a population of cells (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to an essential gene (ExG) locus in the genome of the cell; and (iii) a functional ExG gene comprising (1) a mutation in the native coding sequence of ExG, wherein the mutation confers resistance to inactivation by the guide polynucleotide. (2) introducing a donor polynucleotide comprising an SOI and (3) a sequence for genomic integration at the target locus, where introduction of (i), (ii), and (iii) results in inactivation of ExG in the genome of the cell by a nuclease and integration of the donor polynucleotide at the target locus; (b) cultivating the cells; and (c) selecting one or more surviving cells, where the one or more surviving cells comprise the integrated SOI at the target locus.

[0040] In some embodiments, the disclosure provides a method of introducing a stable episomal vector into a cell, the method comprising: (a) introducing into a population of cells: (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to an essential gene (ExG) locus in the genome of the cell, where introduction of (i) and (ii) results in inactivation of ExG in the genome of the cell by the nuclease; and (iii) an episomal vector comprising: (1) a functional ExG comprising a mutation in a native coding sequence of ExG, the mutation conferring resistance to inactivation by the nuclease; and (2) an autonomous DNA replication sequence; (b) cultivating the cells; and (c) selecting one or more surviving cells, wherein the one or more surviving cells comprise the episomal vector.

[0041] In some embodiments, the mutation in the native coding sequence of ExG removes a protospacer adjacent motif from the native coding sequence, hi some embodiments, the guide polynucleotide is unable to hybridize to a functional ExG of a donor polynucleotide or episomal vector.

[0042] In some embodiments, the nuclease capable of generating a double-stranded break is Cas9. In some embodiments, the Cas9 is capable of generating sticky ends. In some embodiments, the Cas9 comprises the polypeptide sequence of SEQ ID NO: 3 or 4.

[0043] In some embodiments, the guide polynucleotide is an RNA polynucleotide. In some embodiments, the guide polynucleotide further comprises a tracrRNA sequence.

[0044] In some embodiments, the donor polynucleotide is a vector. In some embodiments, the mutation in the native coding sequence of ExG is a substitution mutation, an insertion, or a deletion.

[0045] In some embodiments, the sequences for genomic integration are obtained from a transposon or retroviral vector, hi some embodiments, the episomal vector is an artificial chromosome or a plasmid.

[0046] In some embodiments, two or more guide polynucleotides are introduced into a cell population, each guide polynucleotide forms a complex with a nuclease, and each guide polynucleotide hybridizes to a different region of ExG.

[0047] In some embodiments, the method further comprises introducing the nuclease of (a)(i) and the guide polynucleotide of (a)(ii) into the surviving cells to enrich for surviving cells that contain an SOI integrated into the target locus. In some embodiments, the method further comprises introducing the nuclease of (a)(i) and the guide polynucleotide of (a)(ii) into the surviving cells to enrich for surviving cells that contain an episomal vector. In some embodiments, the nuclease of (a)(i) and the guide polynucleotide of (a)(ii) are introduced into the surviving cells for multiple rounds of enrichment. [Brief description of the drawings]

[0048] [Figure 1A] 1 shows an exemplary cell having a target site and a selection site that are subjected to base editing. Without a selection strategy, only a small percentage of the resulting cell population has the desired "edited" site. With the co-targeting and selection strategy provided herein, the majority of the resulting cell population has the desired "edited" site. [Figure 1B] 1 shows the selection of guide RNAs for targeting HB-EGF by tiling the EGF-like domain of HB-EGF and determining guide RNAs that confer diphtheria toxin resistance. [Figure 1C]Comparison of PCSK9 and BFP editing efficiencies in various cell lines with (control) and without (enriched) diphtheria toxin selection strategy. The cell population with edited PCSK9 or BFP was significantly increased after diphtheria toxin selection. [Diagram 2] BE3 base editor includes nCas9, APOBEC1, and UGI. BE3 can form a complex with targeting gRNA and selecting gRNA. Utilizing both targeting and selecting gRNA results in enrichment of cells with edited targets. [Figure 3A] Slonczewski, JL and Foster, JW, "Chapter 25. Microbial Pathogenesis." Microbiology: An Evolving Science. New York: WW Norton, 2011. Figure 3A shows the mechanism by which diphtheria toxin causes cell death. [Figure 3B] It is described by Mitamura et al., J Biol Chem 270:1015-1019 (1995). Figure 3B is a sequence alignment of the polypeptide sequences of human (hHB-EGF) and mouse (mHB-EGF) HB-EGF proteins. [Figure 4A-4B] We demonstrate the selection of guide RNAs for targeting HB-EGF in HEK293 and HCT116 cells, respectively, by tiling the EGF-like domain of HB-EGF and determining which guide RNAs confer diphtheria toxin resistance. [Figure 4C] Various gRNA designs are shown in Figures 4A and 4B. [Figure 5A] The sequence of gRNA16 (underlined) is shown. [Figure 5B-5C] Shown are editing efficiencies at three different positions in HB-EGF using gRNA16 in HCT116 and HEK293 cells, respectively. [Figure 5D]Figure 1 shows the amino acid mutation pattern of all surviving HEK293 cells upon diphtheria toxin selection. The mutation occurring in the highest percentage of cells (44.13%) codes for only one amino acid change, a glutamate to lysine substitution at position 141. [Figure 6] The structure of HB-EGF is described by Louie et al., Molecular Cell 1(1):67-78 (1997) and shows the E141 residue targeted by gRNA16, shown in FIG. [Figure 7A-7B]

[0036] Figure 1 shows editing efficiency at PCSK9 target sites to generate stop codons with (enriched) and without (control) diphtheria selection in HCT116 and HEK293 cells, respectively. Editing efficiency was increased with diphtheria selection. [Figure 7C] The sequence of the gRNA targeting pCKS9 is shown (underlined). [Figure 7D] Figure 1 shows editing efficiency at DPM2, EGFR, EMX1, and Yas85 target sites to generate stop codons or introduce SNPs, respectively, with (enriched) and without (control) diphtheria selection in HEK293 cells. Editing efficiency was increased with diphtheria selection. [Figure 7E] The sequences of gRNAs targeting DPM2, EGFR, EMX1, and Yas85 are shown. [Figure 8A] The percentage of indels generated at the PCSK9 target site in HEK293 and HCT116 cells with (control) and without (enriched) diphtheria toxin selection is shown. The sequence of the gRNA is the same as that described in Figure 7C. [Figure 8B] The percentage of indels generated at DPM2, EMX1, and Yas85 target sites in HEK293 cells with (control) and without (enriched) diphtheria toxin selection is shown. The sequences of the gRNAs are shown in FIG 7E. The percentage of indels (editing efficiency) increased dramatically with diphtheria toxin selection. [Figure 9A]Illustrated is an embodiment of the method provided herein. A CRISPR-Cas9 complex that targets the diphtheria toxin receptor (DTR) and the gene of interest (GOI) to be edited is introduced into a cell, and the cell expresses the DTR on the cell surface. The cell is then exposed to diphtheria toxin (DTA). Cells that have been successfully introduced with the CRISPR-Cas9 complex have the edited DTR and the desired edited GOI (indicated by an asterisk). These cells do not express DTR and survive DTA treatment. Cells that have not been edited express DTR and die from DTA treatment. [Figure 9B] We present mice with humanized livers that are sensitive to diphtheria toxin that can then be edited and enriched using the selection methods provided herein. [Figure 10A] An exemplary method for biallelic integration of gene of interest (GOI) is shown. In Fig. 10A, wild-type HB-EGF is cleaved by CRISPR-Cas9 complex at the position of intron. HDR template containing splicing acceptor sequence, HB-EGF with diphtheria toxin resistance mutation, and GOI is also introduced. Diphtheria toxin selection results in cells with diphtheria toxin resistance mutation and GOI. [Figure 10B-10C] Figure 10 shows the results of GOI insertion (knock-in) after diphtheria toxin selection. T2A self-cleaving peptide (T2A) carrying mCherry was tested as the GOI. Cells with successful insertion translate mCherry together with the mutant HB-EGF gene, and the cells show mCherry fluorescence. After diphtheria toxin selection, nearly all cells transfected with Cas9, gRNA SaW10, and mCherry HDR template are mCherry positive (Figure 10B), and mCherry expression is uniform throughout the population (Figure 10C). [Fig. 10D-10F]Figure 10A shows the strategy and PCR analysis results of GOI knock-in cells generated by the method described. Figure 10D shows the PCR analysis strategy. PCR1 amplifies the junction region, the forward primer (PCR1_F primer) binds to a sequence in the genome, and the reverse primer (PCR1_R primer) binds to a sequence in the GOI. Only cells with integrated GOI will show a positive band as shown in Figure 10E. PCR2 amplifies the insertion region, the forward primer (PCR2_F primer) binds to a sequence in the 5' end of the insertion, and the reverse primer (PCR2_R primer) binds to a sequence in the 3' end of the insertion. Amplification will only occur if all alleles in the cell have successfully integrated GOI, and the amplification product will show as a single integrant band, as shown in Figure 10F. If any wild type alleles are present, a WT band will be shown, as shown in Figure 10F. Figure 10E shows that insertion was successfully achieved by this method, and Figure 10F shows that there is no wild type allele in the tested cells, indicating biallelic integration. "Condition 1", "Condition 2", and "Condition 3" correspond to different weight ratios of Cas9 plasmid, gRNA plasmid, and knock-in plasmid as described in the table. "Neg" corresponds to negative control 1 as described in Table 2. [Figure 11] Grawunder and Barth (Eds.), Next Generation Antibody Drug Conjugates (ADCs) and Immunotoxins, Springer, 2017; doi:10.1007 / 978-3-319-46877-8. Figure 11 shows an example of an antibody-drug conjugate (ADC) described herein. In an embodiment of the methods provided herein, the ADC is a cytotoxic agent and the receptor for the antibody of the ADC is a receptor. [Figure 12]An exemplary method is shown for the selection of cells with a vector containing a gene of interest (GOI). The CRISPR-Cas9 complex targets the diphtheria toxin receptor (DTR) and generates a knockout of the DTR, which leads to cell death. A vector carrying a DTR (denoted DTR*) that is resistant to the toxin and resistant to Cas9 cleavage, and a GOI, is also introduced into the cells. Selection by diphtheria toxin results in cell death of cells that do not have the edited DTR or vector. Surviving cells carrying the edited genome DTR and a vector containing DTR* and a GOI. The vector may be an episomal vector or may be integrated as a plasmid, transposon, or retroviral vector. [Figure 13] An exemplary method is shown for the selection of cells with a vector containing a gene of interest (GOI). The CRISPR-Cas9 complex targets an essential gene (ExG) and generates a knockout of the ExG, which leads to cell death. A vector containing an ExG (denoted as ExG*) that is resistant to Cas9 cleavage and a GOI is also introduced into the cell. Surviving cells have the edited genome ExG, as well as the vector containing ExG* and the GOI. The vector may be an episomal vector or may be integrated as a plasmid, transposon, or retroviral vector. [Figure 14] Figures 14-22 show maps of the plasmids described in the Examples. Figure 14 shows the plasmid expressing the BE3 base editing enzyme used in Example 3. [Figure 15] 1 shows a plasmid expressing Cas9 used in Example 3. [Figure 16] 1 shows the plasmid expressing the control gRNA used in Example 3. [Figure 17] 1 shows a plasmid expressing gRNA for DPM2 used in Example 3. [Figure 18] 1 shows a plasmid expressing gRNA for EMX1 used in Example 3. [Figure 19] 1 shows a plasmid expressing gRNA for PCSK9 used in Example 3. [Figure 20] 1 shows a plasmid expressing gRNA for SaW10 used in Example 4. [Figure 21] 1 shows a plasmid expressing gRNA for HB-EGF gRNA16 used in Example 3. [Figure 22] 1 shows a donor plasmid for inserting mCherry into a target site used in Example 4. [Figures 23A-23O] A list of essential genes described herein and in Hart et al., Cell 163:1515-1526 (2015) is provided along with the gene accession number for each gene. [Figures 24A-24C] For Example 6. Figure 24A shows a schematic diagram of the sgRNA sites targeted by CBE3 or ABE7.10 to screen for DT resistance mutations. cDNA and hHBEGF show the DNA sequence encoding the EGF-like domain of human HBEGF protein and the corresponding sequence of its amino acids, respectively. mHBEGF shows the aligned amino acid sequence of mouse HBEGF homolog. Matched amino acids in mHBEGF are shown as dots, and non-matched ones are annotated. The position of the amino acid in the human HBEGF protein is shown under mHBEGF. The highlighted sgRNAs were selected to introduce resistance mutations with CBE3 and ABE7.10, respectively. Figure 24B shows the cell viability after DT selection for each combination of base editor and sgRNA. HEK293 cells were transfected with CBE3 or ABE7.10 together with each individual sgRNA, followed by DT treatment. The cell viability of regenerated cells was quantified with AlarmarBlue assay. Figure 24C shows the frequency of resistance alleles in DT-resistant cells after CBE or ABE editing. HEK293 cells were first transfected with either a plasmid encoding CBE and sgRNA10 or a plasmid encoding ABE and sgRNA5, followed by selection with DT, which began 72 hours after transfection. Surviving cells were harvested and analyzed by NGS. The frequency of each allele was analyzed according to the Komor method. Values ​​represent the mean (n=3) of independent biological replicates. [Fig. 25A-25D] Referring to Example 6. Figure 25A shows an alignment of HBEGF homologs from different species. Figure 25B shows the HBEGF protein structure with resistant amino acid substitutions highlighted. The "top" highlighted amino acids are resistant substitutions introduced by the CBE3 / sgRNA10 pair, and the "bottom" highlighted amino acids are resistant substitutions introduced by the ABE7.10 / sgRNA5 pair. Figure 25C shows the indel frequency observed in DT-resistant populations generated using the CBE3 / sgRNA10 pair or the ABE7.10 / sgRNA5 pair. Figure 25D shows cell growth curves of HEK293 wild-type cells (HEK293wt) and DT-resistant cells generated by CBE3 / sgRNA10 (HEK293 CBE3 / sgRNA10), ABE7.10 / sgRNA5 (HEK293 ABE7.10 / sgRNA5), and pHMEJ Xential (HEK293 Xential), respectively. Cell proliferation was measured in 96-well plates and quantified by the IncuCyte S3 Live Cell Analysis System (Essen BioScience). [Figures 26A-26E]Related to Example 7. Figure 26A shows a schematic of the DT-HBEGF co-selection strategy. Figure 26B shows the results of co-selection of cytidine base editing events. HEK293 cells were co-transfected with CBE3, sgRNA10, and sgRNA targeting a second genomic locus and grown with (enriched) or without (non-enriched) DT selection, which began 72 hours after transfection. Genomic DNA was harvested when cells were confluent, and the rate of CT conversion was analyzed by NGS. Figure 26C shows the results of CBE co-selection in different cell lines. CBE3 / sgRNA targeting PCSK9, CBE3 / sgRNA targeting PCSK9, and CBE3 / sgRNA targeting BFP were transfected into HCT116, HEK293, and PC9-BFP cells, respectively. Genomic DNA was extracted from cells that were selected or not selected with DT (20 ng / mL) and analyzed by Amplicon-Seq. Figure 26D shows the results of co-selection of adenosine base editing events. HEK293 cells were transfected with ABE7.10, sgRNA5, and an sgRNA targeting a second genomic locus and grown with (enriched) or without (non-enriched) DT selection, starting 72 hours after transfection and continuing until confluence. Genomic DNA was harvested from these cells and the rate of AG conversion was analyzed by NGS. Figure 26E shows the results of co-selection with SpCas9 editing events. HEK293 cells were co-transfected with SpCas9, sgRNA10, and an sgRNA targeting a second genomic locus and grown with (enriched) or without (non-enriched) DT selection, starting 72 hours after transfection and continuing until confluence. Genomic DNA was harvested from these cells and indel frequencies were analyzed by NGS. Values ​​and error bars reflect the mean ± sd of n = 3 independent biological replicates. Relative fold changes are indicated in the graphs. *P < 0.05, **P < 0.01, ***P < 0.001, Student's paired t-test. [Figures 27A-27E]For Example 8. Figure 27A shows Western blot analysis of p44 / 42MAPK and Phospho-p44 / 42MAPK in cells treated with wild-type HBEGF and HBEGFE141K. Phosphorylation of p44 / 42MAPK represents one major downstream signaling of EGFR activation. Values ​​and error bars reflect the mean ± sd of n = 3 independent biological replicates. Figure 27B shows a schematic diagram of the knock-in enrichment strategy. Figure 27C shows the knock-in efficiency results of various templates and their corresponding designs. HEK293 cells were co-transfected with SpCas9, sgRNAIn3, and each repair template, and then grown with (enriched) or without (non-enriched) DT selection, which began 72 hours after transfection. The mCherry / GFP ratio of each sample was analyzed by flow cytometry. Repair templates were provided in the form of plasmids (pHMEJ, pHR, or pNHEJ), double-stranded DNA (dsHDR, dsHMEJ, dsHR2), or single-stranded DNA (ssHR). These templates were designed to integrate into the target site via either homology-mediated end joining (pHMEJ and dsHMEJ), homologous recombination (pHR, dsHR, ssHR, dsHR2), or non-homologous end joining (pNHEJ). Figure 27D shows a comparison of puromycin and DT enriched knock-in populations. The top panel shows the design of the repair template used in the experiment. The puromycin resistance gene and mCherry gene are fused to the mutant HBEGF gene in the repair template, and they are expected to be co-transcribed and co-translated. The bottom left panel shows the mCherry histograms of HEK293 cell populations edited with or without different treatments. HEK239 cells were transfected with SpCas9, sgRNAIn3, and repair template, and then cultivated (non-enriched) or selected with DT (DT enriched) or puromycin (Puro enriched), starting 72 hours after transfection. Negative controls represent cells transfected with a control sgRNA that does not contain a target locus in the human genome, instead of sgRNAIn3. Cells were analyzed by flow cytometry.The bottom right panel shows the corresponding knock-in efficiency and mean fluorescence intensity of each population. Figure 27E shows the results of PCR analysis of each cell population obtained from the experiment summarized in Figures 27C and 27D. The top panel shows the design of the two PCR analyses. PCR1 is designed to confirm the insertion. The forward and reverse primers were designed to bind to the flanking genomic regions and the insertion region, respectively. If the cell contains the correct insertion, the target band will be amplified. PCR2 is designed to detect wild-type cells in the population. The forward and reverse primers were designed to bind to the left and right flanking genomic regions of the insertion site, respectively. The middle panel shows PCR analysis of genomic DNA of cells obtained from the experiment summarized in Figure 27C, containing the pHMEJ template. The bottom panel shows PCR analysis of genomic DNA of cells obtained from the experiment summarized in Figure 27D. In both analyses, the negative control represents cells transfected with a control sgRNA instead of sgRNAIn3. Values ​​and error bars reflect the mean ± sd of n = 3 independent biological replicates. [Fig. 28A-28F]For Example 9. Figure 28A shows the experimental strategy of co-selecting knock-out and knock-in events with precise knock-in at the HBEGF locus. Figure 28B shows the results of SpCas9 indel co-selection in HEK293 cells. Cells were co-transfected with SpCas9, sgRNAIn3, pHMEJ repair template for the HBEGF locus, and sgRNA targeting a second genomic locus. Cells were then grown with (enriched) or without (non-enriched) DT selection, starting 72 hours after transfection and continuing until confluence. Genomic DNA was extracted from harvested cells and analyzed by NGS. Figure 28C shows the results of co-selecting knock-in events at a second locus, HIST2BC, in HEK293 cells. Cells were co-transfected with SpCas9, sgRNA, and repair templates for both the HBEGF and HIST2BC loci. Both pHR and pHMEJ templates were applied. Different ratios of the amount of sgRNA and template at the HBEGF locus to the amount of sgRNA and template at the HIST2BC locus were applied. N / A indicates that the corresponding component was not used. Cells were grown with (enriched) or without (non-enriched) DT selection, starting 72 hours after transfection, and analyzed by flow cytometry. Values ​​and error bars reflect the mean ± sd of n = 3 independent biological replicates. Relative fold changes are shown in the graphs. *P<0.05, **P<0.01, ***P<0.001, Student's paired t-test. Figure 28D shows a representative histogram showing that the Xential surviving population co-selected for the knockout event maintained mCherry expression. Each target sgRNA was co-transfected into HEK293 cells with SpCas9, sgRNAIn3, and pHMEJ targeting the HBEGF locus. Figure 28E shows a representative scatter plot showing that the Xential surviving population co-selected for the knock-in event maintained mCherry expression.pHMEJ and sgRNA targeting the HIST2BC locus were co-transfected with SpCas9, sgRNAIn3, and pHMEJ targeting the HBEGF locus into HEK293 cells at different weight ratios. DT-selected and non-DT-selected cells were analyzed by flow cytometry. Figure 28F shows the results of Xential co-selection of oligo knock-in events. Oligo templates and sgRNA targeting the CD34 locus were transfected or co-transfected with SpCas9, sgRNAIn3, and pHMEJ targeting the HBEGF locus into HEK293 cells, respectively. Genomic DNA was extracted from selected and non-selected cells and analyzed by Amplicon-Seq. [Figures 29A-29D]Referring to Example 10. Figure 29A shows the results of co-selection of CBE editing events. iPSCs were co-transfected with CBE3, sgRNA10, and sgRNA targeting a second genomic locus and grown with (enriched) or without (non-enriched) DT selection, starting 72 hours after transfection and continuing until confluence. Genomic DNA was then extracted from these cells and analyzed by NGS. Figure 29B shows the results of co-selection of ABE editing events. iPSCs were co-transfected with ABE7.10, sgRNA5, and sgRNA targeting a second genomic locus and grown with (enriched) or without (non-enriched) DT selection, starting 72 hours after transfection and continuing until confluence. Genomic DNA was then extracted from these cells and analyzed by NGS. Figure 29C shows the results of enrichment of knock-in events at the HBEGF locus. iPSCs were co-transfected with SpCas9, sgRNAIn3, and pHMEJ template for the HBEGF locus and grown with (enriched) or without (non-enriched) DT selection, starting 72 hours after transfection. The cells were then analyzed by flow cytometry. The left panel shows flow cytometry scatter plots of non-enriched and enriched samples, and the right panel shows the quantitative frequency of knock-in cells. Values ​​and error bars reflect the mean ± sd of n = 3 independent biological replicates. Relative fold changes are indicated in the graphs. *P<0.05, **P<0.01, ***P<0.001, Student's paired t-test. Figure 29D shows the results of PCR analysis of iPSCs with Xential knock-in. PCR analysis was performed as described in Example 9 to distinguish successful knock-in into HBEGF intron 3 (PCR1) from wild-type sequence (PCR2). Genomic DNA from cells obtained in experiment Fig. 29C was used as PCR template. The negative control represents cells transfected with a control sgRNA instead of sgRNAIn3. [Diagram 30]Related to Example 11. Figure 6 shows the results of co-selection of CBE editing events in primary T cells. Total CD4+ primary T cells were isolated from human blood and electroporated with CBE3 protein, synthetic sgRNA10, and synthetic sgRNA targeting a second genomic locus. These primary T cells were then cultivated with (enriched) or without (non-enriched) DT selection, which began 24 hours after electroporation and continued for 9 days. Genomic DNA was then extracted from these cells and analyzed by NGS. Values ​​and error bars reflect the mean ± sd of n = 3 independent biological replicates. Relative fold changes are indicated in the graph. *P<0.05, **P<0.01, ***P<0.001, Student's paired t-test. [Figure 31A-31C] Related to Example 12. Figure 31A shows a schematic of the in vivo co-enrichment experimental design. The applied adenovirus was designed to introduce sgRNA targeting CBE, sgRNA10, and Pcsk9. Upon reaching the experimental endpoint, mice were terminated and genomic DNA from mouse liver was extracted and analyzed by NGS. Figure 31B shows the enrichment results of CBE editing at the HBEGF locus. Figure 31C shows the results of co-selection of CBE editing events at the Pcsk9 locus. Values ​​and error bars reflect the mean ± sd of n = 3 independent biological replicates. Relative fold changes are shown in the graphs. *P<0.05, **P<0.01, Student's paired t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present disclosure provides methods for introducing site-specific mutations into target cells and for determining the efficacy of enzymes capable of introducing site-specific mutations. The present disclosure also provides methods for providing biallelic sequence integration, for integrating a sequence of interest into a locus in the genome of a cell, and for introducing a stable episomal vector into a cell. The present disclosure further provides a method for generating human cells resistant to diphtheria toxin.

[0050] definition As used herein, "a" or "an" may mean one or more. As used herein in the specification and claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more. As used herein, "another" or "further" may mean at least a second or more.

[0051] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being used to determine the value or the variation that exists between test subjects. Typically, the term is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0052] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0053] As used in the specification and claims, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open ended and do not exclude additional unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, system, host cell, expression vector, and / or composition of the disclosure. Furthermore, the disclosed compositions, systems, host cells, and / or vectors can be used to obtain the disclosed methods and proteins.

[0054] Use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the cited defined term is representative of and an embodiment of the present disclosure that is not limited to the specific examples referenced or cited, unless expressly stated otherwise.

[0055] "Nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide sequence", "oligonucleotide" or "polynucleotide" refers to a polymeric compound comprising covalently linked nucleotides. The term "nucleic acid" includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. In some embodiments, the present disclosure provides a polynucleotide encoding any one of the polypeptides disclosed herein, and is directed, for example, to a polynucleotide encoding a Cas protein or a variant thereof.

[0056] "Gene" refers to a collection of nucleotides that encodes a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can act as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0057] A nucleic acid molecule is "hybridizable" or "hybridized" to another nucleic acid molecule, e.g., cDNA, genomic DNA, or RNA, if a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are known and are exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein. The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Stringency conditions can be adjusted to screen moderately similar fragments, e.g., homologous sequences from distantly related organisms, against highly similar fragments, e.g., genes that duplicate functional enzymes from closely related organisms. For preliminary screening of homologous nucleic acids, a T mLow stringency hybridization conditions corresponding to a higher T can be used, such as 5×SSC, 0.1% SDS, 0.25% milk and no formamide; or 30% formamide, 5×SSC, 0.5% SDS. m For example, 40% formamide and 5× or 6× SCC. High stringency hybridization conditions correspond to a maximum T m For example, 50% formamide corresponds to 5x or 6x SCC. Hybridization requires that two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible.

[0058] The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present disclosure also includes isolated nucleic acid fragments that are complementary to the complete sequences disclosed or used herein and nucleic acid sequences that are substantially similar thereto.

[0059] A DNA "coding sequence" is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. A "suitable regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences can include, but are not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, polyadenylation signals and transcription termination sequences are usually located 3' to the coding sequence.

[0060] A "native coding sequence" typically refers to a wild-type sequence in a genome, and can also refer to a sequence that is substantially similar to the wild-type sequence, e.g., having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence similarity with the wild-type sequence.

[0061] "Open reading frame", abbreviated ORF, means a nucleic acid sequence, either DNA, cDNA or RNA, of a length that includes a translation initiation signal or start codon, e.g., ATG or AUG, and a stop codon, and that can potentially be translated into a polypeptide sequence.

[0062] The term "homologous recombination" refers to the insertion of a foreign DNA sequence into another DNA molecule, for example, the insertion of a vector into a chromosome. In some cases, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector typically contains a sufficiently long region of homology with the sequence of the chromosome to allow complementary binding and integration of the vector into the chromosome. A longer region of complementarity and a greater degree of sequence similarity can increase the efficiency of homologous recombination.

[0063] Methods known in the art can be used to propagate polynucleotides according to the disclosure herein. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in large quantities. As described herein, expression vectors that can be used include, but are not limited to, the following vectors or derivatives thereof: human or animal viruses, such as vaccinia virus or adenovirus, insect viruses, such as baculovirus, yeast vectors, bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors.

[0064] As used herein, "operably linked" means that a polynucleotide of interest, such as a polynucleotide encoding a Cas9 protein, is linked to a regulatory element to allow expression of the polynucleotide sequence. In some embodiments, the regulatory element is a promoter. In some embodiments, the polynucleotide of interest is operably linked to a promoter on an expression vector.

[0065] As used herein, "promoter", "promoter sequence" or "promoter region" refers to a DNA regulatory region / sequence that can bind RNA polymerase and participate in initiating transcription of downstream coding or non-coding sequences. In some examples of the present disclosure, the promoter sequence includes the transcription initiation site and extends upstream to include a minimum number of bases or elements used to initiate transcription at a level detectable above background. In some embodiments, the promoter sequence includes the transcription initiation site and a protein binding domain responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" and "CAT" boxes. Various promoters, including inducible promoters, can be used to drive the various vectors of the present disclosure.

[0066] A "vector" is any means of cloning and / or transferring a nucleic acid into a host cell. A vector can be a replicon to which another DNA segment can be attached resulting in replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., that can replicate under its own control. In some embodiments of the present disclosure, the vector is an episomal vector, i.e., a non-integrated extrachromosomal plasmid capable of autonomous replication. In some embodiments, the episomal vector comprises an autonomous DNA replication sequence, i.e., a sequence that allows the vector to replicate, typically comprising an origin of replication (OriP). In some embodiments, the autonomous DNA replication sequence is a scaffold / matrix attachment region (S / MAR). In some embodiments, the autonomous DNA replication sequence is a viral OriP. An episomal vector can be removed or eliminated from a cell population after many cell generations, for example, by asymmetric partitioning. In some embodiments, the episomal vector is a stable episomal vector and remains in the cell, i.e., is not eliminated from the cell. In some embodiments, the episomal vector is an artificial chromosome or a plasmid. In some embodiments, the episomal vector comprises an autonomous DNA replication sequence. Examples of episomal vectors used in genome engineering and gene therapy are derived from the papovaviridae virus family, including Simian Virus 40 (SV40) and BK virus, the Herpesviridae virus family, including Bovine Papilloma Virus 1 (BPV-1), Kaposi's Sarcoma-Associated Herpes Virus (KSHV), and Epstein-Barr Virus (EBV), and the S / MAR region of the human interferon-β gene. In some embodiments, the episomal vector is an artificial chromosome. In some embodiments, the episomal vector is a mini-chromosome.Episomal vectors are further described in, for example, Van Craenenbroeck et al., Eur J Biochem 267:5665-5678 (2000), and Lufino et al., Mol Ther 16(9):1525-1538 (2008).

[0067] The term "vector" includes both viral and non-viral means for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Many vectors known in the art can be used to manipulate nucleic acids, such as to incorporate response elements and promoters into genes. Possible vectors include plasmids or modified viruses, including, for example, bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives, or Bluescript vectors. For example, insertion of a DNA fragment corresponding to a response element and promoter into a suitable vector can be accomplished by ligating the appropriate DNA fragment into a selected vector having complementary sticky ends. Alternatively, the ends of the DNA molecules can be enzymatically modified, or any site can be produced by ligating nucleotide sequences (linkers) into the DNA termini. Such vectors can be modified to contain a selectable marker gene that provides for selection of cells that have incorporated the marker into the cell genome. Such markers allow for identification and / or selection of host cells that have incorporated the marker and express the protein encoded by the marker.

[0068] Viral vectors, especially retroviral vectors, have been used in a wide range of gene delivery applications in cells and live animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, adenovirus adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors. Retroviral vectors have emerged as tools for gene therapy by facilitating genomic insertion of desired sequences. Retroviral genomes (e.g., murine leukemia virus (MLV), feline leukemia virus (FLV), or any virus belonging to the Retroviridae virus family) contain long terminal repeat (LTR) sequences that flank viral genes. Upon viral infection of a host, the LTRs are recognized by integrase, which integrates the viral genome into the host genome. Retroviral vectors for targeted gene insertion do not have any of the viral genes, but instead have the desired sequence inserted between the LTRs. The LTR is recognized by integrase to integrate the desired sequence into the genome of the host cell. Further details regarding retroviral vectors can be found, for example, in Kurian et al., Mol Pathol 53(4):173-176, and Vargas et al., J Transl Med 14:288 (2016).

[0069] Non-viral vectors include, but are not limited to, plasmids, liposomes, charged lipids, DNA-protein complexes, and biopolymers. In addition to the nucleic acid, the vector may also contain one or more regulatory regions and / or selectable markers useful in selecting, measuring, and monitoring the results of nucleic acid transfer (tissues transferred, duration of expression, etc.).

[0070] Transposons and transposable elements may be contained on a vector. Transposons are mobile genetic elements that contain flanking repeat sequences that are recognized by a transposase, which then removes the transposon from its locus in the genome and inserts it into another genomic locus (commonly referred to as a "cut and paste" mechanism). Transposons are configured for genome engineering by flanking the desired sequence to be inserted with repeat sequences that can be recognized by the transposase. The repeat sequences may be collectively referred to as "transposon sequences." In some embodiments, the transposon sequence and the desired sequence to be inserted may be contained on a vector, the transposon sequence may be recognized by a transposase, and the desired sequence may then be integrated into the genome by the transposase. Transposons are described, for example, in Pray, Nature Education 1(1):204, (2008), Vargas et al., J Transl Med 14:288 (2016), and VandenDriessche et al., Blood 114(8):1461-1468 (2009). Non-limiting examples of transposon sequences include sleeping beauty (SB), piggyBac (PB), and Tol2 transposons.

[0071] The vector can be introduced into the desired host cell by known methods, including but not limited to transfection, transduction, cell fusion, and lipofection. The vector can include various regulatory elements, including promoters. In some embodiments, the vector design can be based on the constructs designed by Mali et al., Nature Methods 10:957-63 (2013). In some embodiments, the present disclosure provides an expression vector comprising any of the polynucleotides described herein, such as an expression vector comprising a polynucleotide encoding a Cas protein or variant thereof. In some embodiments, the present disclosure provides an expression vector comprising a polynucleotide encoding a Cas9 protein or variant thereof.

[0072] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell and is usually in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genomic integrating sequences, phage or nucleotide sequences from any source, linear, circular, or supercoiled single- or double-stranded DNA or RNA, in which multiple nucleotide sequences are combined or recombined into a unique construct that can introduce promoter fragments and DNA sequences for selected gene products with appropriate 3' untranslated sequences into a cell.

[0073] As used herein, "transfection" refers to the introduction of an exogenous nucleic acid molecule, e.g., a vector, into a cell. A "transfected" cell includes an exogenous nucleic acid molecule inside the cell, and a "transformed" cell is one in which the exogenous nucleic acid molecule inside the cell induces a phenotypic change in the cell. The transfected nucleic acid molecule can be integrated into the genomic DNA of the host cell and / or can be maintained by the cell extrachromosomally, either transiently or for extended periods of time. A host cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to as a "recombinant," "transformed," or "transgenic" organism. In some embodiments, the disclosure provides a host cell that includes any of the expression vectors described herein, e.g., an expression vector that includes a polynucleotide encoding a Cas protein or variant thereof. In some embodiments, the disclosure provides a host cell that includes an expression vector that includes a polynucleotide encoding a Cas9 protein or variant thereof.

[0074] The term "host cell" refers to a cell into which a recombinant expression vector has been introduced. The term "host cell" refers not only to the cell into which the expression vector has been introduced (the "parent" cell), but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, e.g., due to mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell."

[0075] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having altered peptide backbones.

[0076] The beginning of a protein or polypeptide is known as the "N-terminus" (or amino-terminus, NH2-terminus, N-terminal part, or amine-terminus), which refers to the free amine (-NH2) group of the first amino acid residue of the protein or polypeptide. The end of a protein or polypeptide is known as the "C-terminus" (or carboxy-terminus, carboxyl-terminus, C-terminal part, or COOH-terminus), which refers to the free carboxyl group (-COOH) of the last amino acid residue of the protein or polypeptide.

[0077] As used herein, "amino acid" refers to a compound containing both a carboxyl (-COOH) and an amino (-NH2) group. "Amino acid" refers to both natural and unnatural (i.e., synthetic) amino acids. Natural amino acids with three-letter and one-letter abbreviations include alanine (Ala; A); arginine (Arg, R); asparagine (Asn; N); aspartic acid (Asp; D); cysteine ​​(Cys; C); glutamine (Gln; Q); glutamic acid (Glu; E); glycine (Gly; G); histidine (His; H); isoleucine (Ile; I); leucine (Leu; L); lysine (Lys; K); methionine (Met; M); phenylalanine (Phe; F); proline (Pro; P); serine (Ser; S); threonine (Thr; T); tryptophan (Trp; W); tyrosine (Tyr; Y); and valine (Val; V).

[0078] "Amino acid substitution" refers to a polypeptide or protein that contains one or more substitutions at an amino acid residue of a wild-type or naturally occurring amino acid with an amino acid that is different to the wild-type or naturally occurring amino acid. The substituted amino acid may be a synthetic or naturally occurring amino acid. In some embodiments, the substituted amino acid is a naturally occurring amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. Substitution variants may be described using an abbreviation system. For example, a substitution mutation in which the fifth amino acid residue has been substituted may be abbreviated as "X5Y," where "X" is the wild-type or naturally occurring amino acid to be replaced, "5" is the amino acid residue position within the amino acid sequence of the protein or polypeptide, and "Y" is the substitution or non-wild-type or non-naturally occurring amino acid.

[0079] An "isolated" polypeptide, protein, peptide, or nucleic acid is a molecule that has been removed from its natural environment. It should also be understood that an "isolated" polypeptide, protein, peptide, or nucleic acid can be formulated with an excipient, such as a diluent or auxiliary, and still be considered isolated.

[0080] The term "recombinant" when used in reference to a nucleic acid molecule, peptide, polypeptide, or protein means those of, or resulting from, a new combination of genetic material not known to exist in nature. Recombinant molecules can be produced by any of the well-known techniques available in the field of recombinant technology, including, but not limited to, polymerase chain reaction (PCR), gene splicing (e.g., using restriction endonucleases), and solid phase synthesis of nucleic acid molecules, peptides, or proteins.

[0081] The term "domain" when used in reference to a polypeptide or protein refers to a distinct functional and / or structural unit in a protein. A domain may be responsible for a specific function or interaction that contributes to the overall role of the protein. Domains may exist in various biological contexts. Similar domains can be found in proteins with different functions. Alternatively, domains with low sequence identity (i.e., less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% sequence identity) may have the same function. In some embodiments, the DNA targeting domain is Cas9 or a Cas9 domain. In some embodiments, the Cas9 domain is a RuvC domain. In some embodiments, the Cas9 domain is an HNH domain. In some embodiments, the Cas9 domain is a Rec domain. In some embodiments, the DNA editing domain is a deaminase or a deaminase domain.

[0082] When used in reference to a polypeptide or protein, the term "motif" generally refers to a set of conserved amino acid residues, typically less than 20 amino acids in length, that may be important for the function of the protein. Certain sequence motifs can mediate common functions in various proteins, such as binding or targeting of proteins to specific subcellular locations. Examples of motifs include, but are not limited to, nuclear localization signals, microbody targeting motifs, motifs that prevent or promote secretion, and motifs that promote protein recognition and binding. Motif databases and / or motif search tools are known to those of skill in the art, including, for example, PROSITE (expasy.ch / sprot / prosite.html), Pfam (pfam.wustl.edu), PRINTS (biochem.ucl.ac.uk / bsm / dbbrowser / PRINTS / PRINTS.html), and Minimotif Miner (cse-mnm.engr.uconn.edu:8080 / MNM / SMSSearchServlet).

[0083] As used herein, a "modified" protein refers to a protein that contains one or more modifications to achieve a desired property in the protein. Exemplary modifications include, but are not limited to, insertions, deletions, substitutions, or fusions with another domain or protein. Modified proteins of the present disclosure include modified Cas9 proteins.

[0084] In some embodiments, the engineered protein is generated from a wild-type protein. As used herein, a "wild-type" protein or nucleic acid is a naturally occurring, unmodified protein or nucleic acid. For example, a wild-type Cas9 protein can be isolated from the organism Streptococcus pyogenes. Wild-type is contrasted with a "mutant," which contains one or more modifications in the amino acid and / or nucleotide sequence of the protein or nucleic acid.

[0085] The term "sequence similarity" or "% similarity" as used herein refers to the degree of identity or correspondence between nucleic acid or amino acid sequences. As used herein, "sequence similarity" refers to nucleic acid sequences in which changes in one or more nucleotide bases result in the substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Sequence similarity" also refers to modifications of nucleic acids that do not substantially affect the functional properties of the resulting transcript, such as the deletion or insertion of one or more nucleotide bases. Thus, it is understood that the present disclosure encompasses more than the specific exemplary sequences. Methods for making substitutions of nucleotide bases are known, as are methods for determining retention of biological activity of the encoded product.

[0086] Moreover, those skilled in the art will recognize that similar sequences encompassed by the present disclosure are also defined by their ability to hybridize under stringent conditions with the sequences exemplified herein. Similar nucleic acid sequences of the present disclosure are nucleic acids whose DNA sequences are at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the DNA sequences of the nucleic acids disclosed herein. Similar nucleic acid sequences of the present disclosure are nucleic acids whose DNA sequences are about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 99%, at least about 99% or about 100% identical to the DNA sequences of the nucleic acids disclosed herein.

[0087] As used herein, "sequence similarity" refers to two or more amino acid sequences in which more than about 40% of the amino acids are identical or in which more than about 60% of the amino acids are functionally identical. Functionally identical or functionally similar amino acids have chemically similar side chains. For example, amino acids can be grouped according to functional similarity as follows: Positively charged side chains: Arg, His, Lys; Negatively charged side chains: Asp, Glu; Polar uncharged side chains: Ser, Thr, Asn, Gln; Hydrophobic side chains: Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp; Others: Cys, Gly, Pro.

[0088] In some embodiments, similar amino acid sequences of the present disclosure have amino acids that are at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% identical.

[0089] In some embodiments, similar amino acid sequences of the disclosure have amino acids that are at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% functionally identical. In some embodiments, similar amino acid sequences of the disclosure have amino acids that are about 40%, at least about 40%, at least about 45%, at least about 45%, about 50%, at least about 50%, at least about 55%, at least about 55%, about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% identical.

[0090] In some embodiments, a similar amino acid sequence of the disclosure has amino acids that are about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% functionally identical.

[0091] As used herein, the term "same protein" refers to a protein having a structure or amino acid sequence substantially similar to the reference protein, performing the same biochemical function as the reference protein, which may include a protein that differs from the reference protein by substitution or deletion of one or more amino acids at one or more sites in the amino acid sequence, i.e., deletion of at least about 60%, at least about 60%, at least about 65%, at least about 65%, about 70%, at least about 70%, at least about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% identical amino acids. In one aspect, "same protein" refers to a protein with an amino acid sequence identical to the reference protein.

[0092] Sequence similarity can be determined by sequence alignment using routine methods in the art, such as BLAST, MUSCLE, Clustal (e.g., ClustalW and ClustalX), and T-Coffee (including variants such as M-Coffee, R-Coffee, and Expresso).

[0093] The term "sequence identity" or "% identity" with respect to nucleic acid or amino acid sequences refers to the percentage of residues in the compared sequences that are identical when the sequences are aligned over a defined comparison window. In some embodiments, only certain portions of two or more sequences are aligned to determine sequence identity. In some embodiments, only certain domains of two or more sequences are aligned to determine sequence similarity. The comparison window can be a segment of at least 10 to more than 1000 residues, at least 20 to about 1000 residues, or at least 50 to 500 residues over which sequences can be aligned and compared. Methods of alignment for determining sequence identity are well known and can be performed using publicly available databases, such as BLAST. "Percent identity" or "% identity" when referring to amino acid sequences can be determined by methods known in the art. For example, in some embodiments, the "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc Nat Acad Sci USA 87:2264-2268 (1990), modified as in Karlin and Altschul, Proc Nat Acad Sci USA 90:5873-5877 (1993). Such algorithms are incorporated into BLAST programs, such as the BLAST+ or NBLAST and XBLAST programs described in Altschul et al., Journal of Molecular Biology, 215:403-410 (1990). BLAST protein searches can be performed using a program, such as the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences that are homologous to the protein molecules of the present disclosure. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Research 25(17):3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0094] In some embodiments, a polypeptide or nucleic acid molecule has 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%, at least 90%, 95%, at least 95%, 97%, at least 97%, 98%, at least 98%, 99% or at least 99% or 100% sequence identity to a reference polypeptide or nucleic acid molecule, respectively (or a fragment of a reference polypeptide or nucleic acid molecule). In some embodiments, a polypeptide or nucleic acid molecule has about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% sequence identity to a reference polypeptide or nucleic acid molecule, respectively (or a fragment of the reference polypeptide or nucleic acid molecule).

[0095] As used herein, "base edit" or "base editing" refers to the conversion of one nucleotide base pair to another. For example, base editing can convert cytosine (C) to thymine (T) or adenine (A) to guanine (G). Thus, base editing can swap a CG base pair to an AT base pair in a double-stranded polynucleotide, i.e., base editing generates a point mutation in the polynucleotide. Base editing is typically performed by a base editing enzyme, which in some embodiments includes a DNA targeting domain and a catalytic domain capable of base editing, i.e., a DNA editing domain. In some embodiments, the DNA targeting domain is Cas9, e.g., a catalytically inactive Cas9 (dCas9) or a Cas9 capable of generating single-strand breaks (nCas9). In some embodiments, the DNA editing domain is a deaminase domain. The term "deaminase" refers to an enzyme that catalyzes a deamination reaction.

[0096] Base editing typically occurs via deamination, which refers to the removal of amine groups from molecules such as cytosine or adenosine. Deamination converts cytosine to uracil and adenosine to inosine. Exemplary cytidine deaminases include, for example, apolipoprotein B mRNA editing complex (APOBEC) deaminase, activation-induced cytidine deaminase (AID), and ACF1 / ASE deaminase. Exemplary adenosine deaminases include, for example, ADAR deaminase and ADAT deaminase (e.g., TadA).

[0097] In an exemplary base editing process, the base editing enzyme contains a modified Cas9 domain (nCas9) capable of generating a single-stranded DNA break (i.e., a "nick"), a cytidine deaminase domain, and a uracil DNA-glycosylase inhibitor domain (UGI). nCas9 is directed by a guide RNA to a target polynucleotide containing a "CG" base pair, where cytidine deaminase converts the cytosine in the "CG" to a uracil to generate a "UG" mismatch. nCas9 also generates a nick in the non-edited strand of the target polynucleotide. The UGI inhibits natural cellular repair that converts the newly converted uracil back to a cytosine, and the natural cellular mismatch repair machinery activated by the nicked DNA strand converts the "UG" mismatch to a "UA" mismatch. Further DNA replication and repair converts the uracil to a thymine, completing base editing of the target polynucleotide. An example of a base editing enzyme is BE3, described in Komor et al., Nature 533(7603):420-424 (2016). Further exemplary base editing processes are described, for example, in Eid et al., Biochem J 475:1955-1964 (2018).

[0098] Methods for generating catalytically dead Cas9 domains (dCas9) are known (see, e.g., Jinek et al., Science 337:816-821 (2012); Qi et al., Cell 152(5):1173-1183 (2013)). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9.

[0099] Non-limiting examples of base editing enzymes are described, for example, in U.S. Pat. Nos. 9,068,179, 9,840,699, and 10,167,457, as well as in Eid et al., Biochem J 475(11):1955-1964(2018), Gehrke et al., Nat Biotechnol 36:977-982(2018), Hess et al., Mol Cell 68:26-43(2017), Kim et al., Nat Biotechnol 35:435-437(2017), Komor et al., Nature 533:420-424(2016), Komor et al., Science Adv 3(8):eaao4774(2017), Nishida et al., Science 353:aaf8729(2016), Rees et al., Nat Commun 8:15790(2017), Shimatani et al., Nat Biotechnol 35:441-443(2017).

[0100] "Cytotoxic agent" or "cytotoxin" as used herein typically refers to any agent that causes cell death by impairing or inhibiting one or more essential cellular processes. For example, cytotoxins, such as diphtheria toxin, shiga toxin, and pseudomonas exotoxin, function by impairing or inhibiting ribosomal function, which stops protein synthesis and leads to cell death. Cytotoxins, such as dolastatins, auristatins, and maytansines, target microtubule function, which disrupts cell division and leads to cell death. Cytotoxins, such as duocarmycin or calicheamicin, target DNA directly and kill cells at any point in the cell cycle. In many cases, cytotoxic agents are introduced into cells by binding to cell surface receptors. Cytotoxic agents may be naturally occurring compounds or derivatives thereof, or the cytotoxic agent may be a synthetic molecule or peptide. In one example, the cytotoxic agent may be an antibody-drug conjugate (ADC) that includes a monoclonal antibody (mAb) attached to a biologically active agent using a chemical linker with a labile bond. ADCs combine the specificity of mAbs with the efficacy of drugs for targeted killing of specific cells, e.g., cancer cells. ADCs (also called "immunotoxins") are further described, for example, in Srivastava et al., Biomed Res Ther 2(1):169-183 (2015), and Grawunder and Barth (Eds.), Next Generation Antibody Drug Conjugates (ADCs) and Immunotoxins, Springer, 2017; doi:10.1007 / 978-3-319-46877-8.

[0101] A "biallelic" site, as used herein, is a locus in a genome that contains two observed alleles. Thus, a "biallelic" modification refers to the modification of both alleles in the genome of a mammalian cell. For example, a biallelic mutation means that a mutation is present in both copies (i.e., the maternal and paternal copies) of a particular gene.

[0102] Methods for introducing site-specific mutations and determining their efficacy In some embodiments, the disclosure provides a method of introducing site-specific mutations in a target polynucleotide in a target cell in a cell population, the method comprising: (a) introducing into the cell population: (i) a base editing enzyme; (ii) a first guide polynucleotide that (1) hybridizes to a gene encoding a cytotoxic agent (CA) receptor and (2) forms a first complex with the base editing enzyme, where the base editing enzyme of the first complex provides a mutation in the gene encoding the CA receptor, where the mutation in the gene encoding the CA receptor forms a CA-resistant cell in the cell population; and (iii) a second guide polynucleotide that (1) hybridizes to the target polynucleotide and (2) forms a second complex with the base editing enzyme, where the base editing enzyme of the second complex provides a mutation in the target polynucleotide; (b) contacting the cell population with a CA; and (c) enriching for target cells comprising the mutation in the target polynucleotide by selecting CA-resistant cells from the cell population.

[0103] In some embodiments, the disclosure provides a method of determining efficacy of a base editing enzyme in a cell population, the method comprising: (a) introducing into the cell population: (i) a base editing enzyme; (ii) a first guide polynucleotide that (1) hybridizes to a gene encoding a cytotoxic agent (CA) receptor and (2) forms a first complex with the base editing enzyme, where the base editing enzyme of the first complex introduces a mutation in the gene encoding the CA receptor, where the mutation in the gene encoding the CA receptor forms CA-resistant cells in the cell population; and (iii) a second guide polynucleotide that (1) hybridizes to a target polynucleotide and (2) forms a second complex with the base editing enzyme, where the base editing enzyme of the second complex introduces a mutation in the target polynucleotide; (b) contacting the cell population with a CA to isolate CA-resistant cells; and (c) determining the ratio of CA-resistant cells to the total cell population to determine the efficacy of the base editing enzyme.

[0104] The method of the present disclosure provides an efficient method for introducing single nucleotide mutations (e.g., C:G to T:A) into various cell lines. Conventional limitations of genome engineering and gene editing strategies suffer from the inability to distinguish successfully edited cells from cells that have not undergone editing, for example, because one or more of the editing components may not be properly introduced or expressed in the cell.Therefore, there is a need in the art to increase editing efficiency by selecting and enriching edited cells.

[0105] The present disclosure also provides rapid and accurate methods for determining editing efficacy in a cell population. Such methods can facilitate the determination of whether editing has occurred without requiring extensive sequence analysis of the target cells. The methods can further allow for the evaluation of multiple guide polynucleotides to determine the most effective guide polynucleotide sequence for a particular purpose. The method of the present disclosure is a "co-targeting enrichment" strategy that dramatically improves the editing efficiency of a base editing enzyme. In a "co-targeting enrichment" strategy, two guide polynucleotides are introduced into a cell: a first guide polynucleotide, e.g., a "selection" polynucleotide that guides the base editing enzyme to a "selection" site, and a second guide polynucleotide, e.g., a "target" polynucleotide that guides the base editing enzyme to a "target" site. In some embodiments, successful editing of the "selection" site results in cells that survive under certain selection conditions (e.g., exposure to a cytotoxic agent, high or low temperature, medium lacking one or more nutrients, etc.). Figure 1A illustrates an embodiment of the present disclosure, showing a starting cell population with a "target" and a "selection" site. Under conditions in which no selection is present, only a small percentage of cells have the desired "edited" site. Under "co-targeted HB-EGF + diphtheria toxin selection," a much higher percentage of cells have the desired "edited" target site.

[0106] In some embodiments, successful editing of the "selection" site allows edited cells to be easily separated from non-edited cells based on physical or chemical properties (e.g., a change in cell shape or size, and / or the ability to produce fluorescence, chemiluminescence, etc.). In some embodiments, cells with an edited "selection" site are more likely to also have an edited "target" site (e.g., due to successful introduction and / or expression of one or more of the editing components). Thus, selection of cells with an edited "selection" site enriches for cells with an edited "target" site, increasing editing efficiency.

[0107] "Site-specific mutations" as described herein include single nucleotide substitutions, e.g., conversion of a cytosine to a thymine, or vice versa, or an adenine to a guanine, or vice versa, in a polynucleotide sequence. In some embodiments, the site-specific mutations are generated by a base editing enzyme. In some embodiments, the site-specific mutations occur via deamination, e.g., by a deaminase, of a nucleotide in the target polynucleotide. In some embodiments, the base editing enzyme comprises a deaminase.

[0108] In some embodiments, site-specific mutations in a target polynucleotide result in a change in the polypeptide sequence encoded by the polynucleotide. In some embodiments, site-specific mutations in a target polynucleotide change the expression of a downstream polynucleotide sequence in a cell. For example, the expression of a downstream polynucleotide sequence may be inactivated, so that the sequence is not transcribed and the encoded protein is not produced, or the sequence does not function as the wild-type sequence. For example, a protein or miRNA coding sequence may be inactivated, so that the protein is not produced.

[0109] In some embodiments, the site-specific mutation in the regulatory sequence increases expression of the downstream polynucleotide. In some embodiments, the site-specific mutation inactivates the regulatory sequence such that it no longer functions as a regulatory sequence. Non-limiting examples of regulatory sequences include promoters, transcription terminators, enhancers, and other regulatory elements described herein. In some embodiments, the site-specific mutation results in a "knockout" of the target polynucleotide.

[0110] In some embodiments, the target cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal or human cell. In some embodiments, the target cell is a human cell. In some embodiments, the human cell is a stem cell. The stem cell can be, for example, a pluripotent stem cell, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs). In some embodiments, the human cell is any differentiated form of a cell described herein. In some embodiments, the eukaryotic cell is a cell derived from a primary cell in culture. In some embodiments, the cell is a stem cell or a stem cell line.

[0111] In some embodiments, the eukaryotic cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte, or a non-parenchymal cell.For example, the eukaryotic cell can be an attached metabolic test human hepatocyte, an attached induction test human hepatocyte, an attached QUALYST TRANSPORTER CERTIFIED human hepatocyte, a suspension test human hepatocyte (for example, 10 donor and 20 donor pooled hepatocytes), a human hepatic Kupffer cell, a human hepatic stellate cell, a dog hepatocyte (for example, single and pooled beagle hepatocytes), a mouse hepatocyte (for example, CD-1 and C57BI / 6 hepatocytes), a rat hepatocyte (for example, Sprague-Dawley, Wistar Han, and Wistar hepatocytes), a monkey hepatocyte (for example, Cynomolgus or Rhesus hepatocytes), a cat hepatocyte (for example, domestic shorthair hepatocytes), and a rabbit hepatocyte (for example, New Zealand White hepatocytes).

[0112] In some embodiments, the disclosed methods include introducing a base editing enzyme into a cell population. In some embodiments, the base editing enzyme comprises a DNA targeting domain and a DNA editing domain. In some embodiments, the DNA targeting domain comprises Cas9. In some embodiments, the Cas9 comprises a mutation in the catalytic domain. In some embodiments, the base editing enzyme comprises a catalytically inactive Cas9 and a DNA editing domain. In some embodiments, the base editing enzyme comprises a Cas9 (nCas9) capable of generating single-stranded DNA breaks and a DNA editing domain. In some embodiments, the nCas9 comprises a mutation at amino acid residue D10 or H840 relative to wild-type Cas9 (numbering is relative to SEQ ID NO:3). In some embodiments, the Cas9 comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:3. In some embodiments, Cas9 comprises a polypeptide at least 90% identical to SEQ ID NO: 3. In some embodiments, Cas9 comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 4. In some embodiments, Cas9 comprises a polypeptide at least 90% identical to SEQ ID NO: 4.

[0113] The CRISPR-Cas system is a recently discovered prokaryotic adaptive immune system that has been modified to enable robust and site-specific genome engineering in various organisms and cell lines. In general, the CRISPR-Cas system is a protein-RNA complex that uses an RNA molecule (e.g., guide RNA) as a guide to localize the complex to the target DNA sequence via base pairing of the guide RNA to the target DNA sequence. Typically, Cas9 may also require a short protospacer adjacent motif (PAM) sequence adjacent to the target DNA sequence to bind to DNA. Once the complex with the guide RNA is formed, Cas9 "searches" the target DNA sequence by binding to a sequence that matches the PAM sequence. Once Cas9 properly recognizes the pair of PAM and guide RNA with the target sequence, the Cas9 protein then acts as an endonuclease to cleave the targeted DNA sequence. Cas9 proteins from different bacterial species can recognize different PAM sequences. For example, Cas9 from S. pyogenes (SpCas9) recognizes a PAM sequence of 5'-NGG-3', where N is any nucleotide. Cas9 proteins can also be engineered to recognize a different PAM than wild-type Cas9. See, e.g., Sternberg et al., Nature 507(7490):62-67 (2014), Kleinstiver et al., Nature 523:481-485 (2015), and Hu et al., Nature 556:57-63 (2018).

[0114] Among known Cas proteins, SpCas9 has been the most widely used tool for genome engineering. SpCas9 protein is a large multi-domain protein that contains two different nuclease domains. As used herein, "Cas9" includes any Cas9 protein and variants thereof, including, for example, the codon-optimized variants and modified Cas9s described in U.S. Pat. Nos. 9,944,912, 9,512,446, and 10,093,910, as well as the Cas9 variants of U.S. Provisional Patent Application No. 62 / 728,184, filed September 7, 2018. Point mutations can be introduced into Cas9 to eliminate nuclease activity and obtain a catalytically inactive or dead Cas9 (dCas9) that still retains its ability to bind DNA in a manner programmed by guide RNA. In principle, when fused to another protein or domain, dCas9 can target the protein to virtually any DNA sequence simply by co-expressing the appropriate guide RNA. See, e.g., Mali et al., Nat Methods 10(10):957-963(2013); Horvath et al., Nature 482:331-338(2012); Qi et al., Cell 152(5):1173-1183(2013). In an embodiment, the point mutation comprises a mutation at D10 and H840 positions of wild-type Cas9 (numbering is relative to the amino acid sequence of wild-type SpCas9). In an embodiment, dCas9 comprises a D10A and H840A mutation.

[0115] Wild-type Cas9 proteins may also be modified such that the Cas9 protein has a nickase activity capable of cleaving only one strand of double-stranded DNA, rather than a nuclease activity that generates a double-stranded break. Cas9 nickases (nCas9) are described, for example, in Cho et al., Genome Res 24:132-141 (2013), Ran et al., Cell 154:1380-1389 (2013), and Mali et al., Nat Biotechnol 31:833-838 (2013). In some embodiments, the Cas9 nickase comprises a single amino acid substitution relative to wild-type Cas9. In some embodiments, the single amino acid substitution is at position D10 of Cas9 (numbering is relative to SEQ ID NO:3). In some embodiments, the single amino acid substitution is H10A (numbering is relative to SEQ ID NO:3). In some embodiments, the single amino acid substitution is at position H840 of Cas9 (numbering is relative to SEQ ID NO: 3). In some embodiments, the single amino acid substitution is H840A (numbering is relative to SEQ ID NO: 3).

[0116] In some embodiments, the base editing enzyme comprises a DNA targeting domain and a DNA editing domain. In some embodiments, the DNA editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) deaminase, activation-induced cytidine deaminase (AID), ACF1 / ASE deaminase, ADAT deaminase, or ADAR deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is APOBEC1.

[0117] As described herein, deaminase enzymes catalyze deamination, e.g., the deamination of cytosine or adenosine. One exemplary family of cytosine deaminases is the APOBEC family, which includes 11 proteins that act to initiate mutagenesis in a controlled and beneficial manner (Conticello et al., Genome Biol 9(6):229(2008)). One family member, activation-induced cytidine deaminase (AID), is responsible for antibody mutations by converting cytosine in ssDNA to uracil in a transcription-dependent, strand-biased manner (Reynaud et al., Nat Immunol 4(7):631-638(2003)). APOBEC3 protects human cells from certain HIV-1 strains through deaminases of cytosine in reverse-transcribed viral ssDNA (Bhagwat et al., DNA Repair(Amst)3(1):85-89(2004)). All of these proteins require Zn for catalytic activity. 2+ Coordination motif (His-X-Glu-X 23-26 -Pro-Cys-X 2-4-Cys) and a bound water molecule. The Glu residue of the motif acts to activate the water molecule to zinc hydroxide for nucleophilic attack in the deamination reaction. Each member of the family preferentially deaminates at its own specific "hot spot", ranging from WRC (W is A or T and R is A or G) in hAID to TTC in hAPOBEC3F (Navaratnam et al., Int J Hematol 83(3):195-200 (2006)). The recent crystal structure of the catalytic domain of APOBEC3G reveals a secondary structure composed of a five-stranded β-sheet core flanked by six α-helices that appears to be conserved across the entire family (Holden et al., Nature 456:121-124 (2008)). An active center loop has been shown to be responsible for both ssDNA binding and determining the identity of the "hot spot" (Chelico et al., J Biol Chem 284(41):27761-27765(2009)). Overexpression of these enzymes has been associated with genomic instability and cancer, thus highlighting the importance of sequence-specific targeting (Pham et al., Biochemistry 44(8):2703-2715(2005)).

[0118] Another exemplary suitable type of nucleic acid editing enzyme and domain is adenosine deaminase. Examples of adenosine deaminase include adenosine deaminase acting on tRNA (ADAT) and adenosine deaminase acting on RNA (ADAR) family. Deaminases of the ADAT family include TadA, a tRNA adenosine deaminase that shares sequence similarity with APOBEC enzymes. Deaminases of the ADAR family include ADAR2, which converts adenosine in double-stranded RNA to inosine to enable base editing of RNA. See, for example, Gaudelli et al., Nature 551:464-471 (2017); Cox et al., Science 358:1019-1027 (2017).

[0119] In some embodiments, the base editing enzyme further comprises a DNA glycosylase inhibitor domain. In some embodiments, the DNA glycosylase is a uracil DNA glycosylase inhibitor (UGI). In general, DNA glycosylases, such as uracil DNA glycosylase, are part of the base excision repair pathway, and upon detection of a U:G mismatch (where "U" is generated from deamination of cytosine), perform an error-free repair and convert the U back to the wild-type sequence, effectively "undoing" the base edit. Thus, adding a DNA glycosylase inhibitor (e.g., a uracil DNA glycosylase inhibitor) inhibits the base excision repair pathway and increases base editing efficiency. Non-limiting examples of DNA glycosylases include OGG1, MAG1, and UNG. The DNA glycosylase inhibitor can be a small molecule or a protein. For example, protein inhibitors of uracil DNA glycosylase are described in Mol et al., Cell 82:701-708 (1995), Serrano-Heras et al., J Biol Chem 281:7068-7074 (2006), and New England Biolabs Catalog Nos. M0281S and M0281L (neb.com / products / m0281-uracil-glycosylase-inhibitor-ugi). Small molecule inhibitors of DNA glycosylases are described, for example, in Huang et al., J Am Chem Soc 131(4):1344-1345(2009); Jacobs et al., PLoS One 8(12):e81667(2013); Donley et al., ACS Chem Biol 10(10):2334-2343(2015); Tahara et al., J Am Chem Soc 140(6):2105-2114(2018).

[0120] Thus, in some embodiments, the base editing enzyme of the disclosure comprises a Cas9 capable of producing a single stranded cut and a cytidine deaminase. In some embodiments, the base editing enzyme of the disclosure comprises nCas9 and a cytidine deaminase. In some embodiments, the base editing enzyme of the disclosure comprises a Cas9 capable of producing a single stranded cut and an adenosine deaminase. In some embodiments, the base editing enzyme of the disclosure comprises nCas9 and an adenosine deaminase. In some embodiments, the base editing enzyme is at least 90% identical to SEQ ID NO:6. In some embodiments, the base editing enzyme comprises a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, the base editing enzyme comprises a polypeptide having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the base editing enzyme is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to SEQ ID NO: 5. In some embodiments, the base editing enzyme is BE3.

[0121] In some embodiments, the methods of the disclosure comprise introducing into a cell population a first guide polynucleotide that hybridizes to a gene encoding a cytotoxic agent (CA) receptor and forms a first complex with a base editing enzyme, wherein the base editing enzyme of the first complex provides a mutation in the gene encoding the CA receptor, and the mutation in the gene encoding the CA receptor forms CA-resistant cells in the cell population.

[0122] In some embodiments, the first guide polynucleotide is an RNA molecule. RNA molecules that bind to CRISPR-Cas components and target them to a specific location within a target DNA are referred to herein as "RNA guide polynucleotides," "guide RNAs," "gRNAs," "small guide RNAs," "single guide RNAs," or "sgRNAs," and may also be referred to herein as "DNA-targeting RNAs." The guide polynucleotide may be introduced into the target cell as an isolated molecule, e.g., an RNA molecule, or introduced into the cell using an expression vector that contains a guide polynucleotide, e.g., a DNA encoding an RNA guide polynucleotide. In some embodiments, the guide polynucleotide is between 10 and 150 nucleotides. In some embodiments, the guide polynucleotide is between 20 and 120 nucleotides. In some embodiments, the guide polynucleotide is between 30 and 100 nucleotides. In some embodiments, the guide polynucleotide is between 40 and 80 nucleotides. In some embodiments, the guide polynucleotide is between 50 and 60 nucleotides. In some embodiments, the guide polynucleotide is between 10 and 35 nucleotides. In some embodiments, the guide polynucleotide is between 15 and 30 nucleotides. In some embodiments, the guide polynucleotide is 20-25 nucleotides.

[0123] In some embodiments, an RNA guide polynucleotide comprises at least two nucleotide segments: at least one "DNA-binding segment" and at least one "polypeptide-binding segment." By "segment" is meant a portion, section, or region of a molecule, e.g., a contiguous stretch of nucleotides of a guide polynucleotide molecule. The definition of "segment" is not limited to a particular number of total base pairs, unless specifically defined otherwise.

[0124] In some embodiments, the guide polynucleotide comprises a DNA binding segment. In some embodiments, the DNA binding segment of the guide polynucleotide comprises a nucleotide sequence that is complementary to a specific sequence in the target polynucleotide. In some embodiments, the DNA binding segment of the guide polynucleotide hybridizes with a gene encoding a cytotoxic agent (CA) receptor in the target cell. In some embodiments, the DNA binding segment of the guide polynucleotide hybridizes with a target polynucleotide sequence in the target cell. Target cells, including various types of eukaryotic cells, are described herein.

[0125] In some embodiments, the guide polynucleotide comprises a polypeptide binding segment. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the DNA targeting domain of the base editing enzyme of the disclosure. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the Cas9 of the base editing enzyme. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the dCas9 of the base editing enzyme. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the nCas9 of the base editing enzyme. Various RNA guide polynucleotides that bind to Cas9 proteins are described, for example, in U.S. Patent Application Publication Nos. 2014 / 0068797, 2014 / 0273037, 2014 / 0273226, 2014 / 0295556, 2014 / 0295557, 2014 / 0349405, 2015 / 0045546, 2015 / 0071898, 2015 / 0071899, and 2015 / 0071906.

[0126] In some embodiments, the guide polynucleotide further comprises a tracrRNA. The "tracrRNA" or trans-activated CRISPR-RNA forms an RNA duplex with the pre-crRNA or pre-CRISPR-RNA, which is then cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. In some embodiments, the guide polynucleotide comprises a crRNA / tracrRNA hybrid. In some embodiments, the tracrRNA component of the guide polynucleotide activates the Cas9 protein. In some embodiments, the activation of the Cas9 protein comprises activating the nuclease activity of Cas9. In some embodiments, the activation of the Cas9 protein comprises the Cas9 protein binding to a target polynucleotide sequence.

[0127] In some embodiments, the sequence of the guide polynucleotide is designed to target a base editing enzyme to a specific location in a target polynucleotide sequence. Various tools and programs are available to facilitate the design of such guide polynucleotides (e.g., the Benchling Base Editor Design Guide (benchling.com / editor#create / crispr), and BE-Designer and BE-Analyzer from CRISPR RGEN Tools (see Hwang et al., bioRxiv dx.doi.org / 10.1101 / 373944, first published July 22, 2018)).

[0128] In some embodiments, the DNA-binding segment of the first guide polynucleotide hybridizes to a gene encoding a cytotoxic agent (CA) receptor, and the polypeptide-binding segment of the first guide polynucleotide forms a first complex with the base editing enzyme by binding to the DNA targeting domain of the base editing enzyme. In some embodiments, the DNA-binding segment of the first guide polynucleotide hybridizes to a gene encoding a cytotoxic agent (CA) receptor, and the polypeptide-binding segment of the first guide polynucleotide forms a first complex with the base editing enzyme by binding to the Cas9 of the base editing enzyme. In some embodiments, the DNA-binding segment of the first guide polynucleotide hybridizes to a gene encoding a cytotoxic agent (CA) receptor, and the polypeptide-binding segment of the first guide polynucleotide forms a first complex with the base editing enzyme by binding to the dCas9 of the base editing enzyme. In some embodiments, the DNA-binding segment of the first guide polynucleotide hybridizes to a gene encoding a cytotoxic agent (CA) receptor, and the polypeptide-binding segment of the first guide polynucleotide binds to the base editing enzyme nCas9, thereby forming a first complex with the base editing enzyme.

[0129] In some embodiments, the first complex is targeted to a gene encoding a CA receptor by a first guide polynucleotide, and a base editing enzyme of the first complex introduces a mutation in the gene encoding the CA receptor. In some embodiments, the mutation in the gene encoding the CA receptor is introduced by a base editing domain of the base editing enzyme of the first complex. In some embodiments, the mutation in the gene encoding the CA receptor forms CA resistant cells in the cell population. In some embodiments, the mutation is a point mutation from cytidine (C) to thymine (T). In some embodiments, the mutation is a point mutation from adenine (A) to guanine (G). The specific location of the mutation in the CA receptor can be guided by design of the first guide polynucleotide, for example, using tools such as the Benchling base editor design guide, BE-Designer, and BE-Analyzer described herein. In some embodiments, the first guide polynucleotide is an RNA polynucleotide. In some embodiments, the first guide polynucleotide further comprises a tracrRNA sequence.

[0130] In some embodiments, the CA is a compound that causes or promotes cell death, as described herein. In some embodiments, the CA is a toxin. In some embodiments, the CA is a natural toxin. In some embodiments, the CA is a synthetic toxin. In some embodiments, the CA is a small molecule, peptide, or protein. In some embodiments, the CA is an antibody-drug conjugate. In some embodiments, the CA is a monoclonal antibody attached to a biologically active agent with a chemical linker having a labile bond. In some embodiments, the CA is a biological toxin. In some embodiments, the toxin is produced by cyanobacteria (cyanotoxins), dinoflagellates (dinotoxins), marine animals such as spiders, snakes, scorpions, frogs, jellyfish, venomous fish, corals, or blue-ringed octopus. Examples of toxins include, for example, diphtheria toxin, botulinum toxin, ricin, apitoxin, shiga toxin, Pseudomonas exotoxin, and mycotoxins. In some embodiments, the CA is diphtheria toxin. In some embodiments, the CA is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises an antibody linked to a toxin. In some embodiments, the toxin is a small molecule, an RNase, or a pro-apoptotic protein.

[0131] In some embodiments, the CA is toxic to one organism, such as a human, but not to another organism, such as a mouse. In some embodiments, the CA is toxic to an organism at one stage of its life cycle (e.g., fetal stage), but not to another stage of the organism's life cycle (e.g., adult stage). In some embodiments, the CA is toxic in one organ of an animal, but not to another organ of the same animal. In some embodiments, the CA is toxic to a subject (e.g., a human or animal) at one condition or state (e.g., diseased), but not to the same subject at another condition or state (e.g., healthy). In some embodiments, the CA is toxic to one cell type, but not to another cell type. In some embodiments, the CA is toxic to a cell at one cell state (e.g., differentiated), but not to the same cell at another cell state (e.g., undifferentiated). In some embodiments, the CA is toxic to a cell at one environment (e.g., low temperature), but not to the same cell at another environment (e.g., high temperature). In some embodiments, the toxin is toxic to human cells, but not to mouse cells.

[0132] In some embodiments, the CA receptor is a biological receptor that binds to CA. CA receptors are protein molecules that bind to CA, typically located on the membrane of cells. For example, diphtheria toxin binds to human heparin-binding EGF-like growth factor (HB-EGF). CA receptors may be specific for one CA, or CA receptors may bind to two or more CAs. For example, monosialoganglioside (GM1) can act as a receptor for both cholera toxin and E. coli heat-labile enterotoxin. Or, two or more CA receptors may bind to one CA. For example, botulinum toxins are believed to bind to different receptors on neuronal and epithelial cells. In some embodiments, the CA receptor is a receptor that binds to CA. In some embodiments, the CA receptor is a G protein-coupled receptor. In some embodiments, the CA receptor is a receptor for an antibody, for example, an antibody of an antibody-drug conjugate. In some embodiments, the CA receptor is a receptor for diphtheria toxin. In some embodiments, the CA receptor is HB-EGF.

[0133] In some embodiments, one or more mutations in a polynucleotide encoding a CA receptor protein confer CA resistance. In some embodiments, a mutation in the CA binding region of a CA receptor confer CA resistance. In some embodiments, a charge reversal mutation of an amino acid at or near the CA binding site of a CA receptor confer CA resistance. Charge reversal mutations include, for example, a negatively charged amino acid such as Glu or Asp, substituted with a positively charged amino acid such as Lys or Arg, or vice versa. In some embodiments, a polarity reversal mutation of an amino acid at or near the CA binding site of a CA receptor confer CA resistance. Polarity reversal mutations include, for example, a polar amino acid such as Gln or Asn, substituted with a non-polar amino acid such as Val or Ile, or vice versa. In some embodiments, a relatively small amino acid residue at or near the CA binding site of a CA receptor is replaced with a "bulky" amino acid residue to block the binding pocket and prevent CA binding, thus conferring CA resistance. Small amino acids include, for example, Gly or Ala, although Trp is generally considered to be a bulky amino acid.

[0134] In some embodiments, the one or more mutations in the polynucleotide encoding the CA receptor change one or more codons in the amino acid sequence of the CA receptor. In some embodiments, the one or more mutations in the polynucleotide encoding the CA receptor change a single codon in the amino acid sequence of the CA receptor. In some embodiments, a single nucleotide mutation in the polynucleotide encoding the CA receptor protein confers CA receptor resistance. In some embodiments, the single nucleotide mutation is a point mutation from cytidine (C) to thymine (T) in the polynucleotide sequence encoding the CA receptor. In some embodiments, the single nucleotide mutation is a point mutation from adenine (A) to guanine (G) in the polynucleotide sequence encoding the CA receptor. In some embodiments, the one or more mutations in the CA receptor are provided by a base editing enzyme described herein. The base editing enzyme is specifically targeted to the CA receptor by a DNA targeting domain (e.g., a Cas9 domain), followed by a base editing domain (e.g., a deaminase domain) to provide the mutation in the CA receptor. In some embodiments, the one or more mutations in the CA receptor are provided by a base editing enzyme comprising nCas9 and a cytidine deaminase. In some embodiments, the one or more mutations in the CA receptor are provided by a base editing enzyme comprising nCas9 and adenosine deaminase. In some embodiments, the one or more mutations in the CA receptor are provided by a base editing enzyme comprising a polypeptide having at least 90% sequence identity to SEQ ID NO:6. In some embodiments, the base editing enzyme is BE3.

[0135] In some embodiments, the CA receptor is a receptor for diphtheria toxin. In some embodiments, the diphtheria toxin receptor is human HB-EGF. Unless otherwise indicated, "HB-EGF" as used herein without organism modifier refers to human HB-EGF. HB-EGF protein from other organisms, such as mouse, is specifically described as "mouse HB-EGF".

[0136] Diphtheria toxin, known as "AB" toxin, is a two-component protein that forms a complex with two subunits, typically linked together by a disulfide bridge, with the "A" subunit typically considered to be the "active" portion and the "B" subunit generally being the "binding" portion. Diphtheria toxin is known to bind to the EGF-like domain of HB-EGF, which is widely expressed in various tissues. Figure 3A shows an exemplary mechanism of action of AB diphtheria toxin on its receptor. As shown in Figure 3A, diphtheria subunit B is responsible for binding to the membrane-bound receptor, HB-EGF. Upon binding, diphtheria toxin enters the cell via receptor-mediated endocytosis. Subsequently, catalytic subunit A is cleaved from subunit B via reduction of the disulfide bond between the two subunits, leaving the endocytic vesicle and catalyzing the addition of ADP-ribose to the elongation factor 2 (EF2) of the ribosome. ADP-ribosylation of EF2 stops protein synthesis, resulting in cell death.

[0137] Unlike human HB-EGF, mouse HB-EGF is resistant to binding and therefore mice are resistant to diphtheria toxin. FIG. 3B shows significant differences in the amino acid sequences of human and mouse HB-EGF proteins. Thus, in some embodiments, one or more mutations in a polynucleotide encoding an HB-EGF protein confer diphtheria toxin resistance. In some embodiments, one or more mutations in a polynucleotide encoding an HB-EGF change one or more codons in the amino acid sequence of HB-EGF. In some embodiments, one or more mutations in a polynucleotide encoding an HB-EGF change a single codon in the amino acid sequence of HB-EGF. In some embodiments, a single nucleotide mutation in a polynucleotide encoding an HB-EGF protein confer diphtheria toxin resistance. In some embodiments, the single nucleotide mutation is a point mutation from cytidine (C) to thymine (T) in the polynucleotide sequence encoding HB-EGF. In some embodiments, the single nucleotide mutation is a point mutation from adenine (A) to guanine (G) in the polynucleotide sequence encoding HB-EGF.

[0138] In some embodiments, a mutation in the diphtheria toxin binding region of HB-EGF confers resistance to diphtheria toxin. In some embodiments, a mutation in the EGF-like domain of HB-EGF confers resistance to diphtheria toxin. In some embodiments, a charge reversal mutation of an amino acid at or near the diphtheria toxin binding site of HB-EGF confers resistance to diphtheria toxin. In some embodiments, the charge reversal mutation is a substitution of a negatively charged residue, such as Glu or Asp, with a positively charged residue, such as Lys or Arg. In some embodiments, the charge reversal mutation is a substitution of a positively charged residue, such as Lys or Arg, with a negatively charged residue, such as Glu or Asp. In some embodiments, a polarity reversal mutation of an amino acid at or near the diphtheria toxin binding site of HB-EGF confers resistance to diphtheria toxin. In some embodiments, the polarity reversal mutation is a substitution of a polar amino acid residue, such as Gln or Asn, with a non-polar amino acid residue, such as Ala, Val, or Ile. In some embodiments, the polarity reversal mutation is a substitution of a non-polar amino acid residue, such as Ala, Val, or Ile, for a polar amino acid residue, such as Gln or Asn. In some embodiments, the mutation is a substitution of a relatively small amino acid residue, such as Gly or Ala, for a "bulky" amino acid residue, such as Trp, at or near the diphtheria toxin binding site of HB-EGF. In some embodiments, the mutation of the small residue to the bulky residue blocks the binding pocket, preventing binding of diphtheria toxin, thereby conferring resistance.

[0139] In some embodiments, a mutation in one or more of amino acids 100-160 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 105-150 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 107-148 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 120-145 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 135-143 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 138-144 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) confers diphtheria toxin resistance. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to ARG141. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to HIS141. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to LYS141. In some embodiments, a mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) from GLU141 to LYS141 confers diphtheria toxin resistance.

[0140] In some embodiments, the one or more mutations in HB-EGF are provided by a base editing enzyme described herein. The base editing enzyme is specifically targeted to HB-EGF by a DNA targeting domain (e.g., a Cas9 domain), followed by a base editing domain (e.g., a deaminase domain) to provide the mutations in HB-EGF. In some embodiments, the one or more mutations in HB-EGF are provided by a base editing enzyme comprising nCas9 and a cytidine deaminase. In some embodiments, the one or more mutations in HB-EGF are provided by a base editing enzyme comprising nCas9 and an adenosine deaminase. In some embodiments, the one or more mutations in HB-EGF are provided by a base editing enzyme comprising a polypeptide having at least 90% sequence identity to SEQ ID NO:6. In some embodiments, the base editing enzyme is BE3.

[0141] In some embodiments, the DNA-binding segment of the second guide polynucleotide hybridizes to a target polynucleotide in a target cell, and the polypeptide-binding segment of the second guide polynucleotide forms a second complex with the base editing enzyme by binding to the DNA targeting domain of the base editing enzyme. In some embodiments, the DNA-binding segment of the second guide polynucleotide hybridizes to a target polynucleotide in a target cell, and the polypeptide-binding segment of the second guide polynucleotide forms a second complex with the base editing enzyme by binding to the Cas9 of the base editing enzyme. In some embodiments, the DNA-binding segment of the second guide polynucleotide hybridizes to a target polynucleotide in a target cell, and the polypeptide-binding segment of the second guide polynucleotide forms a second complex with the base editing enzyme by binding to the dCas9 of the base editing enzyme. In some embodiments, the DNA-binding segment of the second guide polynucleotide hybridizes to a target polynucleotide in a target cell, and the polypeptide-binding segment of the second guide polynucleotide forms a second complex with the base editing enzyme by binding to the nCas9 of the base editing enzyme.

[0142] In some embodiments, the second complex is targeted to the target polynucleotide by a second guide polynucleotide, and a base editor of the second complex introduces a mutation in the target polynucleotide. In some embodiments, the mutation in the target polynucleotide is introduced by a base editing domain of the base editor of the second complex. In some embodiments, the mutation in the target polynucleotide is a point mutation from cytidine (C) to thymine (T). In some embodiments, the mutation in the target polynucleotide is a point mutation from adenine (A) to guanine (G). The specific location of the mutation in the target polynucleotide can be guided by design of the second guide polynucleotide, for example, using tools such as the Benchling base editor design guide, BE-Designer, and BE-Analyzer described herein. In some embodiments, the second guide polynucleotide is an RNA polynucleotide. In some embodiments, the second guide polynucleotide further comprises a tracrRNA sequence.

[0143] In some embodiments, a C to T mutation in the target polynucleotide inactivates expression of the target polynucleotide in a target cell. In some embodiments, a A to G mutation in the target polynucleotide inactivates expression of the target polynucleotide in a target cell. In some embodiments, the target polynucleotide encodes a protein or miRNA. In some embodiments, the target polynucleotide is a regulatory sequence and the C to T mutation alters the function of the regulatory sequence. In some embodiments, the target polynucleotide is a regulatory sequence and the A to G mutation alters the function of the regulatory sequence.

[0144] In some embodiments, the base editing enzyme of the present disclosure is introduced into the cell population as a polynucleotide encoding the base editing enzyme. In some embodiments, the first and / or second guide polynucleotide is introduced into the cell population as one or more polynucleotides encoding the first and / or second guide polynucleotide. In some embodiments, the base editing enzyme, the first guide polynucleotide, and the second guide polynucleotide are introduced into the cell population via a vector. In some embodiments, the polynucleotide encoding the base editing enzyme, the first guide polynucleotide, and the second guide polynucleotide are on a single vector. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleotide encoding the base editing enzyme, the first guide polynucleotide, and the second guide polynucleotide are on one or more vectors. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vector is an adenovirus, an adeno-associated virus, or a lentivirus. Viral transduction with adenovirus, adeno-associated virus (AAV), and lentivirus vectors (administration can be local, targeted, or systemic) has been used as a delivery method for in vivo gene therapy. Methods for introducing vectors, such as viral vectors, into cells (e.g., transfection) are described herein.

[0145] In some embodiments, the base editing enzyme, the first guide polynucleotide, and / or the second guide polynucleotide are introduced to the cell population via a delivery particle. In some embodiments, the base editing enzyme, the first guide polynucleotide, and / or the second guide polynucleotide are introduced to the cell population via a vesicle.

[0146] In some embodiments, the potency of the base editing enzyme can be determined by calculating the ratio of CA-resistant cells to the total cell population. In some embodiments, the number of CA-resistant cells can be counted using techniques known in the art, such as, for example, counting with a hemocytometer, measuring absorbance at a specific wavelength (e.g., 580 nm or 600 nm), and / or measuring the fluorescence of a fluorophore to detect the cell population. In some embodiments, the total cell population is determined, and the ratio of CA-resistant cells to the total cell population is calculated by dividing the total cell population by the CA-resistant cells. In some embodiments, the ratio of CA-resistant cells to the total cell population approximates the base editing potency at the target polynucleotide.

[0147] Site-specific integration methods As described herein, HDR-based DNA double-strand break repair can provide site-specific integration, such as biallelic integration, of a sequence of interest (SOI) desired at a target locus. For applications such as correcting genetic variants, gene therapy, and generating transgenic animals, site-specific integration, particularly biallelic integration, of a genetic modification of interest is highly desirable. Unfortunately, due to the low efficiency of HDR-based DNA double-strand break repair, screening and isolating site-specific integration, particularly biallelic integration, is often difficult and cumbersome, and may require expensive and time-consuming sequencing and analysis. The method of the present disclosure applies the "co-targeting enrichment" strategy described herein to generate site-specific integration of a sequence of interest, and provides a simple and efficient method of screening cells with desired integration. In some embodiments, the site-specific integration is biallelic integration.

[0148] In some embodiments, the disclosure includes a method of providing biallelic integration of a sequence of interest (SOI) into a toxin sensitivity gene (TSG) locus in the genome of a cell, the method comprising: (a) introducing into a cell population: (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to the TSG locus; and (iii) a donor polynucleotide comprising: (1) a mutation in the 5' homology arm, the 3' homology arm, and the native coding sequence of TSG, where the mutation confers resistance to the toxin; and (2) an SOI; wherein introduction of (i), (ii), and (iii) results in integration of the donor polynucleotide into the TSG locus; and (b) contacting the cell population with the toxin and selecting one or more cells that are resistant to the toxin, wherein the one or more cells that are resistant to the toxin comprise a biallelic integration of the SOI.

[0149] FIG. 10A shows an embodiment of the method provided herein. In FIG. 10A, the wild-type sequence of HB-EGF is diphtheria toxin sensitive. The solid boxes in the sequence indicate exons, and the double lines represent introns. Cas9 nuclease is targeted to the intron of HB-EGF by the guide polynucleotide of the CRISPR-Cas complex to generate a double-strand break. The HDR template is introduced into a cell with a splicing acceptor sequence to join the exon on the HDR template with the adjacent genomic exons, the diphtheria toxin resistance mutation in the exon immediately preceding the double-strand break, and the gene of interest (GOI). HDR repairs the double-strand break and inserts the splicing acceptor sequence, the diphtheria toxin resistance mutation, and the GOI at the break site. Thus, only cells with biallelic integration of the HDR template (and thus the GOI) are diphtheria toxin resistant, while cells that are monoallelic or not repaired by HDR are toxin sensitive. Thus, cells that survive contact with the toxin harbor biallelic integration of the GOI.

[0150] In some embodiments, the TSG locus encodes HB-EGF and the toxin is diphtheria toxin. In some embodiments, the nuclease capable of generating double-stranded break is Cas9. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the donor polynucleotide is a HDR template. In some embodiments, the SOI is a gene of interest. In some embodiments, the integration of the donor polynucleotide in the TSG locus is biallelic integration.

[0151] In some embodiments, the disclosure provides a method of integrating a sequence of interest (SOI) into a target locus in the genome of a cell, comprising: (a) administering to a cell population (i) a nuclease capable of generating a double stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to a toxin sensitivity gene (TSG) locus in the genome of the cell, where TSG is an essential gene; (iii) (1) a functional TSG gene comprising a mutation in the native coding sequence of TSG, where the mutation confers resistance to a toxin; and (2) a S The present invention provides a method comprising: introducing a donor polynucleotide comprising an OI and (3) a sequence for genomic integration at the target locus, where introduction of (i), (ii), and (iii) results in inactivation of a TSG in the genome of the cell by a nuclease and integration of the donor polynucleotide at the target locus; (b) contacting the cell population with a toxin; and (c) selecting one or more cells that are resistant to the toxin, where the one or more cells that are resistant to the toxin comprise an SOI integrated into the target locus.

[0152] In some embodiments, the disclosure provides a method of introducing a stable episomal vector into a cell, comprising: (a) introducing into a cell population: (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to a toxin sensitivity gene (TSG) locus in the genome of the cell, where introduction of (i) and (ii) results in inactivation of the TSG in the genome of the cell by the nuclease; and (iii) an episomal vector comprising: (1) a functional TSG gene comprising a mutation in the native coding sequence of the TSG, the mutation conferring resistance to the toxin; (2) an SOI; and (3) an autonomous DNA replication sequence; (b) contacting the cell population with the toxin; and (c) selecting one or more cells that are resistant to the toxin, where the one or more cells that are resistant to the toxin comprise the episomal vector. In some embodiments, the TSG is an essential gene.

[0153] In some embodiments, the nuclease capable of generating double-stranded breaks is Cas9. As described herein, Cas9 is a monomeric protein that includes a DNA targeting domain (which interacts with a guide polynucleotide, e.g., a guide RNA) and a nuclease domain (which cleaves a target polynucleotide, e.g., a TSG locus). The Cas9 protein generates a site-specific break in a nucleic acid. In some embodiments, the Cas9 protein generates a site-specific double-stranded break in DNA. The ability of Cas9 to target a specific sequence in a nucleic acid (i.e., site specificity) is achieved by Cas9 complexing with a guide polynucleotide (e.g., a guide RNA) that hybridizes to a specified sequence (e.g., a TSG locus). In some embodiments, the Cas9 is a Cas9 variant described in U.S. Provisional Patent Application No. 62 / 728,184, filed September 7, 2018.

[0154] In some embodiments, Cas9 is capable of generating sticky ends. Cas9 capable of generating sticky ends is described, for example, in International Publication No. PCTUS 2018 / 061680, filed November 16, 2018. In some embodiments, Cas9 capable of generating sticky ends is a dimeric Cas9 fusion protein. In some embodiments, it is advantageous to use a dimeric nuclease, i.e., a nuclease that is not active until both monomers of the dimer are present in the target sequence, to achieve a higher degree of targeting specificity. The binding and cleavage domains of natural nucleases (such as Cas9), as well as modular binding and cleavage domains that can be fused to generate nuclease-binding specific target sites, are well known to those skilled in the art. For example, a binding domain of an RNA programmable nuclease (e.g., Cas9), or a Cas9 protein with an inactive DNA cleavage domain, can be used as a binding domain (e.g., to bind a gRNA to direct binding to the target site) to specifically bind to a desired target site and fused or conjugated to a cleavage domain, e.g., the cleavage domain of the endonuclease FokI, to generate an engineered nuclease that cleaves the target region. Cas9-FokI fusion proteins are further described, for example, in U.S. Patent Application Publication No. 2015 / 0071899, and in Guilinger et al., "Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification," Nature Biotechnology 32:577-582 (2014).

[0155] In some embodiments, the Cas9 comprises a polypeptide of SEQ ID NO: 3 or 4. In some embodiments, the Cas9 comprises a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 3 or 4. In some embodiments, the Cas9 is SEQ ID NO: 3 or 4.

[0156] In some embodiments, the guide polynucleotide is an RNA polynucleotide. RNA molecules that bind to CRISPR-Cas components and target them to specific locations within a target DNA are referred to herein as "RNA guide polynucleotides," "guide RNAs," "gRNAs," "small guide RNAs," "single guide RNAs," or "sgRNAs," and may also be referred to herein as "DNA-targeting RNAs." The guide polynucleotide may be introduced into a target cell as an isolated molecule, e.g., an RNA molecule, or introduced into a cell using an expression vector that contains a guide polynucleotide, e.g., a DNA encoding an RNA guide polynucleotide. In some embodiments, the guide polynucleotide is between 10 and 150 nucleotides. In some embodiments, the guide polynucleotide is between 20 and 120 nucleotides. In some embodiments, the guide polynucleotide is between 30 and 100 nucleotides. In some embodiments, the guide polynucleotide is between 40 and 80 nucleotides. In some embodiments, the guide polynucleotide is between 50 and 60 nucleotides. In some embodiments, the guide polynucleotide is between 10 and 35 nucleotides. In some embodiments, the guide polynucleotide is between 15 and 30 nucleotides. In some embodiments, the guide polynucleotide is between 20 and 25 nucleotides.

[0157] In some embodiments, an RNA guide polynucleotide comprises at least two nucleotide segments: at least one "DNA-binding segment" and at least one "polypeptide-binding segment." By "segment" is meant a portion, section, or region of a molecule, e.g., a contiguous stretch of nucleotides of a guide polynucleotide molecule. The definition of "segment" is not limited to a particular number of total base pairs, unless specifically defined otherwise.

[0158] In some embodiments, the guide polynucleotide comprises a DNA binding segment. In some embodiments, the DNA binding segment of the guide polynucleotide comprises a nucleotide sequence that is complementary to a specific sequence in the target polynucleotide. In some embodiments, the DNA binding segment of the guide polynucleotide hybridizes with a toxin sensitivity gene (TSG) locus in the cell. Various types of cells, such as eukaryotic cells, are described herein.

[0159] In some embodiments, the guide polynucleotide comprises a polypeptide binding segment. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the DNA targeting domain of a nuclease of the present disclosure. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to Cas9. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to dCas9. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to nCas9. Various RNA guide polynucleotides that bind to Cas9 proteins are described, for example, in U.S. Patent Application Publication Nos. 2014 / 0068797, 2014 / 0273037, 2014 / 0273226, 2014 / 0295556, 2014 / 0295557, 2014 / 0349405, 2015 / 0045546, 2015 / 0071898, 2015 / 0071899, and 2015 / 0071906.

[0160] In some embodiments, the guide polynucleotide further comprises a tracrRNA. The "tracrRNA" or trans-activated CRISPR-RNA forms an RNA duplex with the pre-crRNA or pre-CRISPR-RNA, which is then cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. In some embodiments, the guide polynucleotide comprises a crRNA / tracrRNA hybrid. In some embodiments, the tracrRNA component of the guide polynucleotide activates the Cas9 protein. In some embodiments, the activation of the Cas9 protein comprises activating the nuclease activity of Cas9. In some embodiments, the activation of the Cas9 protein comprises the Cas9 protein binding to a target polynucleotide sequence, e.g., a TSG locus.

[0161] In some embodiments, the guide polynucleotide guides the nuclease to the TSG locus, and the nuclease generates a double-stranded break at the TSG locus. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the nuclease is Cas9. In some embodiments, the double-stranded break at the TSG locus inactivates TSG. In some embodiments, the inactivation of the TSG locus confers toxin resistance to the cell. In some embodiments, the inactivation of the TSG locus confers toxin resistance to the cell, but also disrupts the normal cellular function of the TSG locus. In some embodiments, the TSG locus encodes a gene that performs a cellular function that is independent of toxin sensitivity. For example, the TSG locus can encode a protein that promotes cell growth or division, a receptor for a signaling molecule (e.g., a molecule near the cell), or a protein that interacts with another protein, organelle, or biomolecule to perform normal cellular function.

[0162] In some embodiments, the TSG is an essential gene. An essential gene is a gene of an organism that is considered essential for survival under certain conditions. In some embodiments, disruption or deletion of the TSG results in cell death. In some embodiments, the TSG is an auxotrophic gene, i.e., a gene that produces a particular compound required for growth or survival. Examples of auxotrophic genes include genes involved in nucleotide biosynthesis, such as adenine, cytosine, guanine, thymine, or uracil, or in amino acid biosynthesis, such as histidine, leucine, lysine, methionine, or tryptophan. In some embodiments, the TSG is a gene in a metabolic pathway. In some embodiments, the TSG is a gene in the autophagy pathway. In some embodiments, the TSG is a gene in cell division, such as mitosis, cytoskeleton organization, or in response to stress or stimuli. In some embodiments, the TSG encodes a protein that promotes cell growth or division, a receptor for a signaling molecule (e.g., a molecule near the cell), or a protein that interacts with another protein, organelle, or biomolecule. Exemplary essential genes include, but are not limited to, those listed in Figure 23. Further examples of essential genes are provided, for example, in Hart et al., Cell 163:1515-1526 (2015), Zhang et al., Microb Cell 2(8):280-287 (2015), and Fraser, Cell Systems 1:381-382 (2015).

[0163] Thus, in some embodiments, inactivation of a native TSG (i.e., a TSG in the genome of a cell) (e.g., a double-stranded break in the sequence generated by a nuclease) results in adverse effects on the cell. In some embodiments, inactivation of a native TSG results in cell death. In such cases, an "exogenous" TSG or a portion thereof can be introduced into the cell to compensate for the inactivated native TSG. In some embodiments, the portion of the TSG encodes a polypeptide that performs substantially the same function as the native protein encoded by the TSG. In some embodiments, a portion of the TSG is introduced to complement a partially inactivated TSG. In some embodiments, the nuclease inactivates a portion of the native TSG (e.g., by destroying a portion of the coding sequence of the TSG), and the exogenous TSG includes a destroyed portion of the coding sequence that can be transcribed together with the undestroyed portion of the native sequence to form a functional TSG. In some embodiments, the exogenous TSG or a portion thereof is integrated into the native TSG locus in the genome of the cell. In some embodiments, the exogenous TSG or a portion thereof is integrated into a genomic locus that is different from the TSG locus. In some embodiments, the exogenous TSG or a portion thereof is integrated by a sequence for genomic integration. In some embodiments, the sequence for genomic integration is obtained from a retroviral vector. In some embodiments, the sequence for genomic integration is obtained from a transposon. In some embodiments, the TSG encodes a CA receptor. In some embodiments, the TSG encodes HB-EGF. In some embodiments, the TSG encodes a receptor for an antibody, for example, an antibody of an antibody-drug conjugate.

[0164] In some embodiments, exogenous TSG is introduced into the cell in an exogenous polynucleotide. In some embodiments, exogenous TSG is expressed from an exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is a plasmid. In some embodiments, the exogenous polynucleotide is a donor polynucleotide. In some embodiments, the donor polynucleotide is a vector. Exemplary vectors are provided herein.

[0165] In some embodiments, the exogenous polynucleotide is an episomal vector. In some embodiments, the episomal vector is a stable episomal vector, i.e., an episomal vector that remains in cells. As described herein, the episomal vector comprises an autonomous DNA replication sequence that allows the episomal vector to replicate and remain in cells. In some embodiments, the episomal vector is an artificial chromosome. In some embodiments, the episomal vector is a plasmid.

[0166] In some embodiments, the donor polynucleotide comprises 5' and 3' homology arms. In some embodiments, the donor polynucleotide is a donor plasmid. In some embodiments, the 5' and 3' homology arms of the donor polynucleotide are complementary to a portion of the TSG locus in the genome of the cell. Thus, when optimally aligned, the donor polynucleotide overlaps with one or more nucleotides of TSG (e.g., about or at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides). In some embodiments, when the donor polynucleotide and a portion of the TSG locus are optimally aligned, the nearest nucleotide of the donor polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 100, 1500, 2000, 2500, 5000, 10000 or more nucleotides of the TSG locus. In some embodiments, a donor polynucleotide is introduced into a cell that includes an SOI flanked by 5' and 3' homology arms, where the 5' and 3' homology arms share sequence similarity to either side of the integration site at the TSG locus. In some embodiments, the 5' and 3' homology arms share at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence similarity with either side of the integration site in the TSG locus. In some embodiments, the TSG encodes a CA receptor. In embodiments, the TSG encodes HB-EGF. In some embodiments, the TSG encodes a receptor for an antibody, e.g., an antibody of an antibody-drug conjugate.

[0167] In some embodiments, the 5' and 3' homology arms in the donor polynucleotide facilitate integration of the donor polynucleotide into the genome by homology directed repair (HDR). In some embodiments, the donor polynucleotide is integrated by HDR. In some embodiments, the donor polynucleotide is an HDR template. The HDR pathway is an endogenous DNA repair pathway capable of repairing double-strand breaks. Repair by the HDR pathway is typically high fidelity and relies on homologous recombination with the HDR template having homologous regions (e.g., 5' and 3' homology arms) to the repair site. In some embodiments, the TSG locus is cleaved by a nuclease in a manner that facilitates HDR, for example by generating sticky ends. In some embodiments, the TSG locus is cleaved by a nuclease in a manner that facilitates HDR over low fidelity repair pathways such as non-homologous end joining (NHEJ).

[0168] In some embodiments, the donor polypeptide is integrated by NHEJ. NHEJ pathway is an endogenous DNA repair pathway that can repair double-strand breaks. Generally, NHEJ has high repair efficiency but low fidelity compared to HDR, but if the double-strand break in DNA has a compatible sticky end or overhang, the error is reduced. In some embodiments, the TSG locus is cut by nuclease in a way that reduces the error of NHEJ repair. In some embodiments, the cut in the TSG locus comprises a sticky end.

[0169] In some embodiments, the donor polynucleotide comprises a sequence for genomic integration. In some embodiments, the sequence for genomic integration at the target locus is obtained from a transposon. As described herein, the transposon comprises a transposon sequence that is recognized by a transposase, which then inserts the transposon, which comprises the transposon sequence and a sequence of interest (SOI), into the genome. In some embodiments, the target locus is any genomic locus that is capable of expressing the SOI without disrupting normal cellular function. Exemplary transposons are described herein. Thus, in some embodiments, the donor polynucleotide comprises a functional TSG, which comprises a mutation in the native coding sequence of the TSG, the mutation conferring resistance to a toxin, an SOI, and a transposon sequence for genomic integration at the target locus. In some embodiments, the native TSG of the cell is inactivated by a nuclease, and the donor polynucleotide provides a functional TSG that is toxin-resistant yet capable of compensating for the native cellular function of the native TSG. In some embodiments, the TSG encodes a CA receptor. In embodiments, the TSG encodes HB-EGF, hi some embodiments, the TSG encodes a receptor for an antibody, e.g., an antibody of an antibody-drug conjugate.

[0170] In some embodiments, the donor polynucleotide comprises a sequence for genomic integration. In some embodiments, the sequence for genomic integration at the target locus is obtained from a retroviral vector. As described herein, the retroviral vector comprises a sequence recognized by integrase, typically an LTR, which then inserts the retroviral vector comprising the LTR and SOI into the genome. In some embodiments, the target locus is any genomic locus capable of expressing the SOI without disrupting normal cellular function. Exemplary retroviral vectors are described herein. Thus, in some embodiments, the donor polynucleotide comprises a functional TSG comprising a mutation in the native coding sequence of the TSG, the mutation conferring resistance to a toxin, an SOI, and a retroviral vector for genomic integration at the target locus. In some embodiments, the native TSG of the cell is inactivated by a nuclease, and the donor polynucleotide provides a functional TSG that is toxin-resistant yet capable of compensating for the native cellular function of the native TSG. In some embodiments, the TSG encodes a CA receptor. In embodiments, the TSG encodes HB-EGF. In some embodiments, the TSG encodes a receptor for an antibody, eg, an antibody of an antibody-drug conjugate.

[0171] In some embodiments, an episomal vector is introduced into the cell. In some embodiments, the episomal vector comprises a functional TSG, a SOI, and an autonomous DNA replication sequence, the functional TSG comprising a mutation in the native coding sequence of the TSG, the mutation conferring resistance to a toxin. As described herein, the episomal vector is a non-integrated extrachromosomal plasmid capable of autonomous replication. In some embodiments, the autonomous DNA replication sequence is derived from a viral genome sequence. In some embodiments, the autonomous DNA replication sequence is derived from a mammalian genome sequence. In some embodiments, the episomal vector is an artificial chromosome or a plasmid. In some embodiments, the plasmid is a viral plasmid. In some embodiments, the viral plasmid is an SV40 vector, a BKV vector, a KSHV vector, or an EBV vector. Thus, in some embodiments, the native TSG of the cell is inactivated by a nuclease, and the episomal vector provides a functional TSG that is toxin-resistant yet capable of compensating for the native cellular function of the native TSG. In some embodiments, the TSG encodes a CA receptor. In some embodiments, the TSG encodes HB-EGF. In some embodiments, the TSG encodes a receptor for an antibody, eg, an antibody of an antibody-drug conjugate.

[0172] In some embodiments, a toxin sensitivity gene (TSG) confers toxin sensitivity to a cell, i.e., the cell is susceptible to an adverse reaction, such as growth retardation or death, due to a toxin. In some embodiments, the TSG encodes a receptor that binds to a toxin. In some embodiments, the receptor is a CA receptor. A CA receptor is a protein molecule that binds to a CA, typically located on the membrane of a cell. For example, diphtheria toxin binds to human heparin-binding EGF-like growth factor (HB-EGF). A CA receptor may be specific for one CA, or a CA receptor may bind to two or more CAs. For example, monosialoganglioside (GM1) can act as a receptor for both cholera toxin and E. coli heat-labile enterotoxin. Or, two or more CA receptors may bind to one CA. For example, botulinum toxin is believed to bind to different receptors on neuronal and epithelial cells. In some embodiments, the CA receptor is a receptor that binds to a CA. In some embodiments, the CA receptor is a G protein-coupled receptor. In some embodiments, the CA receptor binds to diphtheria toxin. In some embodiments, the CA receptor is a receptor for an antibody, e.g., an antibody of an antibody-drug conjugate. In some embodiments, the TSG locus comprises a gene encoding heparin-binding EGF-like growth factor (HB-EGF). The mechanism by which HB-EGF and diphtheria toxin cause cell death is described herein and illustrated, for example, in FIG. 3A.

[0173] In some embodiments, the TSG locus comprises an intron and an exon. In some embodiments, the double-stranded break is generated by a nuclease in an intron. In some embodiments, the double-stranded break is generated by a nuclease in an exon. In some embodiments, the mutation in the native coding sequence of TSG, for example, conferring toxin resistance, is in an exon. In some embodiments, the donor polynucleotide comprises a native coding sequence of TSG, including a mutation that confers toxin resistance. In some embodiments, a "native coding sequence" refers to a sequence that is substantially similar to a wild-type sequence encoding a polypeptide, for example, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence similarity with the wild-type sequence.

[0174] In some embodiments, the donor polynucleotide comprises an exon of the native coding sequence of TSG, the exon comprises a mutation that confers toxin resistance, and the donor polynucleotide further comprises a splice acceptor sequence. As used herein, "splice acceptor" or "splice acceptor sequence" refers to a sequence at the 3' end of an intron that promotes the joining of two exons flanking the intron. In some embodiments, the splice acceptor sequence has at least about 90% sequence identity with the splice acceptor sequence of the TSG locus in the genome of the cell. In some embodiments, the exon integrated into the TSG locus from the donor polynucleotide joins with the adjacent exon in the genome of the cell when TSG is transcribed for expression. In some embodiments, the splice acceptor sequence integrated into the TSG locus from the donor polynucleotide promotes the joining of the exon integrated into the TSG locus from the donor polynucleotide with the adjacent exon in the genome of the cell.

[0175] In some embodiments, the 5' and 3' homology arms of the donor polynucleotide are complementary to a portion of the TSG locus in the genome of the cell. Thus, when optimally aligned, the donor polynucleotide overlaps with one or more nucleotides of TSG (e.g., about or at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides). In some embodiments, when the donor polynucleotide and the portion of the TSG locus are optimally aligned, the nearest nucleotide of the donor polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 100, 1500, 2000, 2500, 5000, 10000 or more nucleotides of the TSG locus. In some embodiments, a donor polynucleotide comprising an SOI flanked by 5' and 3' homology arms is introduced into a cell, the 5' and 3' homology arms sharing sequence similarity with either side of the integration site at the TSG locus. In some embodiments, the integration site at the TSG locus is a nuclease cleavage site, i.e., a double-strand break site. In some embodiments, the 5' and 3' homology arms share at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence similarity with either side of the integration site at the TSG locus. In some embodiments, the integration site at the TSG locus is a nuclease cleavage site. In some embodiments, the TSG encodes a CA receptor. In embodiments, the TSG encodes HB-EGF.

[0176] In some embodiments, the TSG encodes HB-EGF and a double-stranded break is generated in an intron of the HB-EGF gene. In some embodiments, the TSG encodes HB-EGF and a double-stranded break is generated in an exon of the HB-EGF gene. In some embodiments, the double-stranded break is in an intron of the HB-EGF gene and the mutation in the native coding sequence of the HB-EGF gene is in an exon of the HB-EGF gene. In some embodiments, the double-stranded break is in an intron of the HB-EGF gene and the mutation in the native coding sequence of the HB-EGF gene is in an exon immediately following the cleaved intron. In some embodiments, the double-stranded break is in an exon of the HB-EGF gene and the mutation in the native coding sequence of the HB-EGF gene is in the same exon of the HB-EGF gene. In some embodiments, the double-stranded break is in an exon of the HB-EGF gene and the mutation in the native coding sequence of the HB-EGF gene is in a different exon of the HB-EGF gene.

[0177] In some embodiments, the 5' and 3' homology arms of the donor polynucleotide share sequence similarity with HB-EGF at the nuclease cleavage site. In some embodiments, the double-stranded break is in an intron of HB-EGF and the 5' and 3' homology arms comprise homology to a sequence of the intron. In some embodiments, the double-stranded break is in an exon of HB-EGF and the 5' and 3' homology arms comprise homology to a sequence of the exon. In some embodiments, the 5' and 3' homology arms of the donor polynucleotide are designed to insert the donor polynucleotide into the double-stranded break site, for example by HDR. In some embodiments, the 5' and 3' homology arms have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence similarity to either side of a nuclease (e.g., Cas9) cleavage site in HB-EGF.

[0178] In some embodiments, the native coding sequence contains one or more alterations relative to the wild-type sequence, but the polypeptide encoded by the native coding sequence is substantially similar to the polypeptide encoded by the wild-type sequence, e.g., the amino acid sequence of the polypeptide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical. In some embodiments, the polypeptides encoded by the native coding sequence and the wild-type sequence have similar structures, e.g., similar overall shapes and folds, as determined by one of skill in the art. In some embodiments, the native coding sequence comprises a portion of the wild-type sequence, e.g., the native coding sequence is substantially similar to one or more exons and / or one or more introns of the wild-type sequence that encodes a protein, such that the exons and / or introns of the native coding sequence can replace the corresponding wild-type exons and / or introns to encode a polypeptide with a sequence and / or structure that is substantially identical to the wild-type polypeptide. In some embodiments, the native coding sequence comprises a mutation relative to the wild-type sequence. In some embodiments, the mutation in the native coding sequence of a TSG is in an exon.

[0179] In some embodiments, the donor polynucleotide comprises a functional TSG, which comprises a mutation in the native coding sequence of the TSG, the mutation conferring resistance to a toxin, an SOI, and a sequence for genomic integration at the target locus. The term "functional" TSG refers to a TSG that encodes a polypeptide substantially similar to the polypeptide encoded by the native coding sequence. In some embodiments, the functional TSG comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence similarity to the native coding sequence of the TSG, and further comprises a mutation in the native coding sequence of the TSG that confers resistance to a toxin. In some embodiments, a polypeptide encoded by a functional TSG has substantially the same structure and performs the same cellular function as a polypeptide encoded by a native coding sequence, except that the polypeptide encoded by the functional TSG is toxin resistant. In some embodiments, a polypeptide encoded by a functional TSG loses its ability to bind to a toxin. In some embodiments, a polypeptide encoded by a functional TSG loses its ability to transport and / or translocate a toxin into a cell.

[0180] In some embodiments, the mutation in the native coding sequence of TSG is a substitution mutation, an insertion, or a deletion. In some embodiments, the mutation is a substitution of a nucleotide in the coding sequence of TSG, which changes a single amino acid in the encoded polypeptide sequence. In some embodiments, the mutation is a substitution of one or more nucleotides, which changes one or more amino acids in the encoded polypeptide sequence. In some embodiments, the mutation is a substitution of one or more nucleotides, which changes an amino acid codon to a stop codon. In some embodiments, the mutation is a nucleotide insertion in the coding sequence of TSG, which results in an insertion of one or more amino acids in the encoded polypeptide sequence. In some embodiments, the mutation is a nucleotide deletion in the coding sequence of TSG, which results in a deletion of one or more amino acids in the encoded polypeptide sequence.

[0181] In some embodiments, the mutation in the native coding sequence of the TSG is a mutation in the toxin-binding region of the protein encoded by the TSG. In some embodiments, the mutation in the toxin-binding region results in the protein losing its ability to bind to a toxin. In some embodiments, the protein encoded by the functional TSG has substantially the same structure and performs the same cellular function as the protein encoded by the native coding sequence, except that the protein encoded by the functional TSG containing the mutation is toxin-resistant. In some embodiments, the protein encoded by the functional TSG loses its ability to bind to a toxin. In some embodiments, the protein encoded by the functional TSG loses its ability to transport and / or translocate a toxin into a cell.

[0182] In some embodiments, the TSG encodes a receptor that binds to a toxin. In some embodiments, the receptor is a CA receptor. In some embodiments, the TSG encodes a receptor that binds to diphtheria toxin. In some embodiments, the TSG encodes a heparin-binding EGF-like growth factor (HB-EGF). In some embodiments, a mutation in the native coding sequence of the TSG renders the cell resistant to diphtheria toxin.

[0183] In some embodiments, the toxin is a natural toxin. In some embodiments, the toxin is a synthetic poison. In some embodiments, the toxin is a small molecule, peptide, or protein. In some embodiments, the toxin is an antibody-drug conjugate. In some embodiments, the toxin is a monoclonal antibody attached to a biologically active agent with a chemical linker having a labile bond. In some embodiments, the toxin is a biological toxin. In some embodiments, the toxin is produced by cyanobacteria (cyanotoxins), dinoflagellates (dinotoxins), marine animals such as spiders, snakes, scorpions, frogs, jellyfish, venomous fish, corals, or blue-ringed octopus. Examples of toxins include, for example, diphtheria toxin, botulinum toxin, ricin, apitoxin, shiga toxin, Pseudomonas exotoxin, and mycotoxins. In some embodiments, the toxin is diphtheria toxin. In some embodiments, the toxin is an antibody-drug conjugate.

[0184] In some embodiments, the toxin is toxic to one organism, such as a human, but not to another organism, such as a mouse. In some embodiments, the toxin is toxic to an organism at one stage of its life cycle (e.g., fetal stage), but not to another stage of the organism's life cycle (e.g., adult stage). In some embodiments, the toxin is toxic in one organ of an animal, but not to another organ of the same animal. In some embodiments, the toxin is toxic to a subject (e.g., a human or animal) at one condition or state (e.g., diseased), but not to the same subject at another condition or state (e.g., healthy). In some embodiments, the toxin is toxic to one cell type, but not to another cell type. In some embodiments, the toxin is toxic to a cell at one cell state (e.g., differentiated), but not to the same cell at another cell state (e.g., undifferentiated). In some embodiments, the toxin is toxic to a cell in one environment (e.g., low temperature), but not to the same cell in another environment (e.g., high temperature). In some embodiments, the toxin is toxic to human cells, but not to mouse cells.

[0185] In some embodiments, a mutation in one or more of amino acids 100-160 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 105-150 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 107-148 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 120-145 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 135-143 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 138-144 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) confers diphtheria toxin resistance. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to ARG141. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to HIS141. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to LYS141. In some embodiments, a mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) from GLU141 to LYS141 confers diphtheria toxin resistance.

[0186] Thus, in some embodiments, the mutation in the native coding sequence of TSG is a mutation at one or more of amino acids 100-160 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation at one or more of amino acids 105-150 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation at one or more of amino acids 107-148 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation at one or more of amino acids 120-145 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation at one or more of amino acids 135-143 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation at one or more of amino acids 138-144 of wild type HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation at amino acid 141 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation from GLU141 to LYS141 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation from GLU141 to HIS141 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation in the native coding sequence of TSG is a mutation from GLU141 to ARG141 of HB-EGF (SEQ ID NO:8). In some embodiments, the mutation from GLU141 to LYS141 in HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin.

[0187] In some embodiments, the functional TSG in the donor polynucleotide or episomal vector is resistant to inactivation by nucleases. In some embodiments, the functional TSG comprises one or more mutations in the native coding sequence of the TSG, the one or more mutations confer resistance to inactivation by nucleases. In some embodiments, the functional TSG does not bind to nucleases. In some embodiments, the TSG that does not bind to nucleases is not easily cleaved by nucleases. As described herein, certain types of nucleases, such as Cas9, may require a PAM sequence at or near the target sequence in addition to recognition of the target sequence by the guide polynucleotide (e.g., guide RNA) via hybridization. In some embodiments, Cas9 binds to the PAM sequence before initiating nuclease activity. In some embodiments, target sequences that do not contain PAM in the target sequence or in adjacent or nearby regions do not bind to nucleases. Thus, in some embodiments, target sequences that do not contain PAM in the target sequence or in adjacent or nearby regions are not cleaved by nucleases and are therefore resistant to inactivation by nucleases. In some embodiments, a functional TSG does not contain a PAM sequence. In some embodiments, a TSG that does not contain a PAM sequence is resistant to inactivation by nucleases.

[0188] In some embodiments, the PAM is within about 30 to about 1 nucleotide of the target sequence. In some embodiments, the PAM is within about 20 to about 2 nucleotides of the target sequence. In some embodiments, the PAM is within about 10 to about 3 nucleotides of the target sequence. In some embodiments, the PAM is within about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 nucleotide of the target sequence. In some embodiments, the PAM is upstream (i.e., in the 5' direction) of the target sequence. In some embodiments, the PAM is downstream (i.e., in the 3' direction) of the target sequence. In some embodiments, the PAM is located within the target sequence.

[0189] In some embodiments, the polypeptide encoded by the functional TSG cannot hybridize with the guide polynucleotide. In some embodiments, the TSG that does not hybridize with the guide polynucleotide is not susceptible to cleavage by a nuclease such as Cas9. As described herein, the guide polynucleotide can hybridize with a target sequence, i.e., be "recognized" by the guide polynucleotide for cleavage by a nuclease such as Cas9. Thus, a sequence that does not hybridize with the guide polynucleotide is not recognized for cleavage by a nuclease such as Cas9. In some embodiments, a sequence that does not hybridize with the guide polynucleotide is resistant to inactivation by a nuclease. In some embodiments, the guide polynucleotide can hybridize with a TSG in the genome of the cell, and the functional TSG on the donor polynucleotide or episomal vector comprises one or more mutations in the native coding sequence of the TSG, such that the guide polynucleotide (1) can hybridize with a TSG in the genome of the cell, and (2) cannot hybridize with the functional TSG on the donor polynucleotide or episomal vector. In some embodiments, a functional TSG that is resistant to inactivation by a nuclease is introduced into a cell simultaneously with a nuclease that targets an ExG in the genome of the cell.

[0190] In some embodiments, the SOI comprises a polynucleotide that encodes a protein. In some embodiments, the SOI comprises a mutated gene. In some embodiments, the SOI comprises a non-coding sequence, e.g., a microRNA. In some embodiments, the SOI is operably linked to a regulatory element. In some embodiments, the SOI is a regulatory element. In some embodiments, the SOI comprises a resistance cassette, e.g., a gene that confers resistance to an antibiotic. In some embodiments, the SOI comprises a marker, e.g., a selection marker or a selectable marker. In some embodiments, the SOI comprises a marker, e.g., a restriction site, a fluorescent protein, or a selection marker.

[0191] In some embodiments, the SOI comprises a mutation of a wild-type gene in the genome of the cell. In some embodiments, the mutation comprises a point mutation, i.e., a substitution of a single nucleotide. In some embodiments, the mutation comprises a substitution of multiple nucleotides. In some embodiments, the mutation introduces a stop codon. In some embodiments, the mutation comprises an insertion of a nucleotide in the wild-type sequence. In some embodiments, the mutation comprises a deletion of a nucleotide in the wild-type sequence. In some embodiments, the mutation comprises a frameshift mutation.

[0192] In some embodiments, the cell population is contacted with a toxin after introducing the nuclease, guide polynucleotide, and donor polynucleotide or episomal vector. Examples of toxins are provided herein. In some embodiments, the toxin is a natural toxin. In some embodiments, the toxin is a synthetic poison. In some embodiments, the toxin is a small molecule, peptide, or protein. In some embodiments, the toxin is an antibody-drug conjugate. In some embodiments, the toxin is a monoclonal antibody linked to a biologically active agent with a chemical linker having a labile bond. In some embodiments, the toxin is a biological toxin. In some embodiments, the toxin is produced by cyanobacteria (cyanotoxins), dinoflagellates (dinotoxins), spiders, snakes, scorpions, frogs, marine animals such as jellyfish, venomous fish, corals, or blue-ringed octopus. Examples of toxins include, for example, diphtheria toxin, botulinum toxin, ricin, apitoxin, shiga toxin, Pseudomonas exotoxin, and mycotoxins. In some embodiments, the toxin is diphtheria toxin, hi some embodiments, the toxin is an antibody-drug conjugate.

[0193] In some embodiments, the toxin is toxic to one organism, such as a human, but not to another organism, such as a mouse. In some embodiments, the toxin is toxic to an organism at one stage of its life cycle (e.g., fetal stage), but not to another stage of the organism's life cycle (e.g., adult stage). In some embodiments, the toxin is toxic in one organ of an animal, but not to another organ of the same animal. In some embodiments, the toxin is toxic to a subject (e.g., a human or animal) at one condition or state (e.g., diseased), but not to the same subject at another condition or state (e.g., healthy). In some embodiments, the toxin is toxic to one cell type, but not to another cell type. In some embodiments, the toxin is toxic to a cell at one cell state (e.g., differentiated), but not to the same cell at another cell state (e.g., undifferentiated). In some embodiments, the toxin is toxic to a cell in one environment (e.g., low temperature), but not to the same cell in another environment (e.g., high temperature). In some embodiments, the toxin is toxic to human cells, but not to mouse cells. In some embodiments, the toxin is diphtheria toxin, hi some embodiments, the toxin is an antibody-drug conjugate.

[0194] In some embodiments, after contacting the cell population with the toxin, one or more cells that are resistant to the toxin are selected. In some embodiments, the one or more cells that are resistant to the toxin are viable cells. In some embodiments, the viable cells have (1) an inactivated native TSG (e.g., inactivated by a double-stranded break generated by a nuclease) and (2) a functional TSG that contains a mutation that confers toxin resistance. Cells that meet only one of the above two conditions are susceptible to cell death: if the native TSG is inactivated, the cells are sensitive to the toxin and die when contacted with the toxin, and if a functional TSG is not introduced, the cells lack the normal cellular function of the TSG and die due to the absence of normal cellular function.

[0195] In embodiments involving the introduction of a donor polynucleotide comprising 5' and 3' homology arms (e.g., homologous sequences of HDR), the surviving cells comprise biallelic integration of a donor polynucleotide comprising an SOI at a native TSG locus, the native TSG being disrupted by integration of the donor polynucleotide, and the cells comprise a functional toxin-resistant TSG. Thus, in such embodiments, one or more cells resistant to the toxin comprise biallelic integration of an SOI. In embodiments involving the introduction of a donor polynucleotide comprising a sequence for genomic integration (e.g., a transposon, a lentiviral vector sequence, or a retroviral vector sequence) at a target locus, the surviving cells comprise integration of a donor polynucleotide comprising an inactivated native TSG and a functional toxin-resistant TSG and an SOI at the target locus. In such embodiments, one or more cells resistant to the toxin comprise an integrated SOI in the target locus. In embodiments involving the introduction of an episomal vector, the surviving cells comprise a stable episomal vector comprising an inactivated native TSG and a functional toxin-resistant TSG and an SOI. In such embodiments, the one or more cells that are resistant to the toxin comprise an episomal vector.

[0196] Methods for Providing Resistance to Diphtheria Toxin In some embodiments, the disclosure provides a method of providing resistance to diphtheria toxin in a human cell, the method comprising introducing into the cell (i) a base editing enzyme and (ii) a guide polynucleotide that targets a heparin-binding EGF-like growth factor (HB-EGF) receptor in the human cell, wherein the base editing enzyme forms a complex with the guide polynucleotide, and the base editing enzyme is targeted to HB-EGF and provides a site-specific mutation in HB-EGF to provide diphtheria toxin resistance in the human cell.

[0197] In some embodiments, the human cell is a human cell line. In some embodiments, the human cell is a stem cell. The stem cell can be, for example, a pluripotent stem cell, such as an embryonic stem cell (ESC), an adult stem cell, an induced pluripotent stem cell (iPSC), a tissue-specific stem cell (e.g., a hematopoietic stem cell), and a mesenchymal stem cell (MSC). In some embodiments, the human cell is any differentiated form of the cells described herein. In some embodiments, the eukaryotic cell is a cell derived from a primary cell in culture. In some embodiments, the cell is a stem cell or a stem cell line. In some embodiments, the human cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte, or a non-parenchymal cell. For example, the eukaryotic cell can be an adherent metabolic test human hepatocyte, an adherent induction test human hepatocyte, an adherent QUALYST TRANSPORTER CERTIFIED human hepatocyte, a suspension test human hepatocyte (e.g., 10 donor and 20 donor pool hepatocytes), a human liver Kupffer cell, or a human hepatic stellate cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a granulocyte, a mast cell, a monocyte, a dendritic cell, a natural killer cell, a B cell, a primary T cell, a cytotoxic T cell, a helper T cell, a CD8+ T cell, a CD4+ T cell, or a regulatory T cell.

[0198] In some embodiments, the human cells are xenografted or transplanted into a non-human animal. In some embodiments, the non-human animal is a mouse, rat, hamster, guinea pig, rabbit, or pig. In some embodiments, the human cells are cells in a humanized organ of a non-human animal. In some embodiments, a "humanized" organ refers to a human organ grown in an animal. In some embodiments, a "humanized" organ refers to an organ produced by an animal, removed of its animal-specific cells, and grafted with human cells. A humanized organ can be immunocompatible with humans. In some embodiments, the humanized organ is a liver, kidney, pancreas, heart, lung, or stomach. Humanized organs are very useful for studying and modeling human diseases. However, most genetic selection tools cannot be converted into a humanized organ in a host animal because most selection markers are harmful to the host animal. Humanized organs are further described, for example, in Garry et al., Regen Med 11(7):617-619, Garry et al., Circ Res 124:23-25(2019), and Nguyen et al., Drug Discov Today 23(11):1812-1817(2018).

[0199] The present disclosure provides a highly advantageous selection method that can be used to humanize cells in an animal host by using diphtheria toxin that is toxic to humans but not to mice. However, the method of the present invention is not limited to diphtheria toxin and can be utilized with any compound that is differentially toxic, i.e., toxic to one organism but not another. The method of the present invention further provides diphtheria toxin resistance by engineering the toxin receptor, which may be desirable in situations because the toxin does not enter the cell, in contrast to previous methods that focus on diphthamide biosynthetic protein 2 (DPH2) (see, e.g., Picco et al., Sci Rep 5:14721).

[0200] In some embodiments, the humanized organ is generated by transplanting human cells into an animal. In some embodiments, the animal is an immunodeficient mouse. In some embodiments, the animal is an immunodeficient adult mouse. In some embodiments, the humanized organ is generated by suppressing one or more animal genes and expressing one or more human genes in an organ of the animal. In some embodiments, the humanized organ is a liver. In some embodiments, the humanized organ is a pancreas. In some embodiments, the humanized organ is a heart. In some embodiments, the humanized organ expresses a human gene encoding a receptor for a cytotoxic agent, i.e., a CA receptor as described herein. In some embodiments, the humanized organ is toxin-sensitive, while the remainder of the animal is toxin-resistant. In some embodiments, the humanized organ expressed human HB-EGF. In some embodiments, the humanized organ is diphtheria toxin-sensitive, while the remainder of the animal is diphtheria toxin-resistant. In some embodiments, the humanized organ is a humanized liver in a mouse, where the humanized liver expresses human HB-EGF and is diphtheria toxin-sensitive, while the remainder of the mouse is HB-EGF-resistant. Thus, upon exposure to diphtheria toxin, only the humanized cells in the mouse liver die.

[0201] In some embodiments, the base editing enzyme comprises a DNA targeting domain and a DNA editing domain. In some embodiments, the DNA targeting domain comprises Cas9. Cas9 proteins are described herein. In some embodiments, Cas9 comprises a mutation in the catalytic domain. In some embodiments, the base editing enzyme comprises a catalytically inactive Cas9 (dCas9) and a DNA editing domain. In some embodiments, nCas9 comprises mutations relative to wild-type Cas9 at amino acid residues D10 and H840 (numbering is relative to SEQ ID NO: 3). In some embodiments, the base editing enzyme comprises a Cas9 capable of generating single-stranded DNA breaks (nCas9) and a DNA editing domain. In some embodiments, nCas9 comprises mutations relative to wild-type Cas9 at amino acid residues D10 or H840 (numbering is relative to SEQ ID NO: 3). In some embodiments, Cas9 comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 3. In some embodiments, Cas9 comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, Cas9 comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 4. In some embodiments, Cas9 comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 4.

[0202] In some embodiments, the DNA editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) deaminase, activation-induced cytidine deaminase (AID), ACF1 / ASE deaminase, ADAT deaminase, or ADAR deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is APOBEC1.

[0203] In some embodiments, the base editing enzyme further comprises a DNA glycosylase inhibitor domain. In some embodiments, the DNA glycosylase is a uracil DNA glycosylase inhibitor (UGI). In general, DNA glycosylases, such as uracil DNA glycosylase, are part of the base excision repair pathway, and upon detection of a U:G mismatch (where "U" is generated from deamination of cytosine), perform an error-free repair and convert the U back to the wild-type sequence, effectively "undoing" the base edit. Thus, adding a DNA glycosylase inhibitor (e.g., a uracil DNA glycosylase inhibitor) inhibits the base excision repair pathway and increases base editing efficiency. Non-limiting examples of DNA glycosylases include OGG1, MAG1, and UNG. The DNA glycosylase inhibitor can be a small molecule or a protein. For example, protein inhibitors of uracil DNA glycosylase are described in Mol et al., Cell 82:701-708 (1995), Serrano-Heras et al., J Biol Chem 281:7068-7074 (2006), and New England Biolabs Catalog Nos. M0281S and M0281L (neb.com / products / m0281-uracil-glycosylase-inhibitor-ugi). Small molecule inhibitors of DNA glycosylases are described, for example, in Huang et al., J Am Chem Soc 131(4):1344-1345(2009); Jacobs et al., PLoS One 8(12):e81667(2013); Donley et al., ACS Chem Biol 10(10):2334-2343(2015); Tahara et al., J Am Chem Soc 140(6):2105-2114(2018).

[0204] Thus, in some embodiments, the base editing enzyme of the disclosure comprises nCas9 and cytidine deaminase. In some embodiments, the base editing enzyme of the disclosure comprises nCas9 and adenosine deaminase. In some embodiments, the base editing enzyme comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:6. In some embodiments, the base editing enzyme comprises a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, the base editing enzyme is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to SEQ ID NO:6. In some embodiments, the polynucleotide encoding the base editing enzyme is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to SEQ ID NO: 5. In some embodiments, the base editing enzyme is BE3.

[0205] In some embodiments, the methods of the disclosure include introducing into a human cell a guide polynucleotide that targets an HB-EGF receptor in the human cell. In some embodiments, the guide polynucleotide forms a complex with a base editing enzyme, which is targeted to HB-EGF by the guide polynucleotide and provides diphtheria toxin resistance in the human cell by providing a site-specific mutation in HB-EGF.

[0206] In some embodiments, the guide polynucleotide is an RNA molecule. The guide polynucleotide may be introduced into the target cell as an isolated molecule, e.g., an RNA molecule, or introduced into the cell using an expression vector containing DNA encoding the guide polynucleotide, e.g., an RNA guide polynucleotide. In some embodiments, the guide polynucleotide is 10-150 nucleotides. In some embodiments, the guide polynucleotide is 20-120 nucleotides. In some embodiments, the guide polynucleotide is 30-100 nucleotides. In some embodiments, the guide polynucleotide is 40-80 nucleotides. In some embodiments, the guide polynucleotide is 50-60 nucleotides. In some embodiments, the guide polynucleotide is 10-35 nucleotides. In some embodiments, the guide polynucleotide is 15-30 nucleotides. In some embodiments, the guide polynucleotide is 20-25 nucleotides.

[0207] In some embodiments, an RNA guide polynucleotide comprises at least two nucleotide segments: at least one "DNA-binding segment" and at least one "polypeptide-binding segment." By "segment" is meant a portion, section, or region of a molecule, e.g., a contiguous stretch of nucleotides of a guide polynucleotide molecule. The definition of "segment" is not limited to a particular number of total base pairs, unless specifically defined otherwise.

[0208] In some embodiments, the guide polynucleotide comprises a DNA binding segment. In some embodiments, the DNA binding segment of the guide polynucleotide comprises a nucleotide sequence that is complementary to a specific sequence in the target polynucleotide. In some embodiments, the DNA binding segment of the guide polynucleotide hybridizes with a gene encoding a cytotoxic agent (CA) receptor in the target cell. In some embodiments, the DNA binding segment of the guide polynucleotide hybridizes with a gene encoding HB-EGF. In some embodiments, the DNA binding segment of the guide polynucleotide hybridizes with a target polynucleotide sequence in the target cell. Target cells, including various types of eukaryotic cells, are described herein.

[0209] In some embodiments, the guide polynucleotide comprises a polypeptide binding segment. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the DNA targeting domain of the base editing enzyme of the disclosure. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the Cas9 of the base editing enzyme. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the dCas9 of the base editing enzyme. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the nCas9 of the base editing enzyme. Various RNA guide polynucleotides that bind to Cas9 proteins are described, for example, in U.S. Patent Application Publication Nos. 2014 / 0068797, 2014 / 0273037, 2014 / 0273226, 2014 / 0295556, 2014 / 0295557, 2014 / 0349405, 2015 / 0045546, 2015 / 0071898, 2015 / 0071899, and 2015 / 0071906.

[0210] In some embodiments, the guide polynucleotide further comprises a tracrRNA. The "tracrRNA" or trans-activated CRISPR-RNA forms an RNA duplex with the pre-crRNA or pre-CRISPR-RNA, which is then cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. In some embodiments, the guide polynucleotide comprises a crRNA / tracrRNA hybrid. In some embodiments, the tracrRNA component of the guide polynucleotide activates the Cas9 protein. In some embodiments, the activation of the Cas9 protein comprises activating the nuclease activity of Cas9. In some embodiments, the activation of the Cas9 protein comprises the Cas9 protein binding to a target polynucleotide sequence.

[0211] In some embodiments, the sequence of the guide polynucleotide is designed to target a base editing enzyme to a specific location in a target polynucleotide sequence. Various tools and programs are available to facilitate the design of such guide polynucleotides (e.g., the Benchling Base Editor Design Guide (benchling.com / editor#create / crispr), and BE-Designer and BE-Analyzer from CRISPR RGEN Tools (see Hwang et al., bioRxiv dx.doi.org / 10.1101 / 373944, first published July 22, 2018)).

[0212] In some embodiments, the DNA-binding segment of the guide polynucleotide hybridizes to a gene encoding HB-EGF, and the polypeptide-binding segment of the guide polynucleotide complexes with the base-editing enzyme by binding to the DNA-targeting domain of the base-editing enzyme. In some embodiments, the DNA-binding segment of the guide polynucleotide hybridizes to a gene encoding HB-EGF, and the polypeptide-binding segment of the guide polynucleotide complexes with the base-editing enzyme by binding to the base-editing enzyme Cas9. In some embodiments, the DNA-binding segment of the guide polynucleotide hybridizes to a gene encoding HB-EGF, and the polypeptide-binding segment of the guide polynucleotide complexes with the base-editing enzyme by binding to the base-editing enzyme dCas9. In some embodiments, the DNA-binding segment of the guide polynucleotide hybridizes to a gene encoding HB-EGF, and the polypeptide-binding segment of the guide polynucleotide complexes with the base-editing enzyme by binding to the base-editing enzyme nCas9.

[0213] In some embodiments, the complex is targeted to HB-EGF by a guide polynucleotide, and a base editor of the complex introduces a mutation in HB-EGF. In some embodiments, the mutation in HB-EGF is introduced by a base editing domain of the base editor of the complex. In some embodiments, the mutation in HB-EGF forms diphtheria toxin resistant cells. In some embodiments, the mutation is a point mutation from cytidine (C) to thymine (T). In some embodiments, the mutation is a point mutation from adenine (A) to guanine (G). The specific location of the mutation in HB-EGF can be guided by design of the guide polynucleotide, for example, using tools such as the Benchling base editor design guide, BE-Designer, and BE-Analyzer described herein. In some embodiments, the guide polynucleotide is an RNA polynucleotide. In some embodiments, the guide polynucleotide further comprises a tracrRNA sequence.

[0214] In some embodiments, the site-directed mutation is in a region of HB-EGF that binds to diphtheria toxin. In some embodiments, a mutation in the EGF-like domain of HB-EGF confers resistance to diphtheria toxin. In some embodiments, a charge-reversal mutation of an amino acid at or near the diphtheria toxin binding site of HB-EGF confers resistance to diphtheria toxin. In some embodiments, the charge-reversal mutation is a substitution of a negatively charged residue, such as Glu or Asp, with a positively charged residue, such as Lys or Arg. In some embodiments, the charge-reversal mutation is a substitution of a positively charged residue, such as Lys or Arg, with a negatively charged residue, such as Glu or Asp. In some embodiments, a polarity-reversal mutation of an amino acid at or near the diphtheria toxin binding site of HB-EGF confers resistance to diphtheria toxin. In some embodiments, the polarity-reversal mutation is a substitution of a polar amino acid residue, such as Gln or Asn, with a non-polar amino acid residue, such as Ala, Val, or Ile. In some embodiments, the polarity reversal mutation is a substitution of a non-polar amino acid residue, such as Ala, Val, or Ile, for a polar amino acid residue, such as Gln or Asn. In some embodiments, the mutation is a substitution of a relatively small amino acid residue, such as Gly or Ala, for a "bulky" amino acid residue, such as Trp, at or near the diphtheria toxin binding site of HB-EGF. In some embodiments, the mutation of the small residue to the bulky residue blocks the binding pocket, preventing binding of diphtheria toxin, thereby conferring resistance.

[0215] In some embodiments, a mutation in one or more of amino acids 100-160 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 105-150 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 107-148 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 120-145 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 135-143 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation in one or more of amino acids 138-144 of wild-type HB-EGF (SEQ ID NO:8) confers resistance to diphtheria toxin. In some embodiments, a mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) confers diphtheria toxin resistance. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to ARG141. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to HIS141. In some embodiments, the mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) is from GLU141 to LYS141. In some embodiments, a mutation at amino acid 141 of wild-type HB-EGF (SEQ ID NO:8) from GLU141 to LYS141 confers diphtheria toxin resistance.

[0216] Thus, in some embodiments, the site-specific mutation is at one or more of amino acids 100-160 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is at one or more of amino acids 105-150 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is at one or more of amino acids 107-148 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is at one or more of amino acids 120-145 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is at one or more of amino acids 135-143 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is at one or more of amino acids 138-144 of wild-type HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is at amino acid 141 of HB-EGF (SEQ ID NO:8). In some embodiments, the site-specific mutation is a GLU141 to LYS141 mutation in HB-EGF (SEQ ID NO: 8). In some embodiments, the site-specific mutation is a GLU141 to HIS141 mutation in HB-EGF (SEQ ID NO: 8). In some embodiments, the site-specific mutation is a GLU141 to ARG141 mutation in HB-EGF (SEQ ID NO: 8). In some embodiments, the GLU141 to LYS141 mutation in HB-EGF (SEQ ID NO: 8) confers diphtheria toxin resistance.

[0217] Selection methods using essential genes The method of the present disclosure is not necessarily limited to the selection using toxin sensitivity genes. Essential genes are genes of an organism that are considered essential for survival under certain conditions. In embodiments, essential genes are used as "selection" sites in the co-targeting enrichment strategy described herein.

[0218] In some embodiments, the disclosure provides a method for integrating and enriching a sequence of interest (SOI) in a mammalian genomic target locus in the genome of a cell, comprising: (a) providing in a population of cells: (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide that forms a complex with the nuclease and is capable of hybridizing to and inactivating an essential gene (ExG) locus in the genome of the cell; and (iii) The present invention provides a method comprising: introducing a donor polynucleotide comprising: (1) a functional ExG gene containing a mutation in a native coding sequence of ExG, the mutation conferring resistance to inactivation by a guide polynucleotide; (2) an SOI; and (3) a sequence for genomic integration at a target locus, wherein introduction of (i), (ii), and (iii) results in inactivation of ExG in the genome of the cell by a nuclease and integration of the donor polynucleotide at the target locus; (b) cultivating the cells; and (c) selecting one or more surviving cells, wherein the one or more surviving cells comprise the integrated SOI at the target locus.

[0219] FIG. 13 shows an embodiment of the method of the present disclosure. In FIG. 13, a CRISPR-Cas complex is introduced into a cell to target ExG, an essential gene for cell survival. A vector containing a gene of interest (GOI) and a modified ExG* that is resistant to targeting by the CRISPR-Cas complex is also introduced into the cell. As a result, cells that have cleaved ExG (indicated by an asterisk in the ExG sequence) and the vector that successfully introduced ExG* can survive, and cells that do not have this vector die as a result of lacking ExG. The guide RNA of the CRISPR-Cas complex can be designed and selected to be close to 100% efficient for the ExG in the genome of the cell and / or multiple guide RNAs can be used to target the same ExG. Alternatively, or in addition, multiple rounds of selecting surviving cells and introducing the CRISPR-Cas complex can be performed to increase the tendency of surviving cells to lack a genomic copy of ExG and survive due to the presence of ExG* (and thus the GOI). Thus, surviving cells are enriched for harboring the GOI.

[0220] In some embodiments, an essential gene is a gene necessary for an organism to survive. In some embodiments, disruption or deletion of an essential gene results in cell death. In some embodiments, an essential gene is an auxotrophic gene, i.e., a gene that produces a particular compound required for growth or survival. Examples of auxotrophic genes include genes involved in nucleotide biosynthesis, such as adenine, cytosine, guanine, thymine, or uracil, or in amino acid biosynthesis, such as histidine, leucine, lysine, methionine, or tryptophan. In some embodiments, an essential gene is a gene in a metabolic pathway. In some embodiments, an essential gene is a gene in the autophagy pathway. In some embodiments, an essential gene is a gene in cell division, such as mitosis, cytoskeleton organization, or response to stress or stimuli. In some embodiments, an essential gene encodes a protein that promotes cell growth or division, a receptor for a signaling molecule (e.g., a molecule near the cell), or a protein that interacts with another protein, organelle, or biomolecule. Exemplary essential genes include, but are not limited to, the genes listed in FIG. 23. Further examples of essential genes are provided, for example, in Hart et al., Cell 163:1515-1526 (2015), Zhang et al., Microb Cell 2(8):280-287 (2015), and Fraser, Cell Systems 1:381-382 (2015).

[0221] In some embodiments, the nuclease capable of generating double-strand breaks is Cas9. In some embodiments, the Cas9 protein generates site-specific breaks in nucleic acids. In some embodiments, the Cas9 protein generates site-specific double-strand breaks in DNA. The ability of Cas9 to target specific sequences in nucleic acids (i.e., site specificity) is achieved by Cas9 complexing with a guide polynucleotide (e.g., a guide RNA) that hybridizes with a designated sequence (e.g., the ExG locus). In some embodiments, the Cas9 is a Cas9 variant described in U.S. Provisional Patent Application No. 62 / 728,184, filed September 7, 2018.

[0222] In some embodiments, Cas9 is capable of generating sticky ends. Cas9 capable of generating sticky ends is described, for example, in International Publication WO 2018 / 061680, filed November 16, 2018. In some embodiments, Cas9 capable of generating sticky ends is a dimeric Cas9 fusion protein. The binding and cleavage domains of natural nucleases (such as Cas9), as well as modular binding and cleavage domains that can be fused to generate nuclease-binding specific target sites, are well known to those skilled in the art. For example, the binding domain of an RNA-programmable nuclease (such as Cas9), or a Cas9 protein with an inactive DNA cleavage domain, can be used as a binding domain (e.g., to bind to a gRNA and direct binding to the target site) to specifically bind to a desired target site, and fused or conjugated to a cleavage domain, such as the cleavage domain of the endonuclease FokI, to generate an engineered nuclease that cleaves the target region. Cas9-FokI fusion proteins are further described, for example, in U.S. Patent Application Publication No. 2015 / 0071899 and in Guilinger et al., "Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification," Nature Biotechnology 32:577-582 (2014).

[0223] In some embodiments, the Cas9 comprises the polypeptide sequence of SEQ ID NO: 3 or 4. In some embodiments, the Cas9 comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 3 or 4. In some embodiments, the Cas9 is SEQ ID NO: 3 or 4.

[0224] In some embodiments, the guide polynucleotide is an RNA polynucleotide. RNA molecules that bind to CRISPR-Cas components and target them to specific locations within a target DNA are referred to herein as "RNA guide polynucleotides," "guide RNAs," "gRNAs," "small guide RNAs," "single guide RNAs," or "sgRNAs," and may also be referred to herein as "DNA-targeting RNAs." The guide polynucleotide may be introduced into a target cell as an isolated molecule, e.g., an RNA molecule, or introduced into a cell using an expression vector that contains a guide polynucleotide, e.g., a DNA encoding an RNA guide polynucleotide. In some embodiments, the guide polynucleotide is between 10 and 150 nucleotides. In some embodiments, the guide polynucleotide is between 20 and 120 nucleotides. In some embodiments, the guide polynucleotide is between 30 and 100 nucleotides. In some embodiments, the guide polynucleotide is between 40 and 80 nucleotides. In some embodiments, the guide polynucleotide is between 50 and 60 nucleotides. In some embodiments, the guide polynucleotide is between 10 and 35 nucleotides. In some embodiments, the guide polynucleotide is between 15 and 30 nucleotides. In some embodiments, the guide polynucleotide is between 20 and 25 nucleotides.

[0225] In some embodiments, an RNA guide polynucleotide comprises at least two nucleotide segments: at least one "DNA-binding segment" and at least one "polypeptide-binding segment." By "segment" is meant a portion, section, or region of a molecule, e.g., a contiguous stretch of nucleotides of a guide polynucleotide molecule. The definition of "segment" is not limited to a particular number of total base pairs, unless specifically defined otherwise.

[0226] In some embodiments, the guide polynucleotide comprises a DNA binding segment. In some embodiments, the DNA binding segment of the guide polynucleotide comprises a nucleotide sequence that is complementary to a specific sequence in the target polynucleotide. In some embodiments, the DNA binding segment of the guide polynucleotide hybridizes with an essential gene locus (ExG) in the cell. Various types of cells, such as eukaryotic cells, are described herein.

[0227] In some embodiments, the guide polynucleotide comprises a polypeptide binding segment. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to the DNA targeting domain of a nuclease of the present disclosure. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to Cas9. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to dCas9. In some embodiments, the polypeptide binding segment of the guide polynucleotide binds to nCas9. Various RNA guide polynucleotides that bind to Cas9 proteins are described, for example, in U.S. Patent Application Publication Nos. 2014 / 0068797, 2014 / 0273037, 2014 / 0273226, 2014 / 0295556, 2014 / 0295557, 2014 / 0349405, 2015 / 0045546, 2015 / 0071898, 2015 / 0071899, and 2015 / 0071906.

[0228] In some embodiments, the guide polynucleotide further comprises a tracrRNA. The "tracrRNA" or trans-activated CRISPR-RNA forms an RNA duplex with the pre-crRNA, or pre-CRISPR-RNA, which is then cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. In some embodiments, the guide polynucleotide comprises a crRNA / tracrRNA hybrid. In some embodiments, the tracrRNA component of the guide polynucleotide activates the Cas9 protein. In some embodiments, the activation of the Cas9 protein comprises activating the nuclease activity of Cas9. In some embodiments, the activation of the Cas9 protein comprises the Cas9 protein binding to a target polynucleotide sequence, for example, the ExG locus.

[0229] In some embodiments, the guide polynucleotide guides the nuclease to the ExG locus, and the nuclease generates a double-stranded break at the ExG locus. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the nuclease is Cas9. In some embodiments, the double-stranded break at the ExG locus inactivates ExG. In some embodiments, the inactivation of the ExG locus disrupts an essential cell function. In some embodiments, the inactivation of the ExG locus prevents cell division. In some embodiments, the inactivation of the ExG locus results in cell death.

[0230] In some embodiments, an "exogenous" ExG or a portion thereof can be introduced into a cell to compensate for the inactivated native ExG. In some embodiments, the exogenous ExG is a functional ExG. The term "functional" ExG refers to an ExG that encodes a polypeptide that is substantially similar to the polypeptide encoded by the native coding sequence. In some embodiments, a functional ExG comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence similarity with the native coding sequence of ExG, and further comprises a mutation in the native coding sequence of ExG that confers resistance to inactivation by nucleases. In some embodiments, the functional ExG is resistant to inactivation by nucleases, and the polypeptide encoded by the functional ExG has substantially the same structure and performs the same cellular function as the polypeptide encoded by the native coding sequence.

[0231] In some embodiments, a portion of ExG encodes a polypeptide that performs substantially the same function as the native protein encoded by ExG. In some embodiments, a portion of ExG is introduced to complement a partially inactivated ExG. In some embodiments, the nuclease inactivates a portion of the native ExG (e.g., by destroying a portion of the coding sequence of ExG), and the exogenous ExG comprises a destroyed portion of the coding sequence that can be transcribed together with the non-destroyed portion of the native sequence to form a functional ExG. In some embodiments, the exogenous ExG or a portion thereof is integrated into the native ExG locus in the genome of the cell. In some embodiments, the exogenous ExG or a portion thereof is integrated into a genomic locus different from the ExG locus.

[0232] In some embodiments, functional ExG does not bind to nucleases. In some embodiments, ExG that does not bind to nucleases is less likely to be cleaved by nucleases. As described herein, certain types of nucleases, such as Cas9, may require a PAM sequence at or near the target sequence in addition to the recognition of the target sequence by a guide polynucleotide (e.g., guide RNA) via hybridization. In some embodiments, Cas9 binds to a PAM sequence before initiating nuclease activity. In some embodiments, a target sequence that does not contain a PAM in the target sequence or in the adjacent or nearby region does not bind to a nuclease. Thus, in some embodiments, a target sequence that does not contain a PAM in the target sequence or in the adjacent or nearby region is not cleaved by a nuclease and is therefore resistant to inactivation by a nuclease. In some embodiments, a mutation in the native coding sequence of ExG removes the PAM sequence. In some embodiments, an ExG that does not contain a PAM sequence is resistant to inactivation by a nuclease.

[0233] In some embodiments, the PAM is within about 30 to about 1 nucleotide of the target sequence. In some embodiments, the PAM is within about 20 to about 2 nucleotides of the target sequence. In some embodiments, the PAM is within about 10 to about 3 nucleotides of the target sequence. In some embodiments, the PAM is within about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 nucleotide of the target sequence. In some embodiments, the PAM is upstream (i.e., in the 5' direction) of the target sequence. In some embodiments, the PAM is downstream (i.e., in the 3' direction) of the target sequence. In some embodiments, the PAM is located within the target sequence.

[0234] In some embodiments, the polypeptide encoded by the functional ExG cannot hybridize with the guide polynucleotide. In some embodiments, the ExG that does not hybridize with the guide polynucleotide is not easily cleaved by a nuclease such as Cas9. As described herein, the guide polynucleotide can hybridize with a target sequence, i.e., the target sequence can be "recognized" by the guide polynucleotide for cleavage by a nuclease such as Cas9. Thus, a sequence that does not hybridize with the guide polynucleotide is not recognized for cleavage by a nuclease such as Cas9. In some embodiments, a sequence that does not hybridize with the guide polynucleotide is resistant to inactivation by a nuclease. In some embodiments, the guide polynucleotide can hybridize to an ExG in the genome of the cell, and the functional ExG on the donor polynucleotide or episomal vector comprises a mutation in the native coding sequence of the ExG such that the guide polynucleotide (1) can hybridize to an ExG in the genome of the cell, and (2) cannot hybridize to the functional ExG on the donor polynucleotide or episomal vector. In some embodiments, the functional ExG that is resistant to inactivation by a nuclease is introduced into the cell simultaneously with a nuclease that targets the ExG in the genome of the cell.

[0235] In some embodiments, the functional ExG comprises one or more mutations relative to the wild-type sequence, but the polypeptide encoded by the native coding sequence is substantially similar to the polypeptide encoded by the wild-type sequence, e.g., the amino acid sequence of the polypeptide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical. In some embodiments, the polypeptide encoded by the functional ExG and the wild-type ExG has a similar structure, e.g., a similar overall shape and fold, as determined by a person skilled in the art. In some embodiments, the functional ExG comprises a portion of the wild-type sequence. In some embodiments, the functional ExG comprises a mutation relative to the wild-type sequence. In some embodiments, the functional ExG comprises a mutation in the native coding sequence of ExG, which mutation confers resistance to inactivation by nucleases.

[0236] In some embodiments, the mutation in the native coding sequence of ExG is a substitution mutation, an insertion, or a deletion. In some embodiments, the substitution mutation is a substitution of one or more nucleotides in a polynucleotide sequence, but the encoded amino acid sequence remains unchanged. In some embodiments, the substitution mutation replaces one or more nucleotides to change a codon for an amino acid to a degenerate codon for the same amino acid. For example, the native coding sequence may contain the sequence "CAT" which codes for histidine, and the mutation may change this sequence to "CAC", which also codes for histidine. In some embodiments, the substitution mutation replaces one or more nucleotides to change an amino acid to a different amino acid, but the different amino acid has similar properties, so that the overall structure of the encoded polypeptide or the overall function of the protein is not affected. For example, the substitution mutation may result in a change of leucine to isoleucine, glutamine to asparagine, glutamate to aspartate, serine to threonine, etc.

[0237] In some embodiments, exogenous ExG or a portion thereof (e.g., an ExG comprising a mutation in the native coding sequence of ExG, which mutation confers resistance to inactivation by nucleases) is introduced into a cell in an exogenous polynucleotide. In some embodiments, the exogenous ExG is expressed from an exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is a plasmid. In some embodiments, the exogenous polynucleotide is a donor polynucleotide. In some embodiments, the donor polynucleotide is a vector. Exemplary vectors are provided herein.

[0238] In some embodiments, the exogenous ExG or a portion thereof on the donor polynucleotide is integrated into the genome of the cell by a sequence for genome integration. In some embodiments, the sequence for genome integration is obtained from a retroviral vector. In some embodiments, the sequence for genome integration is obtained from a transposon.

[0239] In some embodiments, the donor polynucleotide comprises a sequence for genomic integration. In some embodiments, the sequence for genomic integration at the target locus is obtained from a transposon. As described herein, the transposon comprises a transposon sequence that is recognized by a transposase, which then inserts the transposon, which comprises the transposon sequence and a sequence of interest (SOI), into the genome. In some embodiments, the target locus is any genomic locus that is capable of expressing the SOI without disrupting normal cellular function. Exemplary transposons are described herein. Thus, in some embodiments, the donor polynucleotide comprises a functional ExG, which comprises a mutation in the native coding sequence of the ExG, the mutation conferring resistance to inactivation by nucleases, a SOI, and a transposon sequence for genomic integration at the target locus. In some embodiments, the native ExG of the cell is inactivated by a nuclease, and the donor polynucleotide provides a functional ExG that is resistant to inactivation by nucleases yet capable of compensating for the native cellular function of the native ExG.

[0240] In some embodiments, the donor polynucleotide comprises a sequence for genomic integration. In some embodiments, the sequence for genomic integration at the target locus is obtained from a retroviral vector. As described herein, the retroviral vector comprises a sequence recognized by integrase, typically an LTR, which then inserts the retroviral vector comprising the LTR and SOI into the genome. In some embodiments, the target locus is any genomic locus capable of expressing the SOI without disrupting normal cellular function. Exemplary retroviral vectors are described herein. Thus, in some embodiments, the donor polynucleotide comprises a functional ExG comprising a mutation in the native coding sequence of the ExG, the mutation conferring resistance to inactivation by nucleases, an SOI, and a retroviral vector for genomic integration at the target locus. In some embodiments, the native ExG of the cell is inactivated by a nuclease, and the donor polynucleotide provides a functional ExG that is resistant to inactivation by nucleases yet capable of compensating for the native cellular function of the native ExG.

[0241] In some embodiments, the exogenous polynucleotide is an episomal vector. In some embodiments, the episomal vector is a stable episomal vector, i.e., an episomal vector that remains in cells. As described herein, the episomal vector comprises an autonomous DNA replication sequence that allows the episomal vector to replicate and remain in cells. In some embodiments, the episomal vector is an artificial chromosome. In some embodiments, the episomal vector is a plasmid.

[0242] In some embodiments, an episomal vector is introduced into the cell. In some embodiments, the episomal vector comprises a functional ExG, which comprises a mutation in the native coding sequence of ExG, the mutation conferring resistance to inactivation by nucleases, an SOI, and an autonomous DNA replication sequence. As described herein, the episomal vector is a non-integrated extrachromosomal plasmid capable of autonomous replication. In some embodiments, the autonomous DNA replication sequence is derived from a viral genome sequence. In some embodiments, the autonomous DNA replication sequence is derived from a mammalian genome sequence. In some embodiments, the episomal vector is an artificial chromosome or a plasmid. In some embodiments, the plasmid is a viral plasmid. In some embodiments, the viral plasmid is an SV40 vector, a BKV vector, a KSHV vector, or an EBV vector. Thus, in some embodiments, the native ExG of the cell is inactivated by nucleases, and the episomal vector provides a functional ExG that is resistant to inactivation by nucleases but can compensate for the native cellular function of the native ExG.

[0243] In some embodiments, the SOI comprises a polynucleotide that encodes a protein. In some embodiments, the SOI comprises a mutated gene. In some embodiments, the SOI comprises a non-coding sequence, e.g., a microRNA. In some embodiments, the SOI is operably linked to a regulatory element. In some embodiments, the SOI is a regulatory element. In some embodiments, the SOI comprises a resistance cassette, e.g., a gene that confers resistance to an antibiotic. In some embodiments, the SOI comprises a marker, e.g., a selection marker or a selectable marker. In some embodiments, the SOI comprises a marker, e.g., a restriction site, a fluorescent protein, or a selection marker.

[0244] In some embodiments, the SOI comprises a mutation of a wild-type gene in the genome of the cell. In some embodiments, the mutation comprises a point mutation, i.e., a substitution of a single nucleotide. In some embodiments, the mutation comprises a substitution of multiple nucleotides. In some embodiments, the mutation introduces a stop codon. In some embodiments, the mutation comprises an insertion of a nucleotide in the wild-type sequence. In some embodiments, the mutation comprises a deletion of a nucleotide in the wild-type sequence. In some embodiments, the mutation comprises a frameshift mutation.

[0245] In some embodiments, the guide polynucleotide has a targeting efficiency of more than 80%, more than 85%, more than 90%, more than 95%, or about 100% to ExG in the genome of the cell. Targeting efficiency can be measured, for example, by the percentage of cells with inactivated ExG in a population of cells. Guide polynucleotides can be designed and selected for increased efficiency using design tools such as, for example, Chop Chop (chopchop.cbu.uib.no), CasFinder (arep.med.harvard.edu / CasFinder), E-CRISP (e-crisp.org / E-CRISP / designcrispr.html), CRISPR-ERA (crispr-era.stanford.edu / index.jsp).

[0246] In some embodiments, two or more guide polynucleotides are introduced into a cell population, each guide polynucleotide forms a complex with a nuclease, and each guide polynucleotide hybridizes to a different region of ExG. In some embodiments, multiple guide polynucleotides can be used to increase the efficiency of inactivating ExG in the genome of a cell. For example, a first guide polynucleotide can target the 5' region of ExG, a second guide polynucleotide can target an internal region of ExG, and a third guide polynucleotide can target the 3' region of ExG. Although the targeting efficiency of each guide polynucleotide can vary, the overall efficiency can be increased by using two or more guide polynucleotides targeting the same gene, since nuclease cleavage at either the 5', 3', or internal region will inactivate ExG. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 different guide polynucleotides are introduced into a cell population.

[0247] In some embodiments, the surviving cells comprise a mixture of cells containing ExG* and SOI integrated at the target locus or on an episomal vector, and cells containing ExG that have not been inactivated by nuclease, for example due to inherent inefficiency in nucleases or failure to introduce nucleases and / or guide polynucleotides into the cells. Thus, in some embodiments, one or more steps of the method are repeated to enrich for surviving cells containing the desired SOI. Repeated introduction of nucleases and guide polynucleotides may increase the likelihood that ExG in the genome of the cells is inactivated, thereby enriching for surviving cells containing ExG* and SOI integrated at the target locus or on an episomal vector.

[0248] Thus, in an embodiment of the method for integrating an SOI into a target locus, the method further comprises introducing a nuclease capable of generating a double-stranded break and a guide polynucleotide capable of forming a complex with and hybridizing to an ExG in the genome of the cell into one or more selected surviving cells to enrich for surviving cells that contain the SOI integrated into the target locus. In an embodiment of the method for introducing a stable episomal vector into a cell, the method further comprises introducing a nuclease capable of generating a double-stranded break and a guide polynucleotide capable of forming a complex with and hybridizing to an ExG in the genome of the cell into one or more selected surviving cells to enrich for surviving cells that contain the episomal vector.

[0249] In some embodiments, the nuclease and guide polynucleotide are introduced into the viable cell for multiple enrichment rounds. In some embodiments, the nuclease and guide polynucleotide are introduced for 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more than 20 enrichment rounds. Each round of targeting increases the likelihood that the viable cell contains the SOI. That is, it enriches the viable cell that contains the SOI integrated into the target locus or episomal vector.

[0250] array Various polynucleotide and polypeptide sequences are provided herein.

[0251] Polynucleotide sequence of the Cas9 protein from Streptococcus pyogenes (SpCas9; SEQ ID NO:1): [ka] [ka]

[0252] Polynucleotide sequence of the Cas9 protein from Francisella novicida (FnCas9; SEQ ID NO:2): [ka] [ka] [ka]

[0253] Polypeptide sequence of SpCas9 (SEQ ID NO:3): [ka]

[0254] Polypeptide sequence of FnCas9 (SEQ ID NO:4): [ka]

[0255] Polynucleotide sequence of BE3 (SEQ ID NO:5): [ka] [ka] [ka]

[0256] Polypeptide sequence of BE3 (SEQ ID NO:6): [ka]

[0257] Polynucleotide sequence of the HB-EGF locus (SEQ ID NO:7): [ka] [ka] [ka] [ka] [ka]

[0258] Polypeptide sequence of HB-EGF protein (SEQ ID NO:8): [ka]

[0259] Embodiments of the present disclosure are further described in the following sections: [Section 1] 1. A method for introducing site-specific mutations into a target polynucleotide in a target cell in a cell population, comprising: (a) in said cell population, (i) a base editing enzyme; (ii) (1) hybridize with a gene encoding a cytotoxic agent (CA) receptor; (2) A first guide polynucleotide that forms a first complex with the base editing enzyme, a first guide polynucleotide, wherein the base editing enzyme of the first complex provides a mutation in the gene encoding the CA receptor, and the mutation in the gene encoding the CA receptor forms CA resistant cells in the cell population; (iii) (1) hybridizing to the target polynucleotide; (2) a second guide polynucleotide that forms a second complex with the base editing enzyme, The base editing enzyme of the second complex comprises a second guide polynucleotide that provides a mutation in the target polynucleotide; and Introducing (b) contacting the cell population with the CA; (c) enriching the target cells containing the mutation in the target polynucleotide by selecting the CA resistant cells from the cell population; A method comprising: [Section 2] 1. A method for determining the efficacy of a base editing enzyme in a cell population, comprising: (a) in said cell population, (i) a base editing enzyme; (ii) (1) hybridize with a gene encoding a cytotoxic agent (CA) receptor; (2) A first guide polynucleotide that forms a first complex with the base editing enzyme, a first guide polynucleotide, wherein the base editing enzyme of the first complex introduces a mutation in the gene encoding the CA receptor, and the mutation in the gene encoding the CA receptor forms CA resistant cells in the cell population; (iii) (1) hybridizing to the target polynucleotide; (2) a second guide polynucleotide that forms a second complex with the base editing enzyme, the base editing enzyme of the second complex introduces a mutation in the target polynucleotide; and Introducing (b) contacting the cell population with the CA to isolate CA-resistant cells; (c) determining the ratio of the CA resistant cells to the total cell population to determine the efficacy of the base editing enzyme; and A method comprising: [Section 3] The method according to claim 1 or 2, wherein the base editing enzyme comprises a DNA targeting domain and a DNA editing domain. [Section 4] The method of claim 3, wherein the DNA targeting domain comprises Cas9. [Section 5] 5. The method of claim 4, wherein the Cas9 comprises a mutation in the catalytic domain. [Section 6] 6. The method according to any one of items 1 to 5, wherein the base editing enzyme comprises a catalytically inactive Cas9 and a DNA editing domain. [Section 7] The method according to any one of items 1 to 5, wherein the base editing enzyme comprises Cas9 (nCas9) capable of generating single-stranded DNA breaks, and a DNA editing domain. [Section 8] The method of claim 7, wherein the nCas9 comprises a mutation relative to wild-type Cas9 at amino acid residue D10 or H840 (numbering is relative to SEQ ID NO: 3). [Section 9] The method according to any one of items 4 to 8, wherein the Cas9 is at least 90% identical to SEQ ID NO: 3 or 4. [Section 10] 10. The method according to any one of items 3 to 9, wherein the DNA editing domain comprises a deaminase. [Section 11] 11. The method according to item 10, wherein the deaminase is cytidine deaminase or adenosine deaminase. [Section 12] 12. The method according to claim 11, wherein the deaminase is a cytidine deaminase. [Section 13] 12. The method according to claim 11, wherein the deaminase is adenosine deaminase. [Section 14] 14. The method according to any one of items 10 to 13, wherein the deaminase is apolipoprotein B mRNA editing complex (APOBEC) deaminase, activation-induced cytidine deaminase (AID), ACF1 / ASE deaminase, ADAT deaminase, or ADAR deaminase. [Section 15] Item 15. The method according to item 14, wherein the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. [Section 16] 16. The method according to claim 15, wherein the deaminase is APOBEC1. [Section 17] 17. The method according to any one of items 3 to 16, wherein the base editing enzyme further comprises a DNA glycosylase inhibitor domain. [Section 18] Item 18. The method according to item 17, wherein the DNA glycosylase is a uracil DNA glycosylase inhibitor (UGI). [Section 19] The method according to any one of items 1 to 4 or 6 to 18, wherein the base editing enzyme comprises nCas9 and cytidine deaminase. [Section 20] The method according to any one of items 1 to 4 or 6 to 18, wherein the base editing enzyme comprises nCas9 and adenosine deaminase. [Section 21] 20. The method according to any one of items 1 to 12 or 13 to 19, wherein the base editing enzyme comprises a polypeptide sequence that is at least 90% identical to SEQ ID NO:6. [Section 22] The method according to any one of items 1 to 12 or 13 to 19, wherein the base editing enzyme is BE3. [Section 23] 23. The method according to any one of items 1 to 22, wherein the first and / or second guide polynucleotide is an RNA polynucleotide. [Section 24] 24. The method according to any one of items 1 to 23, wherein the first and / or second guide polynucleotide further comprises a tracrRNA sequence. [Section 25] 25. The method according to any one of items 1 to 24 above, wherein the cell population is human cells. [Section 26] 26. The method according to any one of items 1 to 25 above, wherein the mutation in the gene encoding the CA receptor is a point mutation from cytidine (C) to thymine (T). [Section 27] 26. The method according to any one of items 1 to 25 above, wherein the mutation in the gene encoding the CA receptor is a point mutation from adenine (A) to guanine (G). [Section 28] 28. The method according to any one of items 1 to 27, wherein the CA is diphtheria toxin. [Section 29] 29. The method according to claim 28, wherein the cytotoxic agent (CA) receptor is a diphtheria toxin receptor. [Section 30] 30. The method according to item 29, wherein the CA receptor is heparin-binding EGF-like growth factor (HB-EGF). [Section 31] 31. The method according to claim 30, wherein the HB-EGF comprises the polypeptide sequence of SEQ ID NO:8. [Section 32] 32. The method according to claim 31, wherein the base editing enzyme of the first complex provides a mutation in one or more of amino acids 107 to 148 of HB-EGF (SEQ ID NO: 8). [Section 33] 33. The method according to claim 32, wherein the base editing enzyme of the first complex provides a mutation in one or more of amino acids 138 to 144 of HB-EGF (SEQ ID NO: 8). [Section 34] 34. The method of claim 33, wherein the base editing enzyme of the first complex provides a mutation at amino acid 141 of HB-EGF (sequence number 8). [Section 35] 35. The method according to claim 34, wherein the base editing enzyme of the first complex provides a mutation from GLU141 to LYS141 in the amino acid sequence of HB-EGF (sequence number 8). [Section 36] 36. The method according to any one of items 1 to 35, wherein the base editing enzyme of the first complex provides a mutation in a region of HB-EGF that binds to diphtheria toxin. [Section 37] 37. The method according to any one of items 1 to 36, wherein the base editing enzyme of the first complex provides a mutation in HB-EGF that renders the target cell resistant to diphtheria toxin. [Section 38] 38. The method according to any one of items 1 to 37, wherein the mutation in the target polynucleotide is a point mutation from cytidine (C) to thymine (T) in the target polynucleotide. [Section 39] 38. The method according to any one of items 1 to 37, wherein the mutation in the target polynucleotide is a point mutation from adenine (A) to guanine (G) in the target polynucleotide. [Section 40] 40. The method according to any one of items 1 to 39, wherein the base editing enzyme is introduced into the cell population as a polynucleotide encoding the base editing enzyme. [Section 41] The method of claim 40, wherein the polynucleotide encoding the base editing enzyme, the first guide polynucleotide of (ii), and the second guide polynucleotide of (iii) are on a single vector. [Section 42] The method of claim 40, wherein the polynucleotide encoding the base editing enzyme, the first guide polynucleotide of (ii), and the second guide polynucleotide of (iii) are on one or more vectors. [Section 43] 43. The method according to claim 41 or 42, wherein the vector is a viral vector. [Section 44] 44. The method according to claim 43, wherein the viral vector is an adenovirus, a lentivirus, or an adeno-associated virus. [Section 45] 1. A method for providing biallelic integration of a sequence of interest (SOI) into a toxin sensitivity gene (TSG) locus in the genome of a cell, comprising: (a) Within a cell population, (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to the TSG locus; (iii) (1) a mutation in a 5' homology arm, a 3' homology arm, and the native coding sequence of the TSG, the mutation conferring resistance to the toxin; (2) the SOI; A donor polynucleotide comprising: To introduce introducing (i), (ii), and (iii) resulting in integration of the donor polynucleotide into the TSG locus; (b) contacting the cell population with the toxin; (c) selecting one or more cells that are resistant to the toxin; and Including, The one or more cells that are resistant to the toxin comprise the biallelic integration of the SOI. [Section 46] 46. ​​The method of claim 45, wherein the donor polynucleotide is integrated by homology-directed repair (HDR). [Section 47] 46. ​​The method of claim 45, wherein the donor polynucleotide is integrated by non-homologous end joining (NHEJ). [Section 48] 48. The method according to any one of items 45 to 47, wherein the TSG locus comprises an intron and an exon. [Section 49] 49. The method of claim 48, wherein the donor polynucleotide further comprises a splicing acceptor sequence. [Section 50] 50. The method of claim 48 or 49, wherein the nuclease capable of generating a double stranded break generates a break in the intron. [Section 51] 51. The method according to any one of items 48 to 50, wherein the mutation in the native coding sequence of the TSG is in the exon of the TSG locus. [Section 52] 1. A method for integrating a sequence of interest (SOI) into a target locus in the genome of a cell, comprising: (a) Within a cell population, (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to a toxin sensitivity gene (TSG) locus in the genome of the cell, wherein the TSG is an essential gene; and (iii) (1) a functional TSG gene comprising a mutation in the native coding sequence of said TSG, said mutation conferring resistance to said toxin; and (2) the SOI; (3) a sequence for genomic integration at the target locus; and A donor polynucleotide comprising: To introduce As a result of introducing (i), (ii), and (iii), inactivation of the TSG in the genome of the cell by the nuclease; and introducing, which results in integration of the donor polynucleotide into the target locus; (b) contacting the cell population with the toxin; (c) selecting one or more cells that are resistant to the toxin, selecting the one or more cells that are resistant to the toxin, the one or more cells containing the SOI integrated into the target locus; A method comprising: [Section 53] 53. The method according to claim 52, wherein said sequence for genomic integration is obtained from a transposon or a retroviral vector. [Section 54] 54. The method according to any one of items 45 to 53, wherein the functional TSG of the donor polynucleotide is resistant to inactivation by the nuclease. [Section 55] 55. The method according to any one of paragraphs 45 to 54, wherein said mutation in the native coding sequence of said TSG removes a protospacer adjacent motif from said native coding sequence. [Section 56] 56. The method according to any one of items 45 to 55, wherein the guide polynucleotide is not capable of hybridizing with the functional TSG of the donor polynucleotide. [Section 57] 57. The method according to any one of items 45 to 56, wherein the nuclease capable of generating a double-stranded break is Cas9. [Section 58] 58. The method of claim 57, wherein the Cas9 is capable of generating sticky ends. [Section 59] 59. The method according to claim 57 or 58, wherein the Cas9 comprises the polypeptide sequence of SEQ ID NO: 3 or 4. [Section 60] 60. The method according to any one of items 45 to 59, wherein the guide polynucleotide is an RNA polynucleotide. [Section 61] 61. The method according to any one of items 45 to 60, wherein the guide polynucleotide further comprises a tracrRNA sequence. [Section 62] 62. The method according to any one of items 45 to 61, wherein the donor polynucleotide is a vector. [Section 63] 63. The method according to any one of items 45 to 62, wherein the mutation in the native coding sequence of the TSG is a substitution mutation, an insertion or a deletion. [Section 64] 64. The method according to any one of items 45 to 63, wherein the mutation in the native coding sequence of the TSG is a mutation in a toxin-binding domain of a protein encoded by the TSG. [Section 65] 65. The method according to any one of items 45 to 64 above, wherein the TSG locus comprises a gene encoding heparin-binding EGF-like growth factor (HB-EGF). [Section 66] 66. The method according to any one of items 45 to 65 above, wherein the TSG encodes HB-EGF (SEQ ID NO: 8). [Section 67] 67. The method according to any one of items 45 to 66, wherein the mutation in the native coding sequence of the TSG is a mutation in one or more of amino acids 107 to 148 of HB-EGF (SEQ ID NO: 8). [Section 68] 68. The method according to item 67, wherein the mutation in the native coding sequence of the TSG is a mutation in one or more of amino acids 138 to 144 of HB-EGF (SEQ ID NO: 8). [Section 69] 69. The method of claim 68, wherein the mutation in the native coding sequence of the TSG is a mutation at amino acid 141 of HB-EGF (SEQ ID NO: 8). [Section 70] 70. The method of claim 69, wherein the mutation in the native coding sequence of the TSG is a mutation from GLU141 to LYS141 in HB-EGF (SEQ ID NO: 8). [Section 71] 71. The method according to any one of items 65 to 70, wherein the toxin is diphtheria toxin. [Section 72] 72. The method according to any one of paragraphs 65 to 71, wherein the mutation in the native coding sequence of the TSG renders the cell resistant to diphtheria toxin. [Section 73] 73. The method according to any one of items 45 to 72, wherein the toxin is an antibody-drug conjugate and the TSG encodes a receptor for the antibody-drug conjugate. [Section 74] 1. A method of providing resistance to diphtheria toxin in a human cell, comprising administering to the cell (i) a base editing enzyme; (ii) a guide polynucleotide that targets a heparin-binding EGF-like growth factor (HB-EGF) receptor in the human cell; and Including the introduction of The base editing enzyme forms a complex with the guide polynucleotide, and the base editing enzyme is targeted to the HB-EGF and provides diphtheria toxin resistance in the human cell by providing a site-specific mutation in the HB-EGF. [Section 75] 75. The method of claim 74, wherein the base editing enzyme comprises a DNA targeting domain and a DNA editing domain. [Section 76] 76. The method of claim 75, wherein the DNA targeting domain comprises Cas9. [Section 77] 77. The method of claim 76, wherein the Cas9 comprises a mutation in the catalytic domain. [Section 78] 78. The method according to any one of items 74 to 77, wherein the base editing enzyme comprises a catalytically inactive Cas9 and a DNA editing domain. [Section 79] 78. The method according to any one of items 74 to 77, wherein the base editing enzyme comprises Cas9 (nCas9) capable of generating single-stranded DNA breaks, and a DNA editing domain. [Section 80] 80. The method of claim 79, wherein the nCas9 comprises a mutation relative to wild-type Cas9 at amino acid residue D10 or H840 (numbering is relative to SEQ ID NO: 3). [Section 81] 81. The method according to any one of items 76 to 80, wherein the Cas9 is at least 90% identical to SEQ ID NO: 3 or 4. [Section 82] 82. The method according to any one of items 75 to 81, wherein the DNA editing domain comprises a deaminase. [Section 83] 83. The method according to claim 82, wherein the deaminase is selected from cytidine deaminase and adenosine deaminase. [Section 84] 84. The method according to claim 83, wherein the deaminase is a cytidine deaminase. [Section 85] 84. The method according to claim 83, wherein the deaminase is adenosine deaminase. [Section 86] 86. The method according to any one of items 82 to 85, wherein the deaminase is selected from apolipoprotein B mRNA editing complex (APOBEC) deaminase, activation-induced cytidine deaminase (AID), ACF1 / ASE deaminase, ADAT deaminase, and TadA deaminase. [Section 87] 87. The method according to claim 86, wherein the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. [Section 88] 88. The method according to claim 87, wherein the cytidine deaminase is APOBEC1. [Section 89] 89. The method according to any one of items 74 to 88, wherein the base editing enzyme further comprises a DNA glycosylase inhibitor domain. [Section 90] 90. The method according to claim 89, wherein the DNA glycosylase is a uracil DNA glycosylase inhibitor (UGI). [Section 91] The method according to any one of items 74 to 84 or 86 to 90, wherein the base editing enzyme comprises nCas9 and cytidine deaminase. [Section 92] The method according to any one of items 74 to 83 or 85 to 90, wherein the base editing enzyme comprises nCas9 and adenosine deaminase. [Section 93] The method according to any one of Items 74 to 83 or 86 to 91, wherein the base editing enzyme comprises a polypeptide sequence that is at least 90% identical to SEQ ID NO:6. [Section 94] The method according to any one of items 74 to 83 or 86 to 93, wherein the base editing enzyme is BE3. [Section 95] Item 74. The method according to any one of items 74 to 94, wherein the guide polynucleotide is an RNA polynucleotide. [Section 96] Item 74. The method according to any one of items 74 to 95, wherein the guide polynucleotide further comprises a tracrRNA sequence. [Section 97] 97. The method according to any one of items 74 to 96, wherein the site-specific mutation is in one or more of amino acids 107 to 148 of the HB-EGF (SEQ ID NO: 8). [Section 98] 98. The method according to item 97, wherein the site-specific mutation is in one or more of amino acids 138 to 144 of the HB-EGF (SEQ ID NO: 8). [Section 99] 99. The method of claim 98, wherein the site-specific mutation is at amino acid 141 of the HB-EGF (SEQ ID NO: 8). [Section 100] 99. The method according to claim 99, wherein the site-specific mutation is a mutation from GLU141 to LYS141 of the HB-EGF (SEQ ID NO: 8). [Section 101] 101. The method according to any one of items 74 to 100 above, wherein the site-specific mutation is in a region of the HB-EGF that binds to diphtheria toxin. [Section 102] 1. A method for integrating and enriching a sequence of interest (SOI) into a target locus in the genome of a cell, comprising: (a) Within a cell population, (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to an essential gene (ExG) locus in the genome of the cell; and (iii) (1) a functional ExG gene comprising a mutation in the native coding sequence of the ExG, the mutation conferring resistance to inactivation by the guide polynucleotide; and (2) the SOI; (3) a sequence for genomic integration at the target locus; and A donor polynucleotide comprising: To introduce As a result of introducing (i), (ii), and (iii), inactivation of the ExG in the genome of the cell by the nuclease; and resulting in integration of the donor polynucleotide into the target locus; To introduce and (b) cultivating the cells; (c) selecting one or more surviving cells, selecting said one or more surviving cells that contain said SOI integrated at said target locus; A method comprising: [Section 103] 1. A method for introducing a stable episomal vector into a cell, comprising: (a) Within a cell population, (i) a nuclease capable of generating a double-stranded break; (ii) a guide polynucleotide capable of forming a complex with the nuclease and hybridizing to an essential gene (ExG) locus in the genome of the cell, a guide polynucleotide, the introduction of which results in the inactivation of the ExG in the genome of the cell by the nuclease; (iii) (1) a functional ExG comprising a mutation in the native coding sequence of the ExG, the mutation conferring resistance to the inactivation by the nuclease; and (2) an autonomous DNA replication sequence; An episomal vector comprising Introducing (b) cultivating the cells; (c) selecting one or more surviving cells; The one or more viable cells comprise the episomal vector. [Section 104] 104. The method of claim 102 or 103, wherein the mutation in the native coding sequence of ExG removes a protospacer adjacent motif from the native coding sequence. [Section 105] 105. The method according to any one of items 102 to 104, wherein the guide polynucleotide cannot hybridize with the functional ExG of the donor polynucleotide or the episomal vector. [Section 106] 106. The method according to any one of items 102 to 105, wherein the nuclease capable of generating a double-stranded break is Cas9. [Section 107] 107. The method of claim 106, wherein the Cas9 is capable of generating sticky ends. [Section 108] 108. The method of claim 104 or 107, wherein the Cas9 comprises the polypeptide sequence of SEQ ID NO: 3 or 4. [Section 109] 109. The method according to any one of items 102 to 108, wherein the guide polynucleotide is an RNA polynucleotide. [Section 110] The method according to any one of items 102 to 109, wherein the guide polynucleotide further comprises a tracrRNA sequence. [Section 111] 111. The method according to any one of items 102 to 110, wherein the donor polynucleotide is a vector. [Section 112] 112. The method according to any one of items 102 to 111, wherein the mutation in the native coding sequence of ExG is a substitution mutation, an insertion or a deletion. [Section 113] 113. The method according to any one of items 102 or 104 to 112, wherein said sequence for genomic integration is obtained from a transposon or a retroviral vector. [Section 114] 113. The method according to any one of items 103 to 112, wherein the episomal vector is an artificial chromosome or a plasmid. [Section 115] The method according to any one of paragraphs 102 to 114, wherein two or more guide polynucleotides are introduced into the cell population, each guide polynucleotide forms a complex with the nuclease, and each guide polynucleotide hybridizes to a different region of the ExG. [Section 116] 17. The method of any one of paragraphs 102, 104 to 113, or 115, further comprising introducing the nuclease of (a)(i) and the guide polynucleotide of (a)(ii) into viable cells to enrich for viable cells containing the SOI integrated into the target locus. [Section 117] 16. The method according to any one of paragraphs 103 to 112, 114, or 115, further comprising introducing the nuclease of (a)(i) and the guide polynucleotide of (a)(ii) into viable cells to enrich for viable cells containing the episomal vector. [Section 118] 118. The method according to claim 116 or 117, wherein the nuclease of (a)(i) and the guide polynucleotide of (a)(ii) are introduced into the viable cell for multiple enrichment rounds. All references cited herein, e.g., patents, patent applications, articles, textbooks, etc., and the references cited therein, to the extent that they have not already been cited, are incorporated herein by reference in their entirety. EXAMPLES

[0260] Example 1. Experimental Protocol In this example, a protocol for co-targeting enrichment is provided.

[0261] Maintain cell lines expressing heparin-binding EGF receptor in culture and subculture every 2-3 days until transfection. Cells should be >80% confluent on the day of transfection.

[0262] The cells are transfected with a plasmid encoding a base editor or Cas9 and / or with a plasmid encoding a guide RNA targeting HB-EGF and a gene of interest. The DNA-lipid complex for transfection is prepared according to the manufacturer's protocol. Alternatively, mRNA and RNP complexes can be used.

[0263] The complexes are added to the plates containing freshly trypsinized cells that were seeded the day before.

[0264] 72 hours after transfection, the medium is removed and the cells are trypsinized and re-seeded onto new plates with twice the surface area of ​​the previous plates.

[0265] The next day, diphtheria toxin is added to the wells at a concentration of 20 ng / mL. Two days later, a new diphtheria toxin treatment is performed.

[0266] Cell growth is monitored and, if necessary, cells are transferred to larger plates or flasks until all cells of the negative selection are dead.

[0267] After 1-2 weeks, the cells are analyzed by next-generation sequencing to determine the editing efficiency.

[0268] Example 2. Guide RNA screening In this example, guide RNAs (gRNAs) were screened to identify gRNAs that confer diphtheria toxin resistance when co-transfected with BE3. A panel of gRNAs was designed to tile the EGF-like domain of HB-EGF (see FIG. 4C). Each gRNA was co-transfected with BE3 at a transfection weight ratio of 1:4 into HEK293 or HCT116 cells.

[0269] Cells were treated with 20 ng / mL diphtheria toxin on day 3 post-transfection and again on day 5 post-transfection. Cell growth was measured by confluence using an INCUCYTE ZOOM.

[0270] The results shown in Figures 4A and 4B, respectively, indicate that HEK293 and HCT116 cells co-transfected with HB-EGF gRNA16 and BE3 had the highest growth levels among all transfected cells. The results of Sanger sequencing and next-generation sequencing analysis, shown in Figures 5B-5D, revealed that diphtheria toxin resistance in gRNA16-transfected cells was the result of the E141K mutation introduced by BE3 base editing. The sequence of gRNA16 is shown in Figure 5A.

[0271] Example 3. Co-targeted enrichment with BE3 and Cas9 In this example, co-targeting enrichment with diphtheria toxin selection was tested using BE3 and Cas9 by co-transfection of the targeting gRNA identified in Example 2 and gRNA16 to generate diphtheria toxin resistant cells.

[0272] Plasmid construction Cas9 Plasmid: DNA sequences encoding SpCas9, T2A self-cleaving peptide, and puromycin N-acetyltransferase were synthesized by GeneArt and cloned into an expression vector containing a CMV promoter and a BGH polyA tail. See plasmid map in Figure 15.

[0273] BE3 Plasmid. The DNA sequence of Base Editor 3 was synthesized and cloned into pcDNA3.1(+) by GeneArt with restriction sites BamHI and XhoI. See plasmid map in Figure 14.

[0274] gRNA plasmids. The target sequence of the gRNA was introduced into the AarI cleavage site of the template plasmid using a complementary primer pair (5'-AAAC-N20-3' and 5'-ACCG-N20-3'). The template plasmid was synthesized by GeneArt. It contains a U6 promoter driving the gRNA expression cassette, and the rpsL-BSD selection cassette was cloned in the region of the gRNA target sequence flanked by two AarI restriction sites. The primers can be seen in Table 1. The plasmids for gRNA targeting BFP and EGFR are described in Coelho et al., BMC Biology 16:150 (2018) and are shown in Figures 17-23.

[0275] [Table 1]

[0276] [Table 2]

[0277] Cell culture and transfection HEK293T and HCT116 cells obtained from ATCC were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). PC9-BFP cells were maintained in DMEM medium containing 10% FBS.

[0278] Transfections were performed using FUGENE HD transfection reagent (Promega) with a 3:1 transfection reagent to DNA ratio according to the manufacturer's instructions. Transfections in this study were performed in 24-well and 48-well plates. 1.25 × 10 5 and 6.75 x 10 4Cells were seeded in 24-well and 48-well plates, respectively. Transfection was performed with 500ng and 250ng of total DNA for 24-well and 48-well plates, respectively.

[0279] For co-targeting enrichment, Cas9 or BE3 plasmid DNA, targeting gRNA plasmid DNA, and selection gRNA plasmid DNA were transfected at a weight ratio of 8:1:1. The sequence of the targeting gRNA for the PCKS9 site is shown in FIG. 7C, and the sequences of the targeting gRNA for the DPM2, EGFR, EMX1, and Yas85 sites are shown in FIG. 7E. The cells were treated with 20 ng / ml diphtheria toxin 3 days after transfection, and then again 5 days after transfection. When the cells grow to >80% confluence, the cells are harvested for downstream applications. For all cell types used in this study, the cells were harvested 7 days after transfection for genome extraction. For other different cell lines or primary cells, different diphtheria toxin doses and treatment times may be applied to kill all wild-type cells.

[0280] Next-generation sequencing and data analysis Genomic DNA was extracted from cells 72 hours after transfection or treatment using QUICKEXTRACT DNA Extraction Solution (Lucigen) according to the manufacturer's instructions. NGS libraries were prepared by two-step PCR. The first PCR was performed using NEBNEXT Q5 Hot Start HiFi PCR Master Mix (New England Biolabs) according to the manufacturer's instructions. The second PCR was performed using 1 ng of product from the first PCR using KAPA HiFi PCR Kit (KAPABIOSYSTEMS). PCR products were purified using Agencourt AMPure XP (Beckman Coulter) and analyzed by Fragment analyzer.

[0281] The results in Figures 7A and 7B show BE3 base editing efficiency of different cytosines in PCSK9 target sites in HCT116 and HEK293 cells, respectively. The "control" condition shows relatively low base editing efficiency without diphtheria toxin selection, and the "enriched" condition shows significantly higher base editing efficiency when diphtheria toxin selection is used. The results in Figure 7D show increased base editing efficiency at different cytosines in DPM2, EGFR, EMX1, and Yas85 target sites when diphtheria toxin selection is used ("enriched") compared to the "control" condition without diphtheria toxin.

[0282] The results in Figure 8A show Cas9 editing efficiency by measuring the percentage of indels generated at PCSK9 target sites in HEK293 and HCT116 cells. As with base editing, Cas9 editing efficiency was significantly increased under "enriched" conditions with diphtheria toxin selection over "control" conditions without diphtheria toxin selection. The results in Figure 8B show a similar increase in Cas9 editing efficiency at DPM2, EXM1, and Yas85 target sites.

[0283] Example 4.2 Allelic Integration In this example, diphtheria toxin selection was tested to improve knock-in (insertion) efficiency where the gene of interest achieves biallelic integration.

[0284] Donor plasmid for knock-in. The knock-in plasmid for mCherry was synthesized by Genescript. See Figure 23 for the plasmid map, and Figure 10A for the experimental design.

[0285] For knock-in experiments, transfection was performed in 24-well plate format. Cas9 plasmid DNA, gRNA plasmid DNA, and mCherry knock-in (KI) or control plasmid DNA were transfected at different weight ratios under different conditions as shown in Table 2. Cells were treated with 20ng / ml diphtheria toxin 3 days after transfection and then again 5 days after transfection. Cells were then maintained in fresh medium without diphtheria toxin. 13 days after transfection, genomes of all samples were harvested for PCR analysis. 22 days after transfection, cells of transfection condition 3, transfection negative control 1 and 2, and mCherry positive control cell lines were resuspended and analyzed by FACS.

[0286] [Table 3]

[0287] Cells with successful insertion translate mCherry containing the mutant HB-EGF gene and cells show mCherry fluorescence. As shown in Figure 10B, after diphtheria toxin selection, nearly all cells transfected with Cas9, gRNA SaW10, and mCherry HDR template were mCherry positive, while cells without the mCherry donor plasmid did not show any mCherry fluorescence. Figure 10C shows that mCherry expression is uniform across the population (Figure 10C).

[0288] Figures 10E and 10F show the results of PCR analysis using the strategy outlined in Figure 10D. The first PCR reaction (PCR1) amplifies the junction region, with the forward primer (PCR1_F primer) binding to a sequence in the genome and the reverse primer (PCR1_R primer) binding to a sequence in the GOI. Thus, only cells in which the GOI has been integrated will show a positive band in PCR1. The second PCR reaction (PCR2) amplifies the insertion region, with the forward primer (PCR2_F primer) binding to a sequence in the 5' end of the insertion and the reverse primer (PCR2_R primer) binding to a sequence in the 3' end of the insertion. Thus, amplification of PCR2 will only occur if all alleles in the cell have successfully inserted the GOI, and the amplification product will show as a single integrant band. If any wild type alleles are present, a WT band will be shown.

[0289] Figure 10E shows positive bands for all conditions tested, including introduction of Cas9, gRNA, and mCherry donor plasmids, indicating that insertion was successfully achieved. Figure 10F shows single integrant bands for all three conditions, indicating that no wild-type alleles were present in the cells tested, i.e., biallelic integration was achieved.

[0290] Example 5. Detailed Experimental Protocol Experimental protocols are provided for the examples that follow.

[0291] Construction of plasmids and template DNA A plasmid expressing S. pyogenes Cas9 (SpCas9) was constructed by cloning a sequence encoding codon-optimized SpCas9 fused to a nuclear localization signal (NLS) and a self-cleaving puromycin resistance protein (T2A-Puro) synthesized by GeneArt into the pVAX1 vector. Two versions of the SpCas9 plasmid were constructed to drive expression of SpCas9 under the control of the CMV promoter (CMV-SpCas9) or the EF1α promoter (EF1α-SpCas9). Cytidine base editor 3 (CBE3) was synthesized by GeneArt using its published sequence and cloned into the pcDNA3.1(+) vector. Two versions of the plasmid were constructed to drive CBE3 expression under the CMV promoter (CMV-CBE3) or the EF1α promoter (EF1α-CBE3). Similarly, adenine base editor 7.10 (ABE7.10) was synthesized using its published sequence and cloned into the pcDNA3.1(+) vector. Two versions of the plasmid were constructed to control ABE7.10 expression under the CMV promoter (CMV-ABE7.10) or the EF1α promoter (EF1α-ABE7.10). Individual sequence components were ordered from Integrated DNA Technologies (IDT) and assembled using Gibson assembly (New England Biolabs).

[0292] Plasmids expressing different sgRNAs were cloned by replacing the target sequence of the template plasmid. Complementary primer pairs (5'-AAAC-N20-3' and 5'-ACCG-N20-3') containing the target sequence were annealed (95°C for 5 min, followed by a ramp down to 25°C at 1°C / min) and assembled with the AarI-digested template using T4 ligase. All primer pairs are listed in Table 3A. Plasmids expressing sgRNAs targeting BFP, as well as sgRNAs targeting EGFR and CBE3 have been described in previous publications.

[0293] Plasmids acting as repair templates for the HBEGF or HIST2BC loci were ordered from GenScript or modified using Gibson assembly. Individual sequence components were ordered from IDT. Template plasmids for the HBEGF locus were designed to contain a strong splicing acceptor sequence encoded by a polyA sequence, followed by a mutated CDS sequence of HBEGF starting from exon 4, and a self-cleaving mCherry coding sequence. Template plasmids for HIST2BC were designed to contain a GFP coding sequence followed by a self-cleaving blasticidin resistance protein coding sequence. For both loci, pHMEJ and pHR were designed to contain left and right homology arms flanking the insert sequence, and pNHEJ was designed to contain no homology arms. pHMEJ was designed to contain one sgRNA cleavage site flanking each homology arm, whereas pHR did not contain this site. To compare puromycin selection with DT selection, a self-cleaving puromycin resistance protein coding sequence was inserted between the HBEGF exon sequence and the self-cleaving mCherry coding sequence (pHMEJ_PuroR).

[0294] Double-stranded DNA (dsDNA) templates were prepared by PCR amplification of plasmid pHMEJ using the primers listed in Table 3B, followed by purification using MAGBIO magnetic SPRI beads. PCR amplification was performed using high-fidelity PHUSION polymerase. ssDNA templates were prepared with the primers listed in Tables 3A-3E using the GUIDE-IT™ Long ssDNA Production System (Takara Bio Inc.). The final products were purified with MAGBIO magnetic SPRI beads and analyzed by Fragment Analyzer (Agilent). Templates for the CD34 locus were ordered from IDT as PAGE-purified oligonucleotides.

[0295] [Table 4]

[0296]

Table 5

[0297]

Table 6

[0298]

Table 7

[0299]

Table 8

[0300]

Table 9

[0301]

Table 10

[0302]

Table 11

[0303]

Table 12

[0304] Cell culture HEK293 (ATCC, CRL-1573), HCT116 (ATCC, CCL-247), and PC9-BFP cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Human induced pluripotent stem cells (iPSCs) were maintained in a Cellartis DEF-CS 500 Culture System (Takara Bio Inc.) according to the manufacturer's instructions. All cell lines were cultured at 37°C and 5% CO2. Cell lines were authenticated by STR profiling and tested negative for mycoplasma.

[0305] T cell isolation, activation, and propagation Blood from healthy donors was obtained from AstraZeneca's blood donation center (Moelndal, Sweden). Peripheral blood mononuclear cells were isolated from fresh blood using Lympoprep (STEMCELL Technologies) density gradient centrifugation, and total CD4+ T cells were enriched by negative selection using the EasySep Human CD4+ T Cell Enrichment Kit (STEMCELL Technologies). Enriched CD4+ T cells were further purified to an average purity of 98% by fluorescence-activated cell sorting (FACSAria III, BD Biosciences) based on the exclusion of CD8+CD14+CD16+CD19+CD25+ cell surface markers. The following antibodies were purchased from BD Biosciences: CD4-PECF594 (RPA-T4), CD25-PECy7 (M-A251), CD8-APCCy7 (RPA-T8), CD14-APCCy7 (MφP-9), CD16-APCCy7 (3G8), CD19-APCCy7 (SJ25-C1), CD45RO-BV510. (UCHL1). Cell sorting was performed using a FACSAria III (BD Biosciences).

[0306] CD4+ T cells were propagated in RPMI-1640 medium containing the following supplements: 1% (v / v) GlutaMAX-I, 1% (v / v) non-essential amino acids, 1 mM sodium pyruvate, 1% (v / v) L-glutamine, 50 U / mL penicillin and streptomycin, and 10% heat-inactivated FBS (all from Gibco, Life Technologies). T cells were activated using the T Cell Activation / Expansion kit (130-091-441, Miltenyi). 1 × 10 6 Cells / mL were activated at a bead-to-cell ratio of 1:2, 2 x 10 cells per well. 5 Cells were seeded in round-bottom tissue culture treated 96-well plates for 24 hours. Cells were pooled prior to electroporation.

[0307] Cell transfection 24 hours before transfection, 1.25 × 10 5 or 6.75 x 10 4 HEK293, HCT116, and PC9-BFP cells were seeded in 24-well or 48-well plates, respectively. Transfection was performed with FuGENE HD Transfection Reagent (Promega) using a transfection reagent to plasmid DNA ratio of 3:1. For the 24-well plate format, the amount and weight ratio of transfected DNA are listed in Tables 4 and 5. For the 48-well plate format, the amount of DNA was reduced by half.

[0308] [Table 13]

[0309] [Table 14]

[0310] iPSCs were transfected with FuGENE HD using a transfection reagent to DNA ratio of 2.5:1 and a reverse transfection protocol. For transfection, 4.2 × 10 cells were transfected per well of a 48-well format. 4 of cells were seeded directly onto the prepared transfection complexes described in Table 6.

[0311] [Table 15]

[0312] CD4+ T cells were electroporated with ribonucleoprotein complexes (RNPs) using 10 μL of the Neon transfection kit (MPK1096, ThermoFisher). CD3 protein was produced using a method previously described. An additional purification step was performed on a HiLoad 26 / 600 Superdex 200pg column (GE Healthcare) with a mobile phase containing 20 mM Tris-Cl pH 8.0, 200 mM NaCl, 10% glycerol, and 1 mM TCEP. Purified CBE3 protein was concentrated to 5 mg / mL in a Vivaspin protein concentration spin column (GE Healthcare) at 4°C and then flash frozen in small aliquots in liquid nitrogen. RNPs were prepared as follows: 20 μg of CBE3 protein, 2 μg of target sgRNA, and 2 μg of selected sgRNA (TrueGuide Synthetic gRNA, Life Technologies), and 2.4 μg of electroporation enhancer oligonucleotide (Sigma) (Table 3E) were mixed and incubated for 15 min. Cells were washed with PBS and 5 × 10 7 Resuspended in buffer R at a concentration of 5 × 10 cells / mL. 5Cells were electroporated with RNP using the following settings: voltage: 1600V, width: 10ms, pulse number: 3. After electroporation, cells were incubated overnight in 1mL RPMI medium supplemented with 10% heat-inactivated FBS in a 24-well plate. The next day, cells were harvested, centrifuged at 300×g for 5 min, resuspended in 1mL complete growth medium containing 500U / mL IL-2 (Prepotech), and split into 5 wells of a round-bottom 96-well plate.

[0313] Diphtheria toxin (DT) treatment Transfected HEK293, HCT116, and PC9-BFP cells were selected with 20ng / mL DT on days 3 and 5 after transfection. iPSCs were treated with 20ng / mL DT on day 3 after transfection. Growth medium supplemented with DT was replaced daily until the death of negative control cells. Transfected CD4+ T cells were treated with 1000ng / mL DT on days 1, 4, and 7 after electroporation.

[0314] Alamar Blue Assay Cell viability was analyzed using AlamarBlue cell viability reagent (ThermoFisher) according to the manufacturer's instructions.

[0315] PCR analysis PCR analysis was performed to distinguish between successful knock-ins into HBEGF intron 3 (PCR1) and wild-type sequences (PCR2). PCR reactions were performed in a volume of 20 μL using 1.5 μL of extracted genomic DNA as template. PHUSION (ThermoFisher) was used with a primer concentration of 0.5 μM according to the manufacturer's recommended protocol. The primer pair PCR1_fwd and PCR1_rev were used for PCR1 to detect knock-in junctions (annealing temperature; 62° C., extension time: 1 min), and the primer pair PCR2_fwd and PCR2_rev were used for PCR2 to detect wild-type HBEGF introns (annealing temperature; 64.5° C., extension time: 5 s). The sequences of the primer pairs are provided in Table 3D. For PCR2, the extension time was set at 5 seconds to favor amplification of the wild-type HBEGF intron 3 product (280 bp) over the integrant PCR product (2229 bp).

[0316] Flow cytometry analysis The frequency of cells expressing mCherry and GFP was assessed using a BD Fortessa flow cytometer (BD Biosciences), and flow cytometry data were analyzed with FlowJo software (Three Star).

[0317] Genomic DNA extraction and next-generation amplicon sequencing Genomic DNA was extracted from cells 3 days after transfection or complete DT selection using QuickExtract DNA extraction solution (Lucigen) according to the manufacturer's instructions. Amplicons of interest were analyzed from genomic DNA samples on the NextSeq platform (Illumina). Genomic sites of interest were amplified in the first round of PCR using primers containing NGS forward and reverse adapters (Table 3C). The first PCR was set up with 0.5 μM primers and 1.5 μL genomic DNA using NEBNext Q5 Hot Start HiFi PCR Master Mix (New England Biolabs) in a 15 μL reaction. PCR was performed with the following cycle conditions: 98°C for 2 min, 98°C for 10 s, annealing temperature of each pair of primers for 20 s (calculated using the NEB Tm Calculator), and 5 cycles of 65°C for 10 s, followed by 25 cycles of 98°C for 10 s, 98°C for 20 s, and 65°C for 10 s, followed by a final 65°C extension for 5 min. PCR products were purified using the HighPre PCR Clean-up System (MAGBIO Genomics) and the exact PCR product size and DNA concentration were analyzed with a Fragment Analyzer (Agilent). In the second round of PCR, unique Illumina indexes were added to the PCR products using KAPA HiFi HotStart Ready Mix (Roche). In the second PCR step, index primers were added and 1 ng of purified PCR product from the first PCR was used as template in a 50 μL reaction. PCR was performed with the following cycling conditions: 72°C for 3 min, 98°C for 30 s, followed by 10 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 3 min, followed by a final extension at 72°C for 5 min. Final PCR products were purified using the HighPre PCR Clean-up System (MAGBIO Genomics) and analyzed on a Fragment analyzer (Agilent).Libraries were quantified using a Qubit 4 Fluorometer (Life Technologies) and pooled and sequenced on a NextSeq instrument (Illumina).

[0318] Bioinformatics NGS sequencing data were demultiplexed using bcl2fastq software, and individual FASTQ files were analyzed using a Perl implementation of a Matlab script described in a previous publication. For quantification of indel or base edit frequency, sequencing reads were scanned for matches to two 10 bp sequences flanking an intervening window where indels or base edits may occur. If no match was found (allowing a maximum of 1 bp mismatch on each side), the read was excluded from the analysis. If the length of the intervening window was longer or shorter than the reference sequence, the sequencing read was classified as an insertion or deletion, respectively. The frequency of insertion or deletion was calculated as the percentage of reads classified as insertion or deletion within the total reads analyzed. If the length of this intervening window matched the reference sequence exactly, the read was classified as not containing an indel. For these reads, the frequency of each base at each locus was calculated within the intervening window and used as the frequency of base edits.

[0319] Cytidine base editing and DT processing of mice humanized for hHBEGF expression All mouse experiments were approved by the AstraZeneca internal committee for animal research and by the Gothenburg Ethics Committee for Experimental Animals for laboratory animals (license number: 162-2015+), which comply with the EU Directive on the protection of animals used for scientific purposes. Experimental mice were generated as double heterozygotes by breeding Alb-Cre mice (The Jackson Laboratory) to iDTR mice (expression of the transgene human HBEGF is blocked by STOP sequences flanking loxP) on a C57BL / 6NCrl genetic background. Mice were housed in negative pressure IVC caging in a temperature-controlled room (21 °C) with a 12:12 h light-dark cycle (dawn: 5:30 am, lights on: 6:00 am, dusk: 5:30 pm, lights off: 6 pm) and humidity-controlled (45–55%). Mice had free access to a regular chow diet (R36, Lactamin AB) and water.

[0320] For base editing, 6 male and 6 female 6-month-old mice were randomized into two groups, with equal numbers of male and female mice in each group. CBE3, sgRNA10, and mouse Pcsk9 (1 × 10 per mouse) were randomized into two groups, with equal numbers of male and female mice in each group. 9 Adenoviral vectors expressing sgRNA targeting IFU particles were injected intravenously. Two weeks after virus administration, all mice received DT (200 ng / kg) intraperitoneally. Control mice were terminated 24 hours after DT injection. Experimental mice were terminated 11 days after DT injection. Four mice were terminated prior to the experimental endpoint because they reached the humane endpoint of the ethics license. At necropsy, liver tissue was collected for morphological and molecular analysis.

[0321] Example 6. Amino acid substitutions in HBEGF In this example, base editing was used to scan for mutations in the human EGF-like domain that confer diphtheria toxin (DT) resistance to cells.

[0322] Detailed experimental protocol is described in Example 5. In summary, to screen sgRNAs, each sgRNA was co-transfected with CBE3 or ABE7.10 at a weight ratio of 1:4. Transfection was performed using FuGENE HD Transfection Reagent (Promega) according to the manufacturer's instructions, with a transfection reagent to plasmid DNA ratio of 3:1. Cells were treated with 20ng / mL diphtheria toxin 3 days after transfection, and then again 5 days after transfection. Cell viability was analyzed using AlamarBlue cell viability reagent (Thermo Fisher) according to the manufacturer's instructions. Genomic DNA was extracted from viable cells and analyzed by next generation sequencing (NGS) by Amplicon-Seq.

[0323] Fourteen single guide RNAs (sgRNAs) tiling exon sequences encoding the human EGF-like domain, covering all regions encoding amino acids different from the mouse EGF-like domain (Figure 24A). Each sgRNA was transiently expressed in HEK293 cells with either cytidine base editor 3 (CBE3) or adenosine base editor 7.10 (ABE7.10). The corresponding mutation, C to T (by CBE3) or A to G (by ABE7.10), was introduced into the editing window of each sgRNA. Edited cells were treated with a lethal dose of DT (20 ng / μl for HEK293 cells) 72 hours after transfection and cell growth was monitored. The results in Figure 24B show that CBE3 in combination with sgRNA7 or sgRNA10 induced effective resistance mutations to DT in HBEGF, and ABE7.10 induced resistance in combination with sgRNA5 or sgRNA10.

[0324] The combination of ABE7.10 / sgRNA5 or CBE3 / sgRNA10 was selected for further analysis. Genomic DNA from resistant cells was collected and the corresponding targeted loci were analyzed by Amplicon-Seq using next generation sequencing (NGS). The majority of mutations introduced by the combination of CBE3 and sgRNA10 in resistant cells resulted in Glu141Lys substitution in HBEGF. Approximately 90% of the variants introduced by the combination of ABE7.10 / sgRNA5 resulted in Tyr123Cys conversion in HBEGF (see Figure 24C and Figure 25A-C). No impaired growth was observed in the edited cells compared to wild-type cells, indicating that there are no adverse effects introduced by the edited HBEGF variants (Figure 25D).

[0325] Collectively, these data demonstrated that DT resistance could be induced by modifying a single amino acid in the HBEGF protein using base editing without altering cell proliferation, and thus the DT-HBEGF system could be effectively applied to select genome editing events in cells.

[0326] Example 7. Enrichment of cytidine and adenosine base editing In this example, the DT-HBEGF selection system was tested for enrichment of base editing events at a second, unrelated genomic locus. Figure 26A provides an overview of the DT-HBEGF co-selection strategy.

[0327] Detailed experimental protocol is described in Example 5. In summary, for co-targeting enrichment, Cas9 / CBE3 / ABE7.10 plasmid DNA, targeting sgRNA plasmid DNA, and selection sgRNA plasmid DNA were transfected at a weight ratio of 8:1:1. Transfection was performed using FuGENE HD transfection reagent (Promega) according to the manufacturer's instructions, with a transfection reagent to plasmid DNA ratio of 3:1. Cells were treated with 20ng / mL diphtheria toxin 3 days after transfection, and then again 5 days after transfection. Genomic DNA was extracted from surviving cells and analyzed by next generation sequencing (NGS) by Amplicon-Seq.

[0328] First, CBE co-selection in HEK293 cells was performed. sgRNAs targeting five different genomic loci were tested: DPM2 (dolichylphosphate mannosyltransferase subunit 2), EGFR (epidermal growth factor receptor), EMX1 (blank spiracles homeobox 1), PCSK9 (proprotein convertase subtilisin / kexin type 9), and DNMT3B (DNA methyltransferase 3β). Each of these sgRNAs was co-transfected into cells with CBE3 and sgRNA10 as described in Example 6, and selected cells were enriched with DT (20 ng / μL) starting 72 hours after transfection. Genomic DNA was then harvested from cells with or without selection and analyzed by NGS.

[0329] Notably, a significant increase in CT conversion rates was observed across all tested sites in DT-selected cells compared to unselected cells, with fold changes ranging from 4.1-fold to 7.0-fold (Figure 26B). For the DPM2 site, DT selection increased the total conversion rate from 20% to 94% (Figure 26B). Similar improvements in editing efficiency were also observed when the method was applied to other cell lines: a 12.8-fold increase in CT conversion rates at the PCSK9 locus in HCT116 cells and a 4.9-fold increase at the integrated BFP locus in DT-treated PC9 cells compared to untreated cells (Figure 26C).

[0330] Similar co-selection experiments were performed for enrichment of ABE editing events. Five sgRNAs were tested, including one targeting EMX1 and four others targeting two different sites in novel genomic loci: CTLA4 (cytotoxic T-lymphocyte-associated protein 4), IL2RA (interleukin-2 receptor subunit alpha), and AAVS1 (adeno-associated virus integration site 1). Each of these sgRNAs was co-transfected with ABE7.10 and sgRNA5 into HEK293 cells as described in Example 6. After 72 hours, the selected sgRNAs were The selected cells were treated with DT (20 ng / μl). Genomic DNA was extracted from both selected and unselected cells and analyzed by Amplicon-Seq. A dramatic improvement in AG conversion rates was observed across all tested targets in selected cells compared to unselected cells, from 5.7-fold to 12.7-fold. At the targeted loci CTLA4 and IL2RA, the total conversion rates increased from 4.6% to 39% and 11.5% to 77.4%, respectively (Figure 26D).

[0331] In addition to co-selection for base editing events, we also tested the possibility of co-selected indels generated by SpCas9. The four sgRNAs used in CBE co-selection (respectively targeting DPM2, EMX1, PCSK9, and DNMT3B) were tested in experiments for genome editing co-selection. Each sgRNA was co-transfected with the SpCas9 / sgRNA10 combination (described above in Example 6) into HEK293 cells to generate indels, and Amplicon-Seq was performed after selection. It was observed that the indel rate increased to over 90% across all four targets (DPM2, EMX1, PCSK9, and DNMT3B). Notably, the editing efficiency of the PCKS9 site increased from 30% to 98% through DT selection (Figure 26E).

[0332] Example 8. Efficient enrichment of biallelic knock-in events at the HBEGF locus In this example, experiments were performed to enhance the knock-in efficiency of a gene of interest or to achieve biallelic knock-in of a gene of interest.

[0333] Detailed experimental protocol is described in Example 5. In summary, for knock-in experiments, Cas9 plasmid DNA, sgRNAIn3 plasmid DNA, and template DNA were transfected at a weight ratio of 4:1:10. Transfection was performed using FuGENE HD transfection reagent (Promega) according to the manufacturer's instructions, with a transfection reagent to plasmid DNA ratio of 3:1. 22 days after transfection, cells were evaluated with BD Fortessa (BD Biosciences), and flow cytometry data was analyzed with FlowJo software (Three Star). Genomic DNA was also extracted from cells, and PCR analysis was performed to distinguish successful knock-in into HBEGF intron 3 (PCR1) from wild-type sequence (PCR2).

[0334] It was hypothesized that cells could be rendered DT-resistant by knocking in intron 3 of HBEGF, a cassette containing a strong splicing acceptor combined with a cDNA sequence containing all remaining exons downstream of exon 3 and containing a mutation that prevents DT binding. Based on the base editing screen described in Example 6 and the presence of a similar substitution in mouse HBEGF, the Glu141Lys amino acid substitution was inserted (see FIG. 25A). To further exclude the possibility of a detrimental effect of this substitution on cell compatibility, the recombinant Glu141Lys-substituted HBEGF protein showed that it was still functional in inducing p44 / p42 MAPK phosphorylation, with no significant difference observed compared to wild-type HBEGF, suggesting that its primary function in EGFR activation was maintained (FIG. 27A).

[0335] Next, a knock-in strategy was designed to introduce DT-resistant HBEGF linked to the gene of interest. First, an sgRNA (sgRNAIn3) targeting the middle region of intron 3 of HBEGF was selected, which has low predicted non-target sites and is efficient in inducing indels at the target site. The repair template was also designed to contain the remainder of the mutant HBEGF exon sequence, encoding a splice acceptor and a Glu141Lys substitution, and linked to the gene of interest (e.g., mCherry or GFP) by a T2A self-cleaving peptide (Figure 27B). With this design, wild-type cells or edited cells exhibiting small indels in intron 3 will not gain DT resistance, while cells containing the desired knock-in will be DT-resistant.

[0336] Repair templates were tested in various formats, including plasmid, double-stranded DNA (dsDNA), and single-stranded DNA (ssDNA), to determine knock-in efficiency. Templates were designed with or without homology arms or flanking sgRNAs and were expected to integrate into the HBEGF locus by non-homologous end joining (NHEJ), homologous recombination (HR), or homology-mediated end joining (HMEJ) (Figure 27C). Each template was co-transfected with SpCas9 and sgRNAIn3 into HEK293 cells to generate knock-in cells. Selection was performed as described above. Because expression of mCherry or GFP genes is linked to the mutated HBEGF gene, only cells containing the correct insertion were expected to express functional fluorescent protein. The percentage of knock-in cells (fluorescent cells) was quantified by flow cytometry analysis.

[0337] Notably, it was observed that mCherry or GFP positive cells arose regardless of the template applied, and the percentage of knock-in cells dramatically increased after selection under all conditions (Figure 27C). Notably, cells repaired with a plasmid template containing homology arms and sgRNA (pHMEJ) or a plasmid template containing only homology arms (pHR) achieved nearly 100% knock-in after selection (Figure 27C). Among all the templates tested, pHMEJ was shown to be the most efficient, with only 34.8% of knock-in cells obtained without selection (Figure 27C). These observations are consistent with additional results showing biallelic variation in base editing selection (Figure 24B), suggesting that cells may require biallelic knock-in to survive DT treatment. Two pairs of primers were designed to check the genomic status of edited cells, one pair amplifying the 5' junction of the knock-in sequence (PCR1) and the other pair amplifying the wild-type sequence of the HBEGF intron (PCR2). PCR analysis was performed on cells repaired with pHMEJ template with or without selection, respectively. Although both samples showed bands of homologous knock-in (PCR1), only wild-type bands were detected in the unselected sample (Figure 27E), indicating that all cells obtained biallelic knock-in after DT selection.

[0338] The DT selection method was further compared with the traditional antibiotic-dependent selection method for enrichment of knock-in events. A novel pHMEJ template was designed to contain both DT resistance mutations and puromycin resistance genes, and the expression of these two selection markers was linked by a P2A self-cleaving peptide (Figure 27D). To test this novel template for knock-in, knock-in cells were enriched with either DT or puromycin, followed by flow cytometry analysis. Interestingly, nearly 100% of mCherry-positive cells were observed in both populations, but DT-enriched cells showed dramatically higher mean fluorescence intensity compared to puromycin-enriched cells (Figure 27D). This observation, together with PCR analysis (Figure 27E), indicated that DT selection, but not puromycin selection, enriched cells containing biallelic knock-ins.

[0339] This genetic engineering strategy is referred to herein as "Xential" (recombination (X) in a locus essential for cell viability).

[0340] Example 9. Enrichment of knock-out and knock-in events by Xential co-selection In this example, Xential knock-in was tested for enrichment of knock-out or knock-in events at a second, unrelated locus.

[0341] Detailed experimental protocol is described in Example 5. In summary, for the Xential co-selection experiment, the amount of each transfected plasmid is listed in Table 7 below. Transfection was performed using FuGENE HD Transfection Reagent (Promega) according to the manufacturer's instructions, with a transfection reagent to plasmid DNA ratio of 3:1. Cells were treated with 20ng / ml diphtheria toxin 3 days after transfection, and then again 5 days after transfection. 22 days after transfection, cells were evaluated with BD Fortessa (BD Biosciences), and flow cytometry data was analyzed with FlowJo software (Three Star). Genomic DNA was also extracted from cells, and the same PCR and Amplicon-Seq analyses were performed as described for the previous examples.

[0342] [Table 16]

[0343] First, enrichment of knockout events was tested. The same four sgRNAs (targeting DPM2, EMX1, PCSK9, and DNMT3B, respectively) tested in the previous indel enrichment experiment described in Example 7 (Figure 26E) were used. Each sgRNA was co-delivered with SpCas9, sgRNAIn3, and pHMEJ templates into HEK293 cells, and DT selection was performed as described in Figure 28A. Genomic DNA was extracted from these cells and analyzed by Amplicon-Seq. Significant improvement in editing efficiency (ranging from 4.4-fold to 14.3-fold improvement) was observed for all targets in selected cells compared to unselected cells. Notably, editing efficiency at the EMX1 locus increased from 22% to 88% by DT selection (Figure 28B). All surviving cells maintained mCherry expression, indicating that the edited cells maintained the precise knock-in at the HBEGF locus (Figure 28D).

[0344] We then tested Xential for co-selection of knock-in events. Two forms of repair template plasmids were designed (one pHR and one pHMEJ) to introduce a C-terminal GFP tag into histone protein H2B (HIST2BC) using the same sgRNA. SpCas9, sgRNA, and two templates targeting HIST2BC and HBEGF were co-delivered into HEK293 cells, and knock-in efficiency was analyzed by the percentage of GFP (HIST2BC) or mCherry (HBEGF). Either form of the provided template resulted in significantly improved knock-in efficiency after DT selection. In the case of the pHR template, the efficiency improved by 6.4-fold, and in the case of the pHMEJ template, the efficiency improved by 5.3-fold to reach 48% (Figure 28C). By reducing the ratio of the amount of sgRNA and template at the HBEGF locus to that at the HIST2BC locus, the knock-in efficiency at the HIST2BC locus can be increased in the selected cells, indicating that the enrichment fold is adjustable (Figure 28C). The percentage of GFP-positive cells in the enriched cells increased from 23% to 42% to 48% by applying increasing weight ratios of the repair plasmid at the HIST2BC locus to the repair plasmid at the HBEGF locus, from 1:1 to 3:1 and 1:1 to 4:1, respectively, while the percentage of mCherry-positive cells remained at nearly 100% (Figure 28E). This method also demonstrated the enrichment of the efficiency of oligo-mediated knock-in at the CD34 locus. When co-selection was applied, a 26-fold increase in the percentage of knock-in cells was observed, suggesting the flexibility of template usage in knock-in-mediated co-selection (Figure 28F).

[0345] Example 10. Enrichment of base editing and knock-in events in iPSCs In this example, experiments were performed using DT-HBEGF selection to enrich for base editing events and precise knock-in events in iPSCs.

[0346] Detailed experimental protocol is described in Example 5. In summary, for CBE / ABE co-selection of iPSCs, iPSC / CBE3 / ABE7.10 plasmid DNA, targeting sgRNA plasmid DNA, and selection sgRNA plasmid DNA were transfected at a weight ratio of 8:1:1. For Xential knock-in in iPSCs, Cas9 plasmid DNA, sgRNAIn3 plasmid DNA, and template plasmid DNA were transfected at a weight ratio of 4:1:10. Transfection was performed using FuGENE HD transfection reagent (Promega) according to the manufacturer's instructions, with a transfection reagent to plasmid DNA ratio of 2.5:1 and reverse transfection protocol. Three days after transfection, cells were treated with 20ng / ml diphtheria toxin. Growth medium supplemented with DT was replaced daily until the negative control cells died. Xential knock-in cells were assayed on a BD Fortessa (BD Biosciences) and flow cytometry data were analyzed with FlowJo software (Three Star). Genomic DNA was also extracted from the cells and the same PCR and Amplicon-Seq analyses were performed as described for the previous examples.

[0347] Two sgRNAs were selected for CBE and ABE co-selection, one targeting EMX1, a locus that has been widely tested in other genome editing studies, and the other targeting CTLA4, a gene that has been extensively studied for its role in immune signaling. Each sgRNA was co-transfected into iPSCs with the CBE3 / sgRNA10 or ABE7.10 / sgRNA5 pair. Selection was performed by DT treatment (20ng / μL) starting 72 hours after transfection. Genomic DNA was extracted at confluence and the targeted loci were analyzed by Amplicon-Seq using NGS. Notably, a dramatic increase in editing efficiency upon DT selection was observed at all tested sites for both CBE and ABE. The increase in CBE editing efficiency ranges from 19-fold to 60-fold across these two sites, while the increase in ...

Claims

1. 1. An in vitro method for introducing site-specific mutations into a target polynucleotide in a target cell in a cell population, comprising: (a) introducing into the cell population (i) a base editing enzyme; and (ii) (1) hybridizing with a gene encoding a cytotoxic agent (CA) receptor; (2) A first guide polynucleotide that forms a first complex with the base editing enzyme, a first guide polynucleotide, wherein the base editing enzyme of the first complex provides a mutation in the gene encoding the CA receptor, and the mutation in the gene encoding the CA receptor forms CA resistant cells in the cell population; (iii) (1) hybridizing with the target polynucleotide; (2) A second guide polynucleotide that forms a second complex with the base editing enzyme, The base editing enzyme of the second complex provides a mutation in the target polynucleotide; and Introducing (b) contacting the cell population with the CA; (c) enriching the target cells containing the mutation in the target polynucleotide by selecting the CA resistant cells from the cell population; A method comprising:

2. 1. An in vitro method for determining the efficacy of a base editing enzyme in a cell population, comprising: (a) introducing into the cell population (i) a base editing enzyme; and (ii) (1) hybridizing with a gene encoding a cytotoxic agent (CA) receptor; (2) A first guide polynucleotide that forms a first complex with the base editing enzyme, a first guide polynucleotide, wherein the base editing enzyme of the first complex introduces a mutation in the gene encoding the CA receptor, and the mutation in the gene encoding the CA receptor forms CA resistant cells in the cell population; (iii) (1) hybridizing with a target polynucleotide; (2) A second guide polynucleotide that forms a second complex with the base editing enzyme, a second guide polynucleotide, wherein the base editing enzyme of the second complex introduces a mutation in the target polynucleotide; and Introducing (b) contacting the cell population with the CA to isolate CA-resistant cells; (c) determining the potency of the base editing enzyme by determining the ratio of the number of CA resistant cells to the total number of cells in the cell population of step (a); and A method comprising:

3. The method of claim 1 or 2, wherein the base editing enzyme comprises a DNA targeting domain and a DNA editing domain, and the DNA targeting domain comprises Cas9 comprising a mutation in a catalytic domain.

4. The method of any one of claims 1 to 3, wherein the base editing enzyme comprises a catalytically inactive Cas9 and a DNA editing domain.

5. The method of any one of claims 1 to 3, wherein the base editing enzyme comprises a mutation relative to wild-type Cas9 at amino acid residue D10 or H840 (numbering is relative to SEQ ID NO: 3), a Cas9 capable of generating a single-stranded DNA break (nCas9), and a DNA editing domain.

6. The method of any one of claims 3 to 5, wherein the Cas9 is at least 90% identical to SEQ ID NO: 3 or 4.

7. The method of any one of claims 3 to 6, wherein the DNA editing domain comprises a deaminase selected from cytidine deaminase and adenosine deaminase.

8. 8. The method of claim 7, wherein the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase, activation-induced cytidine deaminase (AID), ACF1 / ASE deaminase, ADAT deaminase, or ADAR deaminase, or the deaminase is APOBEC1.

9. The method of any one of claims 3 to 8, wherein the base editing enzyme further comprises a DNA glycosylase inhibitor domain, or the base editing enzyme further comprises a uracil DNA glycosylase inhibitor (UGI) domain.

10. The method according to any one of claims 3 to 9, wherein the base editing enzyme comprises nCas9 and a deaminase selected from cytidine deaminase and adenosine deaminase.

11. 11. The method of claim 10, wherein the base editing enzyme comprises a polypeptide sequence that is at least 90% identical to SEQ ID NO:

6.

12. 12. The method of claim 11, wherein the base editing enzyme is BE3.

13. 13. The method of any one of claims 1 to 12, wherein the first and / or second guide polynucleotide further comprises a tracrRNA sequence.

14. The method of any one of claims 1 to 13, wherein the cell population is human cells.

15. 15. The method of any one of claims 1 to 14, wherein the mutation in the gene encoding the CA receptor is a cytidine (C) to thymine (T) point mutation or an adenine (A) to guanine (G) point mutation.

16. 16. The method of any one of claims 1 to 15, wherein the CA is diphtheria toxin and the cytotoxic agent (CA) receptor is a receptor for diphtheria toxin.

17. The method of claim 16, wherein the CA receptor is a heparin-binding EGF-like growth factor (HB-EGF) comprising the polypeptide sequence of SEQ ID NO:

8.

18. 17. The method of claim 16, wherein the base editing enzyme of the first complex provides a mutation at one or more of amino acids 107-148 of HB-EGF (SEQ ID NO: 8); or the base editing enzyme of the first complex provides a mutation at one or more of amino acids 138-144 of HB-EGF (SEQ ID NO: 8); or the base editing enzyme of the first complex provides a GLU141 to LYS141 mutation in HB-EGF (SEQ ID NO: 8).

19. 19. The method of any one of claims 1 to 18, wherein the base editing enzyme of the first complex provides a mutation in a region of HB-EGF that binds to diphtheria toxin.

20. 20. The method of any one of claims 1 to 19, wherein the mutation in the target polynucleotide is a point mutation from cytidine (C) to thymine (T) in the target polynucleotide, or a point mutation from adenine (A) to guanine (G) in the target polynucleotide.

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