WO2022238958A1 - Multiplex gene editing - Google Patents

Abstract

Provided herein are methods and compositions for gene editing at multiple sites. The methods comprise delivering to cells two or more programmable gene editing systems and an X-family DNA polymerase, wherein the programmable gene editing systems generate cleaved genomic loci and the X-family DNA polymerase catalyzes addition of nucleotides to 3' termini of the cleaved genomic loci, such that chromosomal translocations are reduced between the cleaved genomic loci, thereby facilitating multiplex gene editing.

Description

1

MULTIPLEX GENE EDITING

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63/187,755, filed May 12, 2021 , the disclosure of which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on May 10, 2022 is named 100867-727793_CT162-

PCT1_Sequence_Listing_ST25.txt, and is about 18,000 bytes in size.

FIELD

[0003] The present disclosure relates to the field of CRISPR-based multiplex gene editing.

BACKGROUND

[0004] Multiplex gene editing, in which multiple gene editing systems are introduced simultaneously into cells, have greatly expanded the scope and efficiency of gene editing applications. Chromosomal rearrangements, however, can result from the simultaneous generation of several double-strand breaks at multiple genomic loci. As such, there is a need for methods of reducing chromosomal translocations during multiplex gene editing.

SUMMARY

[0005] Various aspects of the present disclosure relate to methods for gene editing at multiple gene loci. In various aspects, the methods herein comprise delivering to a cell: (a) two or more CRISPR-Cas nuclease systems or nucleic acids encoding two or more CRISPR-Cas nuclease systems, wherein each CRISPR-Cas nuclease system is targeted to a different genomic locus; and (b) an X-family DNA polymerase or a nucleic acid 2 encoding an X family DNA polymerase; wherein the two or more CRISPR-Cas nuclease systems generate cleaved genomic loci and the X-family DNA polymerase catalyzes addition of nucleotides to 3’ termini of the cleaved genomic loci, such that chromosomal translocations are reduced between the cleaved genomic loci, thereby facilitating editing at multiple genomic loci.

[0006] In various aspects, the addition of nucleotides may increase nucleotide insertion frequencies, increase insertion lengths, and/or decrease nucleotide deletion frequencies as compared to a comparable cell in which the X-family DNA polymerase is not delivered.

[0007] In various aspects, chromosomal translocations may be reduced by at least 5% when two genomic loci are edited as compared to a comparable cell in which the X-family DNA polymerase is not delivered.

[0008] In any of the methods provided herein, each CRISPR-Cas nuclease system may comprise a CRISPR-Cas nuclease and a gRNA. In various aspects, the CRISPR- Cas nuclease may be a type II CRISPR-Cas nuclease ora type V CRISPR-Cas nuclease. As a non-limiting example, the CRISPR-Cas nuclease may be SpCas9, SluCas9, SaCas9, ora variant thereof with increased fidelity. In any of the methods provided herein, the gRNA may be a single molecule gRNA (sgRNA).

[0009] In any of the methods provided herein, each of the CRISPR-Cas nuclease systems may be delivered as a ribonucleoprotein (RNP) complex. Alternatively, each of the CRISPR-Cas nuclease systems may be delivered as a CRISPR-Cas nuclease and a gRNA. Alternatively, each of the CRISPR-Cas nuclease systems may be delivered as mRNA encoding the CRISPR-Cas nuclease and a gRNA. As another option, each of the CRISPR-Cas nuclease systems may be delivered as DNA encoding the CRISPR-Cas nuclease and DNA encoding the gRNA.

[0010] In any of the methods provided herein, the X-family DNA polymerase may be a terminal deoxyribonucleotidyl transferase (TdT), polymerase lambda (Pol l), or polymerase mu (Pol m). In some aspects, the X-family DNA polymerase is a short isoform of TdT (TdtS) (e.g., having an amino acid sequence of SEQ ID NO: 34). 3

[0011] In any of the methods provided herein, the X-family DNA polymerase may be delivered as mRNA, protein, or DNA.

[0012] In any of the methods provided herein, two CRISPR-Cas nuclease systems are delivered to the cell. In other methods, three CRISPR-Cas nuclease systems are delivered to the cell. In still other methods, four CRISPR-Cas nuclease systems are delivered to the cell. In various aspects, five CRISPR-Cas nuclease systems may be delivered to the cell.

[0013] In any of the methods herein, delivering may comprise electroporation.

[0014] In any of the methods herein, the cell may be a mammalian cell. In some aspects, the cell is other than an immature, pre-B or pre-T lymphoid cell, a leukemia cell, a lymphoma cell, or an immortalized cell derived therefrom. In some aspects, the cell may be a primary T cell or an NK cell.

[0015] In any of the methods provided, the cell may be in vitro, ex vivo, in situ, or in vivo.

[0016] Further aspects of the present disclosure are directed to a cell comprising (a) two or more CRISPR-Cas nuclease systems and (b) a short isoform of terminal deoxyribonucleotidyl transferase (TdTS), wherein the CRISPR-Cas nuclease systems and the TdTS are exogenous to the cell.

[0017] In various aspects, each CRISPR-Cas nuclease systems of (a) may be a ribonucleoprotein (RNP) complex. In other aspects, each CRISPR-Cas nuclease system of (a) may comprise CRISPR-Cas mRNA and gRNA.

[0018] In various aspects, (b) may comprise mRNA encoding TdTs or may comprise TdTS protein.

[0019] In other aspects, each CRISPR-Cas nuclease system of (a) comprises DNA encoding CRISPR-Cas nuclease and DNA encoding gRNA and (b) comprises DNA encoding TdTS.

[0020] Further aspects of the present disclosure are directed to a kit for multiplex gene editing, the kit comprising (a) two or more CRISPR-Cas nuclease systems, and (b) a short isoform of terminal deoxyribonucleotidyl transferase (TdTS). In various aspects, each CRISPR-Cas nuclease systems of (a) in the kits provided herein may be a RNP complex. 4

In other aspects, each CRISPR-Cas nuclease system of (a) may comprise CRISPR-Cas mRNA and gRNA. In various aspects, (b) may comprise mRNA encoding TdTS or may comprise TdTS protein. In various aspects, the kits provided are as described herein each CRISPR-Cas nuclease system of (a) comprises DNA encoding CRISPR-Cas nuclease and DNA encoding gRNA and (b) comprises DNA encoding TdTS.

[0021 ] In further aspects, any of the kits provided herein may further comprise at least one reagent for gene editing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1A presents editing efficiencies with NPM1 or ALK gRNAs in Jurkat or T cells. Plotted is the percent of indels in Jurkat or T cells for each gRNA.

[0023] FIG. 1B presents indel spectra for T cells (upper) and Jurkat cells (lower). Plotted are the frequencies of deletions or insertions, with darker shading indicating higher frequencies.

[0024] FIG. 2A presents editing efficiencies after multiplex editing at PD1 and CD70 loci in Jurkat and T cells.

[0025] FIG. 2B presents indel spectra at PD1 and CD70 loci in T cells (left) and Jurkat cells (right). Plotted are the frequencies of insertions or deletions, with darker shading indicating higher frequencies.

[0026] FIG. 3A diagrams translocation variants of PD1/CD70.

[0027] FIG. 3B presents the percentage of T1-T4 translocation variants of PD1/CD70 in Jurkat and T cells.

[0028] FIG. 4 presents Western images showing the levels of TdT and alpha-tubulin in Jurkat and T cells.

[0029] FIG. 5 presents Western images showing the levels of TdT and alpha-tubulin in Jurkat cells edited to knock out TdT (labeled T1 , T11 , T14), and wild type Jurkat and T cells.

[0030] FIG. 6A presents indel spectra at PD1 and CD70 loci in wild type Jurkat (TdT+), edited Jurkat (TdT-), and T cells. 5

[0031] FIG. 6B presents editing efficiencies at PD1 and CD70 loci in Jurkat (TdT+), Jurkat (TdT-), and T cells.

[0032] FIG. 6C presents the percentage of each translocation variant of PD1/CD70 in Jurkat (TdT+), Jurkat (TdT-), and T cells.

[0033] FIG. 7 shows the kinetics of expression of TdT short isoform (TdTS), TdT long isoforms (TdTL1 , TdTL2, TdTL3), and Cas9 after delivery of the corresponding mRNA to T cells.

[0034] FIG. 8A shows the levels of TdTS after delivery of increasing amounts of TdTS mRNA to T cells and the level in Jurkat (TdT+) cells.

[0035] FIG. 8B presents the relative levels of TdTS to alpha-tubulin in under the conditions described in FIG. 8A.

[0036] FIG. 9A presents indel spectra at PD1 and CD70 loci in T cells provided with TdTS mRNA or control mRNA.

[0037] FIG. 9B presents the percentage of each translocation variant of PD1/CD70 in T cells provided with TdTS mRNA or control mRNA.

[0038] FIG. 9C presents editing efficiencies at PD1 and CD70 loci in T cells provided with TdTS mRNA or control mRNA.

[0039] FIG. 10A presents indel spectra at PD1 and CD70 loci in T cells provided with short or long isoforms of TdT.

[0040] FIG. 10B shows the percentage of each translocation variant of PD1/CD70 in T cells provided with short or long isoforms of TdT.

[0041 ] FIG. 11 A presents editing efficiencies at PD1 and CD70 loci in T cells provided with TdTS mRNA or control mRNA and Cas9 RNP.

[0042] FIG. 11B presents editing efficiencies at PD1 and CD70 loci in T cells provided with TdTS mRNA and Cas9 mRNA + sgRNA.

[0043] FIG. 12A shows indel spectra at the CD70 locus in T cells provided with TdTS mRNA and Cas9 RNP.

[0044] FIG. 12B shows indel spectra at PD1 locus in T cells provided with TdTS mRNA and Cas9 RNP. 6

[0045] FIG. 12C present indel spectra at the CD70 locus in T cells provided with TdTS mRNA and Cas9 mRNA + sgRNA.

[0046] FIG. 12D present indel spectra at the PD1 locus in T cells provided with TdTS mRNA and Cas9 mRNA + sgRNA.

[0047] FIG. 13A shows the percentage of each translocation variant of PD1/CD70 in T cells provided with TdTS mRNA and Cas9 RNP.

[0048] FIG. 13B shows the percentage of each translocation variant of PD1/CD70 in T cells provided with TdTS mRNA and Cas9 mRNA + sgRNA.

DETAILED DESCRIPTION

[0049] The present disclosure provides means for multiplex gene editing in which two or more programmable gene editing systems targeted to different genomic loci and an X- family DNA polymerase are delivered to cells. The endonucleases of the two or more programmable gene editing systems generate double stranded DNA breaks at the targeted genomic loci and the X-family DNA polymerase adds nucleotides to the 3’ termini of the double strand DNA breaks, leading to insertion-rich indel patterns. The insertion- rich indel patterns may reduce chromosomal translocations by a) disrupting the ability of the endonuclease to recognize and recut DNA after mutagenic scarring, b) facilitating preferential end-joining to its intrachromosomal partner through synapsis of the double strand DNA break, and c) shifting end joining pathway selection from microhomology- mediate end joining (MMEJ) to non-homologous end joining (NHEJ). The addition of nucleotides by the X-family DNA polymerase at the nuclease-induced double strand DNA breaks reduces the iterative rounds of nuclease recutting by subverting error-free repair and restoration of the DNA’s original configuration. As such, ectopic supplementation the X-family DNA polymerase also serves as an adjuvant for editing efficiency.

(I) Methods for Multiplex Gene Editing

[0050] One aspect of the present disclosure encompasses methods for gene editing at multiple genomic loci, the method comprising delivering to a cell (a) two or more programmable gene editing systems or nucleic acid encoding two or more programmable 7 gene editing systems, wherein each programmable gene editing system is targeted to a different genomic locus, and (b) an X-family DNA polymerase or nucleic acid encoding an X family DNA polymerase, wherein the two or more programmable gene editing systems generate cleaved genomic loci and the X-family DNA polymerase catalyzes addition of nucleotides to 3’ termini of the cleaved genomic loci, such that chromosomal translocations are reduced between the cleaved genomic loci, thereby facilitating editing at multiple genomic loci.

Programmable Gene Editing Systems

[0051 ] The method comprises delivery of two or more programmable editing systems such that two or more genomic loci can be edited simultaneously. Programmable gene editing systems are proteins or ribonucleoprotein complexes that can be programmed or engineered to target precise sequences in the genome and introduce double-stranded breaks at the targeted DNA loci. Suitable programmable gene editing systems include RNA-guided CRISPR-Cas systems, zinc finger nucleases, transcription activator-like effector-based nucleases, homing endonucleases, and hybrid nucleases.

[0052] CRISPR-Cas Systems. The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein) system is a naturally occurring defense mechanism in prokaryotes and archaea that has been repurposed as a RNA-guided DNA-targeting platform used for gene editing. A CRISPR-Cas system comprises a noncoding guide RNA (gRNA) to target the DNA and a CRISPR-Cas nuclease that cleaves the DNA.

[0053] The gRNA drives sequence recognition and specificity of the CRISPR-Cas system through Watson-Crick base pairing typically with a ~20 nucleotide (nt) sequence in the target locus, wherein the target sequence is adjacent to a specific short DNA motif referred to as a protospacer adjacent motif (PAM). The gRNA forms an RNA-duplex structure that is bound by the CRISPR-Cas nuclease to form the catalytically active CRISPR-Cas complex, which can then cleave the target locus. Once the CRISPR-Cas complex is bound to DNA at a target site, two independent nuclease domains within the 8

CRISPR-Cas nuclease cleave opposite strands of the DNA leaving a double-strand break (DSB).

[0054] CRISPR-Cas systems include Types I, II, III, IV, V, and VI systems. In some embodiments, the CRISPR-Cas system can be a Type II CRISPR-Cas system (e.g., Cas9). In other embodiments, the CRISPR-Cas system can be a Type V CRISPR-Cas system (e.g., Cas12a, previously termed Cpf1). The CRISPR-Cas9 nuclease or the CRISPR-Cas12a nuclease used in the methods described herein can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99% sequence identity to a wild-type CRISPR-Cas9 nuclease or CRISPR-Cas12a nuclease.

[0055] The Type-ll CRISPR-Cas system component can be from a Type-IIA, Type- IIB, or Type-IIC system. Cas9 and its orthologs are encompassed. Non-limiting exemplary species that the Cas9 nuclease or other components are from include Streptococcus pyogenes, Streptoccoccus lugdunensis, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter 9 racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, or Acaryochloris marina. In some embodiments, the Cas9 protein can be from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein can be from S. lugdunensis (SluCas9). In some embodiments, the Cas9 protein can be from Staphylococcus aureus (SaCas9). In some embodiments, a suitable Cas9 protein for use in the present disclosure is any disclosed in W02019/183150 and WO2019/118935, each of which is incorporate herein by reference.

[0056] In some embodiments, the CRISPR-Cas nuclease can be engineered for increased fidelity. As used herein, the term “fidelity” when used in reference to a CRISPR/Cas system comprising a Cas nuclease and gRNA refers to the specificity of the system for a target site in a DNA molecule (e.g., genomic DNA molecule) that is homologous (e.g., perfect match) to the gRNA spacer sequence. In some embodiments, a CRISPR-Cas system with increased fidelity has reduced activity at off-target sites in the DNA molecule, i.e. , sites that are an imperfect match to the gRNA spacer sequence. [0057] In some embodiments, the CRISPR-Cas system can comprise a Cas variant (e.g., a SpCas9 functional derivative, a SluCas9 functional derivative, a SaCas9 functional derivative) comprising one or more mutations for increased fidelity. In some embodiments, the one or more mutations result in reduced activity of the CRISPR-Cas system at off-target sites in the DNA molecule, for example, compared to a system comprising an unmodified version of the Cas nuclease (e.g., wild-type SpCas9 nuclease, wild-type SluCas9 nuclease, wild-type SaCas9 nuclease). In some embodiments, the CRISPR-Cas system has substantially equivalent activity for inducing cleavage at an on- target site in the DNA molecule, for example, as compared to the system comprising an unmodified version of the Cas nuclease. Methods of making Cas variants with increased fidelity are known in the art. For example, in some embodiments, a method of structure- guided engineering is used to make a Cas variant with increased fidelity. 10

[0058] In some embodiments, the CRISPR-Cas system described herein comprises a Cas9 nuclease comprising one or more mutations for increased fidelity. In some embodiments, the Cas9 nuclease is derived from S. pyogenes, wherein the Cas nuclease comprises one or more mutations relative to wild-type SpCas9 for increased fidelity. In some embodiments, the Cas9 nuclease is derived from S. aureus, wherein the Cas nuclease comprises one or more mutations relative to wild-type SaCas9 for increased fidelity. In some embodiments, the Cas9 nuclease is derived from S. lugdunensis, wherein the Cas nuclease comprises one or more mutations relative to wild-type SluCas9 for increased fidelity.

[0059] A suitable Cas9 nuclease with increased fidelity for use in the present disclosure includes anyone described in US2019/0010471; US2018/0142222; US 9,944,912; W02020/057481 ; US2019/0177710; US2018/0100148; US 10,526,591 ; and US20200149020; each of which is incorporated herein by reference in their entirety. [0060] In some embodiments, the CRISPR-Cas nuclease can be engineered by one or more amino acid substitutions, deletions, and/or insertions to have improved targeting specificity, improved fidelity, altered PAM specificity, decreased off-target effects, and/or increased stability. Non-limiting examples of one or more mutations that improve targeting specificity, improve fidelity, and/or decrease off-target effects include N497A, R661A, Q695A, K810A, K848A, K855A, Q926A, K1003A, R1060A, and/or D1135E (with reference to the numbering system of SpCas9).

[0061] The CRISPR-Cas nuclease can be linked to one or more nuclear localization signal (NLS) at or within 50 amino acids from the N-terminal end and/or the C-terminal end. NLSs are well known in the art. For example, the NLS can be the SV40 Large T- antigen NLS, nucleoplasmin NLS, c-Myc NLS, or derivatives thereof. The linkage between the CRISPR nuclease and the NLS can be a direct or it can be indirect via an intervening linker sequence. Suitable linker sequences are well known in the art.

[0062] Exemplary CRISPR-Cas nucleases include the Cas9 proteins as published in Fonfara et al. , “Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems,” Nucleic Acids Research, 2014, 42: 2577-2590. The CRISPR-Cas gene naming system has undergone extensive 11 rewriting since the Cas genes were discovered. Fonfara et al. also provides PAM sequences for the Cas9 polypeptides from various species.

[0063] A gRNA comprises at least (i) a spacer sequence that hybridizes to a target sequence and (ii) a CRISPR repeat sequence (crRNA). In Type II CRISPR-Cas systems, the gRNA also comprises a tracrRNA (trans-activating RNA) sequence. In Type II CRISPR-Cas systems, the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. A type II system gRNA can be a single molecule (i.e. , single molecule gRNA or sgRNA) or can comprise two separate molecules (e.g., crRNA and tracrRNA). In Type V CRISPR-Cas systems, a crRNA sequence forms a duplex. In both systems, the duplex binds the CRISPR-Cas nuclease such that the gRNA and the CRISPR-Cas nuclease form a complex. The gRNA, thus, directs the activity of the CRISPR-CAS nuclease and provides specificity in targeting the CRISPR-Cas nuclease to the targeted genomic location.

[0064] Each gRNA comprises a spacer sequence that defines the target sequence of a target nucleic acid (or target locus). The “target sequence” is adjacent to a PAM sequence in the target DNA and is cleaved by the CRISPR-Cas nuclease. The “target nucleic acid” is a double-stranded molecule; one strand comprises the target sequence and is referred to as the “PAM strand,” and the other complementary strand is referred to as the “non-PAM strand.” One of skill in the art recognizes that the gRNA spacer sequence hybridizes to the reverse complement of the target sequence, which is located in the non-PAM strand of the target nucleic acid. The spacer of a gRNA interacts with a target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus varies depending on the target sequence of the target locus.

[0065] In some embodiments, the spacer sequence of the gRNA may be from about 15 to about 30 nucleotides long, or about 18 to 22 nucleotides long. In some embodiments, the spacer sequence is 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence is 18, 19, 20, or 21 nucleotides long. In some embodiments, the spacer sequence is 20 nucleotide long. (See, e.g., Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471 , 12

602-607 (2011 )). In general, the spacer sequence has at least about 90%, at least about 95%, or at least about 99% sequence identity to the target sequence in the target nucleic acid. In certain embodiments, the spacer sequence has 100% sequence identity to the target sequence.

[0066] In some embodiments, a single molecule gRNA in a Type II CRISPR-Cas system can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may comprise one or more hairpins.

[0067] A double molecule gRNA in a Type II CRISPR-Cas system can comprise a first strand comprising, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence (crRNA), and a second strand comprising a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3’ tracrRNA sequence and an optional tracrRNA extension sequence.

[0068] A gRNA in a Type V CRISPR-Cas system can comprise, in the 5' to 3' direction, a minimum CRISPR repeat sequence (crRNA) and a spacer sequence.

[0069] The gRNA, in some embodiments, can comprise one or more uracil residues at the 3’ end of the gRNA sequence. For example, the gRNA may comprise one (U), two (UU), three (UUU), four (UUUU) or more uracils at the 3’ end of the gRNA sequence. In some embodiments, the gRNA comprises 5, 6, 7, or 8 uracils at the 3’ end of the gRNA sequence. In some embodiments, the gRNA comprises 1 to 8, 2 to 8, 3 to 8, or 4 to 8 uracils at the 3’ end of the gRNA sequence.

[0070] The gRNA can be unmodified or modified. For example, modified gRNAs may comprise one or more 2'-0-methyl phosphorothioate nucleotides. For example, the gRNA may comprise 2'-0-methyl-phosphorothioate residues at the 5' end/or the 3' end. 13

In some embodiments, the gRNA comprises three 2'-0-methyl-phosphorothioate residues at the 5' end and 2'-0-methyl-phosphorothioate residues at the 3' end.

[0071 ] Zinc finger nucleases (ZFNs). Among the various types of modular nucleases are ZFNs, which are proteins comprised of an engineered zincfinger DNA binding domain linked to the catalytic domain of the type II endonuclease Fokl. Because Fokl functions only as a dimer, a pair of ZFNs are engineered to bind to cognate target “half-site” sequences on opposite DNA strands and with precise spacing between them to enable the catalytically active Fokl dimer to form. Upon dimerization of the Fokl domain, which itself has no sequence specificity per se, a DNA double-strand break is generated between the ZFN half-sites as the initiating step in genome editing.

[0072] The DNA binding domain of each ZFN is typically comprised of 3-6 zinc fingers of the abundant Cys2-His2 architecture, with each finger primarily recognizing a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interaction with a fourth nucleotide also can be important. Alteration of the amino acids of a finger in positions that make key contacts with the DNA alters the sequence specificity of a given finger. Thus, a four-finger zinc finger protein will selectively recognize a 12 bp target sequence, where the target sequence is a composite of the triplet preferences contributed by each finger, although triplet preference can be influenced to varying degrees by neighboring fingers. An important aspect of ZFNs is that they can be readily re-targeted to almost any genomic address simply by modifying individual fingers. In most applications of ZFNs, proteins of 4-6 fingers are used, recognizing 12-18 bp respectively. Hence, a pair of ZFNs will typically recognize a combined target sequence of 24-36 bp, not including the typical 5-7 bp spacer between half-sites. The binding sites can be separated further with larger spacers, including 15-17 bp. A target sequence of this length is likely to be unique in the human genome, assuming repetitive sequences or gene homologs are excluded during the design process. Nevertheless, the ZFN protein-DNA interactions are not absolute in their specificity so off-target binding and cleavage events do occur, either as a heterodimer between the two ZFNs, or as a homodimer of one or the other of the ZFNs. The latter possibility has been effectively eliminated by engineering the dimerization interface of the Fokl domain to create “plus” and “minus” variants, also 14 known as obligate heterodimer variants, which can only dimerize with each other, and not with themselves. Forcing the obligate heterodimer prevents formation of the homodimer. This has greatly enhanced specificity of ZFNs, as well as any other nuclease that adopts these Fokl variants.

[0073] A variety of ZFN-based systems have been described in the art, modifications thereof are regularly reported, and numerous references describe rules and parameters that are used to guide the design of ZFNs; see, e.g., Segal et al. , Proc Natl Acad Sci, 1999 96(6):2758-63; Dreier et al., J Mol Biol., 2000, 303(4):489-502; Liu et al., J Biol Chem., 2002, 277(6):3850-6; Dreier et al., J Biol Chem., 2005, 280(42):35588-97; and Dreier et al., J Biol Chem. 2001 , 276(31 ):29466-78.

[0074] Transcription activator-like effector nucleases (TALENS). Another type of modular nucleases are TALENs, which like ZFNs comprise an engineered DNA binding domain linked to the Fokl nuclease domain. As such, a pair of TALENs operate in tandem to achieve targeted DNA cleavage. The major difference from ZFNs is the nature of the DNA binding domain and the associated target DNA sequence recognition properties. The TALEN DNA binding domain derives from TALE proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALES are comprised of tandem arrays of 33-35 amino acid repeats, with each repeat recognizing a single base pair in the target DNA sequence that is typically up to 20 bp in length, giving a total target sequence length of up to 40 bp. Nucleotide specificity of each repeat is determined by the repeat variable diresidue (RVD), which includes just two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are predominantly recognized by the four RVDs: Asn-Asn, Asn-lle, His-Asp and Asn-Gly, respectively. This constitutes a much simpler recognition code than for zinc fingers, and thus represents an advantage over the latter for nuclease design. Nevertheless, as with ZFNs, the protein- DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefitted from the use of obligate heterodimer variants of the Fokl domain to reduce off- target activity.

[0075] Additional variants of the Fokl domain have been created that are deactivated in their catalytic function. If one half of either a TALEN or a ZFN pair contains an inactive 15

Fokl domain, then only single-strand DNA cleavage (nicking) will occur at the target site, rather than a DSB. The outcome is comparable to the use of CRISPR-Cas nickase mutants in which one of the Cas9 cleavage domains has been deactivated.

[0076] A variety of TALEN-based systems have been described in the art, and modifications thereof are regularly reported; see, e.g., Boch, Science, 2009 326(5959): 1509-12; Mak et al., Science, 2012, 335(6069)716-9; and Moscou et al., Science, 2009, 326(5959): 1501.

[0077] Homing endonucleases (HEs). HEs are sequence-specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity - often at sites unique in the genome. There are at least six known families of HEs as classified by their structure, including GIY-YIG, His-Cis box, H-N-H, PD-(D/E)xK, and Vsr- like that are derived from a broad range of hosts, including eukarya, protists, bacteria, archaea, cyanobacteria and phage. As with ZFNs and TALENs, HEs can be used to create a DSB at a target locus as the initial step in genome editing. The large target sequence of HEs and the specificity that they offer have made them attractive candidates to create site-specific DSBs.

[0078] A variety of HE-based systems have been described in the art, and modifications thereof are regularly reported; see, e.g., the reviews by Steentoft et al., Glycobiology, 2014, 24(8):663-80; Belfort and Bonocora, Methods Mol Biol., 2014, 1123:1-26; and Hafez and Hausner, Genome, 2012, 55(8):553-69, which are each incorporated by reference in their entirety.

[0079] Hybrid nucleases. Nucleases can be engineered to comprise a fusion of a meganuclease (Mega) domain, a TALE DNA binding domain, and/or a homing endonuclease domain. The MegaTAL platform and Tev-mTALEN platforms utilize fusion of TALE DNA binding domains and catalytically active HEs, taking advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of the HE; see, e.g., Boissel et al., Nucleic Acids Res., 2014, 42: 2591-2601 ; Kleinstiveret al., G3, 2014, 4:1155-65; and Boissel and Scharenberg, Methods Mol. Biol., 2015, 1239: 171-96, which are each incorporated by reference in their entirety. The MegaTev architecture is the fusion of a meganuclease with the nuclease domain derived 16 from the GIY-YIG homing endonuclease l-Tevl (Tev). The two active sites are positioned ~30 bp apart on a DNA substrate and generate two DSBs with non-compatible cohesive ends; see, e.g., Wolfs et al. , Nucleic Acids Res., 2014, 42, 8816-29, incorporated herein by reference in its entirety. It is anticipated that other combinations of existing nuclease- based approaches will evolve and be useful in achieving the targeted genome modifications described herein.

[0080] As further example, fusion of the TALE DNA binding domain to a catalytically active HE, such as l-Tevl, takes advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of l-Tevl, with the expectation that off-target cleavage can be further reduced.

X-family DNA Polymerase

[0081] The methods disclosed herein further comprises delivering to the cells an X- family DNA polymerase or functional fragment thereof or a nucleic acid encoding an X family polymerase or functional fragment thereof. The X-family of DNA polymerases have similar three-dimensional structures and include terminal deoxyribonucleotidyl transferase (TdT), polymerase lambda (Pol l), and polymerase mu (Pol m). Pol l and Pol m are responsible for polymerizing nucleotide addition at the site of a dsDNA break as part of the normal cellular DNA repair processes. TdT uniquely provides junctional diversity during VJD recombination in T- and B- cell precursors by adding nucleotides to the 3’ termini of dsDNA breaks in a template-free manner. In humans, terminal transferase is encoded by the DNTT gene. In several species including humans, there are multiple isoforms of TdT resulting from alternative mRNA splicing. The short form (TdTS) consists of 509 amino acids; long form 1 (TdTL1) contains exon 12 and consists of 518 amino acids; long form 2 (TdTL2) contains exon 7 and consists of 527 amino acids; and long form 3 (TdTL3) contains both exons 7 and 13 and consists of 536 amino acids. [0082] In some embodiments, the X-family DNA polymerase is Pol l. In other embodiments, the X-family DNA polymerase is Pol m. In certain embodiments, the X- family DNA polymerase is TdT, for example human TdT. In some embodiment, the X- 17 family DNA polymer is a long isoform of human TdT. In other embodiments, the X-family

DNA polymerase is a combination of TdTS and either of TdTL1 , TdTL2, or TdTL3. In specific embodiments, the X-family DNA polymerase is the short isoform (TdTS) of human TdT. The sequence of the 509 amino acid hTdTS protein is presented below.

MDPPRASHLSPRKKRPRQTGALMASSPQDIKFQDLVVFILEKKMGTTRRAFLMELARR

KGFRVENELSDSVTHIVAENNSGSDVLEWLQAQKVQVSSQPELLDVSWLIECIRAGKP

VEMTGKHQLVVRRDYSDSTNPGPPKTPPIAVQKISQYACQRRTTLNNCNQIFTDAFDIL

AENCEFRENEDSCVTFMRAASVLKSLPFTIISMKDTEGIPCLGSKVKGIIEEIIEDGESSE

VKAVLNDERYQSFKLFTSVFGVGLKTSEKWFRMGFRTLSKVRSDKSLKFTRMQKAGF

LYYEDLVSCVTRAEAEAVSVLVKEAVWAFLPDAFVTMTGGFRRGKKMGHDVDFLITSP

GSTEDEEQLLQKVMNLWEKKGLLLYYDLVESTFEKLRLPSRKVDALDHFQKCFLIFKLP

RQRVDSDQSSWQEGKTWKAIRVDLVLCPYERRAFALLGWTGSRQFERDLRRYATHE

RKMILDNHALYDKTKRIFLKAESEEEIFAHLGLDYIEPWERNA (SEQ ID NO: 34).

Encoding Nucleic Acids

[0083] The two or more programmable gene editing systems and/or the X-family DNA polymerase can be provided to the cells as encoding nucleic acids. In some embodiments, proteins of the gene editing systems and/or the X-family DNA polymerase can be coded by mRNA. In some embodiments, proteins of the gene editing systems and/or the X-family DNA polymerase can be coded by DNA. In embodiments in which the programmable gene editing systems are CRISPR-Cas systems, the two or more gRNAs can be encoded by DNA.

[0084] In embodiments in which the encoding nucleic acid is mRNA, the mRNA can be 5’ capped and/or 3’ polyadenylated. The mRNA can be single stranded and linear. In embodiments in which the encoding nucleic acid is DNA, the DNA can be single stranded, double stranded, linear, or circular. In embodiments in which the gene editing system is a CRISPR-Cas system, the DNA encoding the CRISPR-Cas nuclease generally is codon optimized for expression in the cells of interest.

[0085] In embodiments in which the encoding nucleic acid is mRNA, the mRNA can be synthesized by chemical means, as described in the art. While chemical synthetic procedures are continually expanding, purification of such RNAs by procedures such as 18 high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs are more readily generated enzymatically in vitro. Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability and the like.

[0086] In other embodiments, the encoding nucleic acid can be provided as part of vector system. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, wherein additional nucleic acid segments can be ligated into the viral genome. Viral vectors can be RNA or DNA. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0087] In some instances, vectors can be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to as "recombinant expression vectors", or more simply "expression vectors", which serve equivalent functions.

[0088] The term "operably linked" means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology, 1990, 185, Academic Press, San Diego, CA. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those 19 that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue- specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.

[0089] Suitable expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus (AAV), SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXT1 , pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors can be used so long as they are compatible with the host cell.

[0090] In some examples, a vector can comprise one or more transcription and/or translation control elements. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector. Non-limiting examples of suitable eukaryotic promoters (i.e. , promoters functional in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 a promoter (EF1a), chicken beta-actin promoter (CBA), ubiquitin C promoter (UBC), a hybrid construct comprising the cytomegalovirus enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-l promoter. In other embodiments, the promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), a tissue specific promoter, or a cell type specific promoter. 20

[0091] In other embodiments, DNA encoding gRNAs for two or more CRISPR-Cas systems can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Examples of suitable Pol III promoters include, but are not limited to, mammalian U6, U3, H1 , and 7SL RNA promoters. In some embodiments, DNA encoding an array of two or more gRNAs can be operably linked to a Pol III promoter.

Delivering to the Cells

[0092] As detailed above, the two or more programmable gene editing systems can be delivered to the cells as proteins or RNP complexes, encoding mRNA, or encoding DNA and the X-family DNA polymerase can be delivered to the cells as protein, encoding RNA, or encoding DNA.

[0093] In some embodiments, two programmable gene editing systems are delivered to the cells. In other embodiments, three programmable gene editing systems are delivered to the cells. In further embodiments, four programmable gene editing systems are delivered to the cells. In still other embodiments, five programmable gene editing systems are delivered to the cells. In additional some embodiments, six or more programmable gene editing systems are delivered to the cells.

[0094] The two or more programmable gene editing systems and the X-family DNA polymerase can be delivered to the cells by a variety of methods. Suitable methods include electroporation (e.g., nucleofection), lipofection, sonoporation, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation conjugates, lipidmucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and agent- enhanced uptake of DNA. In specific embodiments, the gene editing system can be delivered to the cells of interest via electroporation.

[0095] In embodiments in which the two or more programmable gene editing systems are CRISPR-Cas systems, the two or more CRISPR-Cas systems can comprise the same CRISPR-Cas nuclease (e.g., SpCas9) or different CRISPR-Cas nucleases (e.g., SpCas9 and SaCas9, etc.), wherein each gRNA is engineered to complex with a specific Cas9 protein. The number of CRISPR-Cas systems is determined by the number of different gRNAs. In some embodiments, two different gRNAs are provided to the cells. In other 21 embodiments, three different gRNAs are provided to the cells. In still other embodiments, four different gRNAs are provided to the cells. In additional embodiments, five different gRNAs are provided to the cells. In further embodiments, six or more different gRNAs are provided to the cells.

[0096] In embodiments in which the programmable gene editing systems are CRISPR- Cas systems, the two or more CRISPR-Cas systems can be delivered to the cells as (i) RNP complexes, each comprising a CRISPR-Cas nuclease and a gRNA; (ii) mRNA encoding the CRISPR-Cas nuclease and two or more gRNAs; (iii) mRNA encoding the CRISPR-Cas nuclease and DNA encoding the two or more gRNAs; or (iv) DNA encoding the CRISPR-Cas nuclease and DNA encoding the two or more gRNAs.

[0097] In embodiments in which the CRISPR-Cas system is delivered as RNP, the weight ratio of each gRNA to the CRISPR-Cas nuclease can range from about 1 :1 to about 10:1. In various embodiments, the weight ratio of each gRNA to the CRISPR-Cas nuclease can be about 1 :1 , about 1.5:1 , abut 2:1 , about 2.5:1 , about 3:1 , about 3.5:1 , about 4:1 , about 4.5:1, about 5:1 , about 5.5:1 , about 6:1 , about 6.5:1 , about 7:1 , about 7.5:1 , about 8:1 , about 8.5:1 , about 9:1 , about 9.5:1 , or about 10:1.

[0098] In the above-mentioned embodiments in which the programmable gene editing systems are CRISPR-Cas systems, the X-family DNA polymerase generally is delivered as encoding mRNA. In some embodiments, however, the X-family DNA polymerase can be delivered as encoding DNA or purified protein.

[0099] In still further embodiments, the X-family DNA polymerase can be fused to the CRISPR-Cas nuclease. The X-family DNA polymerase or a functional fragment thereof can be fused to the amino terminus, carboxy terminus, or both of the CRISPR-Cas nuclease. The fusion can be direct or via a linker, which are well known in the art. The X-family DNA polymerase/CRISPR-Cas fusion can be delivered to the cells as a protein, encoding mRNA, or encoding DNA.

Cells

[00100] A variety of cells are suitable for use in the methods disclosed herein. In general, the cells are mammalian cells. In specific embodiments, the cells are human 22 cells. Generally, the cells are other than immature, pre-B or pre-T lymphoid cells, leukemia cells, lymphoma cells, or immortalized cells derived from any of the foregoing. [00101] In some embodiments, the cells are primary cells isolated directly from human (or animal) tissue. In various embodiments, the cells used in the methods or compositions disclosed herein can be CD34+ HSPCs, T cells, NK cells, monocytes, macrophages, microglial cells, neutrophils, eosinophils, basophils, dendritic cells, or adipocytes. In certain embodiments, the cells can be T cells, e.g., human T cells. The T cells can be helper T cells or cytotoxic T cells. In other embodiments, the cells can be NK cells, e.g., human NK cells.

[00102] In further embodiments, the primary cells can be, without limit, adipocytes, astrocytes, blood cells, chondrocytes, endothelial cells, epithelial cells, fibroblasts, hair cells, hepatocytes, keratinocytes, melanocyte, myocytes, neurons, osteoblasts, skeletal muscle cells, smooth muscle cells, stem cells, or synoviocytes.

[00103] In still other embodiments, the cells can be stem cells (e.g., embryonic stem cells, fetal stem cells, amniotic stem cells, or umbilical cord stem cells). In certain embodiments, the stem cells can be adult stem cells isolated from bone marrow, adipose tissue, or blood. In still other embodiments, the stem cells can be induced pluripotent stem cells (e.g., human iPSCs).

[00104] In other embodiments, the cells may be hematopoietic stem and progenitor cells (HSPCs) or hematopoietic stem cells (HSCs). HSPCs give rise to all blood cell types, including erythroid (erythrocytes or red blood cells (RBCs)), myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, and dendritic cells), and lymphoid (T-cells, B-cells, NK-cells). Blood cells are produced by the proliferation and differentiation of a very small population of pluripotent HSCs that also have the ability to replenish themselves by self-renewal. During differentiation, the progeny of HSCs progress through various intermediate maturational stages, generating multi-potential and lineage-committed progenitor cells prior to reaching maturity. Bone marrow (BM) is the major site of hematopoiesis in humans and, under normal conditions, only small numbers of HSPCs can be found in the peripheral blood (PB). Treatment with cytokines (in particular granulocyte colony-stimulating factor; G-CSF), some 23 myelosuppressive drugs used in cancer treatment, and compounds that disrupt the interaction between hematopoietic and BM stromal cells can rapidly mobilize large numbers of stem and progenitors into the circulation. The cell surface glycoprotein CD34 is routinely used to identify and isolate HSPCs.

[00105] In other embodiments, the cells can be mesenchymal stem cells (e.g., multipotent stromal cells that can differentiate into a variety of cell types). Mesenchymal stem cells (MSCs) are adult stem cells found in the bone marrow, or isolated from other tissues such as cord blood, peripheral blood, fallopian tube, and fetal liver and lung. As multipotent stem cells, MSCs differentiate into multiple cell types including adipocytes, chondrocytes, osteocytes, and cardiomyocytes. Mesenchymal stem cells are a distinct entity to the mesenchyme, embryonic connective tissue, which is derived from the mesoderm and differentiates to form hematopoietic stem cells (HPCs).

[00106] In some embodiments, the cells used in the methods disclosed herein can be in vitro. In other embodiments, the cells used in the methods can be ex vivo. In still other embodiments, the cells used in the methods can be in situ. In yet another embodiment, the cells used in the methods can be in vivo.

Indel Profiles and Chromosomal Translocations

[00107] Upon delivery of the two or more programmable gene editing systems and the X-family DNA polymerase, the gene editing systems introduce double stranded DNA breaks at the targeted genomic loci, thereby generating cleaved genomic loci and the X- family DNA polymerase catalyzes addition of nucleotides to 3’ termini of the cleaved genomic loci, which we have found to lead to a reduction of chromosomal translocations between the two or more cleaved genomic loci.

[00108] In general, when two or more genomic loci are edited simultaneously in the presence of the X-family DNA polymerase, chromosomal translocations can be reduced by at least about 5%. In embodiments in which two loci are edited simultaneously in the presence of the X-family DNA polymerase, such as TdTS, the percent of chromosomal translocations can be reduced by at least about 5%, by at least about 10%, by at least about 15%, by at least about 20%, by at least about 35% ,by at least about 50% (2-fold 24 reduction), by at least about 2.2-fold, by at least about 2.5-fold, by at least about 3-fold, by at least about 3.5-fold, by at least about 4-fold, or at least more than 4-fold. In some embodiments, each type of translocation variant (e.g., monocentric, dicentric, acentric) can be reduced a comparable amount. In other embodiments, at least one type of variant can be reduced more than the other variants.

[00109] The addition of nucleotides by the X-family DNA polymerase produces insertion-rich indel profiles. In particular, cells comprising an X-family DNA polymerase, such as TdTS, exhibit increased percentages of nucleotide insertions, increased percentages of longer insertions, and decreased percentages of deletions at the targeted chromosomal loci as compared to cells lacking the X-family DNA polymerase.

[00110] The percentage of nucleotide insertions of more than one nucleotide can be increased by at least about 40% as compared to cells lacking the X-family DNA polymerase. In some embodiments, the percentage of insertions can be increased about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. The length of insertions can be increased from about 1 to about 8 to 10 nucleotides, whereas cells lacking the X-family DNA polymerase rarely exhibit insertions longer than one nucleotide. The percentage of nucleotide deletions can be decreased by at least about 30% as compared to cells lacking the X-family DNA polymerase. In some embodiments, the percentage of deletions can be decreased about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%.

[00111] While the efficiency of editing varies between different genomic loci, the efficiency does not differ significantly between cells with or without the X-family DNA polymerase. In general, the efficiency of editing is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

(II) Compositions and Kits

[00112] Another aspect of the present disclosure encompasses compositions and kits for multiplex gene editing. For example, the present disclosure provides cells comprising two or more programmable gene editing systems and an X-family DNA polymerase as described above in section (I). The two or more programmable gene editing systems and 25 an X-family DNA polymerase can exist as DNA, RNA, protein, or combinations thereof. In some embodiments, the cells comprise two or more CRISPR-Cas systems and TdT. In other embodiments, the cells comprise two or more CRISPR-Cas systems and the short isoform of TdT (TdTS). Suitable cells are detailed above in section (l)(e).

[00113] Also provided herein are kits for multiplex gene editing. In general, the kits comprise two or more programmable gene editing systems and an X-family DNA polymerase as described above in section (I), as well as reagents for gene editing. Reagents for gene editing include appropriate buffers, dNTPs, divalent salts, and the like. The kits can also include reagents and/or primers/probes for analyzing indel patterns and/or chromosomal translocations. In some embodiments, the kits comprise two or more CRISPR-Cas systems and TdT. In other embodiments, the kits comprise two or more CRISPR-Cas systems and the short isoform of TdT (TdTS). The kits can provide the two or more CRISPR-Cas systems as RNPs, RNA, and/or DNA, and can provide the TdT or TdTS as RNA, DNA, or protein.

[00114]

[00115] DEFINITIONS

[00116] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[00117] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[00118] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e. , to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111 .03. 26

[00119] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[00120] Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.

[00121] As used herein, the terms “complementary” or “complementarity” refer to the association of double-stranded nucleic acids by base pairing through specific hydrogen bonds. The base paring may be standard Watson-Crick base pairing (e.g., 5’-A G T C-3’ pairs with the complementary sequence 3’-T C A G-5’). The base pairing also may be Hoogsteen or reversed Hoogsteen hydrogen bonding. Complementarity is typically measured with respect to a duplex region and thus, excludes overhangs, for example. Complementarity between two strands of the duplex region may be partial and expressed as a percentage (e.g., 70%), if only some (e.g., 70%) of the bases are complementary. The bases that are not complementary are “mismatched.” Complementarity may also be complete (i.e. , 100%), if all the bases in the duplex region are complementary.

[00122] A “gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[00123] The terms “nuclease” and “endonuclease” are used interchangeably herein, and refer to an enzyme that cleaves both strands of a double-stranded nucleic acid sequence.

[00124] The terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double- stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an 27 analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T.

[00125] The term “nucleotide” refers to deoxyribonucleotides or ribonucleotides. The nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. A nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety. A nucleotide analog may be a naturally occurring nucleotide (e.g., inosine, pseudo uridine, etc.) or a non-naturally occurring nucleotide. Non-limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deaza purines). Nucleotide analogs also include dideoxy nucleotides, 2’-0-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.

[00126] The term "sequence identity" as used herein, indicates a quantitative measure of the degree of identity between two sequences of substantially equal length. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: 28 genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website.

EXAMPLES

[00127] The following examples illustrate various non-limiting embodiments of the present disclosure.

Example 1. General Methods

[00128] Media/Culturing. For primary T cells, PBMCs (AllCells) were thawed and cultured in Expansion Media [T cell CTS™ OpTmizer™ T Cell Expansion Media (Thermo Fisher) supplemented with 5% Human Serum AB (Vally Biomedical), 50 ng/ml IL-2 (Miltenyi Biotec) and 10 ng/ml IL-7 (Cellgenix). Upon thawing, T cells were activated with T Cell TransAct™ (Miltenyi) for 3 days. T Cell TransAct™ was subsequently removed from culture and the cells were seeded at a density of 5 x 105 cells/ml. Jurkat cells, Clone E6-1 (ATCC TIB-152) were grown in RPMI-1640 + GlutaMax (Thermo Fisher) supplemented with 10% Fetal Bovine Serum (Thermo Fisher) and 1% Penicillin- Streptomycin (Thermo Fisher). Cells were seeded at a density of 1 x 105 cells/ml and passaged before exceeding 1 x 106 cells/ml. Preculturing of cells prior to nucleofection was followed according to recommendations by Lonza.

[00129] CRISPR Editing of Genomic Loci in Jurkat and Primary T cells. Chemically modified (three 2'-0-methyl-phosphorothioate residues at each 5’ and 3’ end) guides (Table 1) were used to edit primary T cells and Jurkat cells (ATCC® TIB-152™). For Cas9 editing by RNP, an RNP mixture of S. pyogenes Cas9 (Biomay) and sgRNA (Agilent or Biospring) was prepared at a molar ratio of 1 :5 (Cas9:sgRNA) with final concentrations of 20 pmol Cas9 and 100 pmol sgRNA. Where indicated, cells were also edited using S. pyogenes Cas9 mRNA (0.28 pmol) combined with 28 pmol of sgRNA (molar ratio of 1 :100). In one instance, the CD70 locus was edited with 0.28 pmol of mRNA encoding 29

Staphylococcus lugdunensis (Slu) combined with 28 pmol of guide “CD70_SluCas9_gRNA_3”. Gene-editing experiments that included mRNA-encoding TdT isoforms were performed by including the relevant mRNA directly in the RNP mixture.

[00130] Primary T cells and Jurkat cells were nucleofected using the Lonza4D nucleofector and the P3 primary cell and SE Cell line kits, respectively. Cells were counted using a Cellometer K2 or Cellaca MX HighThroughput Cell Counter (Nexcelom). T cells and Jurkat cells were resuspended in P3/SE buffer with supplement at a concentration of 5x104/pl and 1x104/mI, respectively. A total of 1x106 T cells or and 2x105 Jurkat cells were combined with RNP complex and transferred to a Lonza nucleofection cuvette for all editing experiments. T cells were nucleofected using program EO-115 and Jurkat cells nucleofected using program CL-120. Following electroporation, Jurkat cells were immediately recovered in RPMI media. T cells were recovered in serum-free media for 1 hour post-nucleofection before being transferred to media supplemented with serum. After 48 hours, cells were recovered, and genomic DNA was harvested using the QIAcube HT DNA extraction system and QIAamp 96 DNA QIAcube HT Kit or DNeasy Blood and Tissue Kit (Qiagen).

[00131] Nucleofection of TdT mRNA for Kinetics of TdT Expression in Primary T cells. For kinetics of TdT expression experiments, nucleofection of TdT-encoding mRNA was 30 performed as described above by adding mRNA directly to the P3 cellular mixture. In these experiments, a total of 5x106 T cells were transferred to a large Lonza nucleofection cuvette. Cells were recovered directly in T cell expansion media and 1 million T cells were harvested at the indicated time points for preparation of protein lysates.

[00132] PCR and Tracking of Indels by Decomposition (TIDE). Isolated genomic DNA was subjected to PCR to determine indel frequency by TIDE analysis (Brinkman et al. 2014, Nucleic Acids Research 42 (22): e168-e168). PCR for relevant regions was performed (1 cycle at 95°C for 3 min; 34 cycles at 98°C for 20 sec, 69°C for 15 sec, 72°C for 1 min; 1 cycle at 72°C for 3 min) according to the manufacturer’s recommendations for each commercial PCR polymerase. Resulting amplicons were examined on cast 1.2% or 2% pre-cast agarose gels. PCR clean-up of the resulting amplicons was performed using SPRIselect magnetic beads (Beckman Coulter). Sanger sequencing results were input into Tsunami along with guide sequences and resulting indel frequencies and identities were calculated by the software. Indel frequencies and editing efficiency was plotted using Prism (GraphPad). Indels were plotted as heatmaps with the indel identities on one axis and the corresponding frequency indicated by color intensity. Total editing frequencies were plotted as bar graphs.

[00133] Droplet Digital PCR ( ddPCR ). ddPCR assays were designed to detect four translocations denoted (T1 , T2, T3, and T4) from simultaneous editing of the PD1 and CD70 loci. All primers and probes were ordered from IDT. Primer pairs and a HEX- labeled reference probe were designed to count all copies of the reference amplicon within the RAG1 locus. Paired primers and a FAM-labeled probe (Table 2) were designed to polymerize amplicon across the breakpoint of the predicted translocation. Amplification reactions were formulated in 2x ddPCR Supermix for Probes (No dUTP) (BioRad). Primers and probes were introduced at a final concentration of 1.8 mM and 0.5 pM, respectively. 100-200 ng of genomic DNA from cells harvested 48-hours post- nucleofection was introduced to each ddPCR reaction along with 0.1 Unit of BamH1-HF to reduce viscosity generated by high DNA concentration. Individual reactions comprising 25 pi of ddPCR amplification mixture, were placed into an Automated Droplet Generator (BioRad). For each sample, roughly 15,000-20,000 nanoliter-sized droplets were 31 generated. Subsequently, the droplets were transferred to a 96-well plate for PCR in a thermal cycler (1 cycle at 95°C for 10 min; 40 cycles at 90°C for 30 sec, 57°C for 1 min, 72°C for 1 min; 1 cycle at 98°C for 10 min; 4°C hold). Following PCR amplification, droplets were read by QX200 Droplet Reader and analyzed using the Quantasoft™ software (BioRad), which analyzes each droplet individually using a two-color detection system (set to detect FAM and HEX). The ratio between translocation specific events (FAM) over events of reference amplicon (HEX) (x100) is the percentage of translocation. No template controls (NTCs) were included for each translocation assessed to determine background fluorescence. Data was plotted in the form of bar graphs on Prism (GraphPad).

32

[00134] Capillary Western for Immunodetection of hTdT and Tubulin. Protein lysates were prepared from cell pellets using RIPA Lysis and Extraction Buffer (Thermo Fisher) supplemented with Halt™ Protease Inhibitor Cocktail (100X) (Thermo Fisher). Protein levels were quantitated with the Pierce™ BCA Protein Assay Kit (Thermo Fisher) on the Epoch2 microplate reader (BioTek). Immunodetection of TdT and tubulin levels was determined via Simple Western technology, an automated capillary-based size sorting and immunolabeling system (ProteinSimpleTM). For TdT, the Anti-Rabbit Detection Module (ProteinSimpleTM) was used with the Anti-hTdT RabMab antibody [EPR2976Y] (ab76544) (Abeam) at 1.45 pg/ml. Tubulin was detected with the Anti-Mouse Detection Module (ProteinSimpleTM) and the monoclonal Anti-a-Tubulin clone B-5-1-2 (Millipore Sigma) at a 1 :10,000 dilution. Capillary westerns were run on the JESS device with the 12-230 kDa kits (ProteinSimpleTM) and all procedures were performed according to manufacturer’s protocol. The JESS device was associated with Compass software for device settings and raw data recording (ProteinSimple/Biotechne).

[00135] Creation of TdT Knockout Jurkat Cells. TdT-/- Jurkat cells were created by CRISPR editing of the DNTT locus. Briefly, the DNTT gene was singly edited with each 33 guide in Table 3 according to the same method detailed in “CRISPR Editing of Genomic Loci in Jurkat and Primary T cells” using S. pyogenes Cas9 for RNP formation. Editing efficiencies were established by TIDE analysis of Sanger sequencing using the primers (Table 4) and amplification protocol (1 cycle at 95°C for 3 min; 34 cycles at 98°C for 20 sec, 68°C for 45 sec, 72°C for 1 min; 1 cycle at 72°C for 3 min). Diminution of TdT expression was confirmed using “Immunodetection by Capillary Western” described above. Bulk-edited cultures displayed near-complete loss of TdT expression and were therefore used for downstream editing and translocation analysis following editing of the PD1 and CD70 loci.

[00136] In vitro transcription of mRNA. Plasmids encoding TdT isoforms and Cas9 nucleases from S. pyogenes and S. lugdunensis were synthesized by ATUM to contain an upstream T7 promoter, a downstream polyA tail, and a single Sap I restriction site adjacent to the polyA tail. Linear templates were created by digesting plasmid with Sap I 34

(New England Biolabs) and purified by phenol/chloroform extraction. In vitro transcription and purification of mRNA was performed with the MegaScript T7 Transcription Kit (Thermo Fisher).

Example 2. Jurkat and T cells Exhibit Similar Editing Efficiencies but Different Indel Profiles

[00137] Several gRNAs were designed to target the NPM1 gene locus or the ALK gene locus in human cells (Table 1). RNPs comprising SpCas9 protein and gRNA were delivered by electroporation to Jurkat and T cells as described above in Example 1. After an appropriate period of time, the edited loci were analysed by PCR and TIDE (as described above) to determine the efficiency of editing and the mutational profile (or indel spectrum) of the types and distribution of indels after repair of the dsDNA break introduced by Cas9. As shown in FIG. 1A, the editing efficiencies were similar between the two cell types at each of the loci. However, the indel profiles of T cells mainly comprised single nucleotide insertions (+1) and deletions, whereas those of Jurkat cells mainly comprised nucleotide insertions of +1 or greater (FIG. 1B).

Example 3. Jurkat Cells Exhibit Reduced Frequency of Chromosomal Translocations During Multiplex Editing at Two Loci

[00138] Simultaneous editing at two gene loci was then examined in Jurkat cells and T cells to determine whether increased insertions during editing could reduce chromosomal translocations during multiplex editing. Guide RNAs were designed to target PD1 and CD70 loci (Table 1). Multiplex editing was performed by delivering RNPs comprising SpCas9:PD1_4gRNA and SluCas9:CD70_3gRNA to Jurkat and T cells. Similar to the results presented in Example 2, the editing efficiencies were similar between the two cell types (FIG. 2A), and the Jurkat cells exhibited increased frequencies of insertions of +1 or more nucleotides at these two loci (FIG. 2B). Moreover, the Jurkat cells exhibited reduced frequencies of chromosomal translocations (FIG. 3B) as detected by droplet digital PCR (ddPCR) as described in Example 1. FIG. 3A diagrams the various translocations variants. 35

[00139] It has been reported that TdT is not active in mature lymphocytes (primary T cells) but is reactivated in some stable cell lines originating from T cells (e.g., Jurkat cells). FIG. 4 presents a Western blot confirming that Jurkat, but not T cells, express TdT.

Example 4. Editing of Jurkat Cells to Knock Out TdT

[00140] To determine whether TdT affects the indel profiles and chromosomal rearrangements in Jurkat cells, the DNTT gene locus was disrupted such that no TdT was expressed. For this, several gRNAs that target various exons were designed, as shown above in Table 3. After editing, TdT knock out was confirmed at the level of DNA and protein. TdT levels were very low in the bulk edited pool of cells (FIG. 5). Clonal outgrowth identified Jurkat (TdT-) cells.

[00141] Multiplex editing at PD1 and CD70 loci was performed in the edited Jurkat (TdT-) cells, wild type Jurkat (TdT+) cells, and primary T cells essentially as described above in Example 3. As shown in FIG. 6A and 6B, the indel profile of the Jurkat (TdT-) cells mirrored that of T cells in that the profiles comprised mainly single nucleotide insertions and deletions. The Jurkat (TdT-) cells also exhibited translocation frequencies similar to those of T cells (FIG. 6C).

Example 5. Delivery of TdT mRNA to T Cells

[00142] TdT exists in several isoforms, e.g., short isoform, long isoform 1 (which includes exon 12), long isoform 2 (which included exon 7), and long isoform 3 (which included exons 7 and 12). T cells were electroporated with 6 pmol of mRNA encoding the various TdT isoforms, as well as Cas9 mRNA. The kinetics of TdT expression relative to alpha-tubulin expression are presented in FIG. 7. Surprisingly, it was discovered that the short isoform (TdTS) persisted at higher levels for longer periods of time than the long isoforms. Lower levels of TdTS mRNA were introduced into T cells (FIG. 8A). Even at the lowest dose of 1.5 pmol, the relative abundance of TdTS to alpha-tubulin in T cells was higher than that in wild type Jurkat cells at 12 hours post electroporation (FIG. 8B). 36

Example 6. Providing T Cells with TdTS mRNA Creates Insertion Heavy Indel Profiles and Suppresses Translocations

[00143] T cells were electroporated with Cas9 editing reagents (RNPs) targeting PD1 and CD70, as described above, and 3 pmol of TdTS mRNA (with no mRNA or GFP mRNA serving as controls). The T cells provided with TdTS mRNA exhibited indel profiles with increased frequency of insertions of +1 and greater (FIG. 9A) and exhibited a three-fold decrease in translocations as compared to control cells (FIG. 9B). The editing efficiency was high at both loci under the various conditions (FIG. 9C). Unexpectedly, it was found that providing the long isoforms of TdT did not change the indel profile (FIG. 10A) or the translocate rate in T cells (FIG. 10B).

Example 7. Indel Patterns in T Cells Edited with Cas9^RNP and TdTS^mRNA versus Cas9^mRNA+sgRNA and TdTS^mRNA

[00144] Cas9 was delivered as RNP complex (Cas9protein + sgRNA) or as nucleic acids (Cas9mRNA + sgRNA) to determine whether the means of Cas9 delivery could affect editing efficiency, indel profile, and/or translocation rate. T cells were multiplex edited at PD1 and CD70 in the absence or presence of TdTSmRNA. The editing efficiency did not differ between the two types of Cas9 delivery (FIGs. 11 A, 11 B). Delivery of TdTS resulted in insertion heavy indels (FIGS. 12A-12D) and reduced frequency of translocations (FIGS. 13A, 13B). Delivery of Cas9mRNA + sgRNA appeared to increase the frequency of insertions and created longer insertions (FIGS. 12C, 12D). It was speculated that this effect may be due to co-expression of Cas9 and TdTS at about the same time, suggesting that mRNA delivery of both may be the preferred delivery mechanism.

Example 8. Multiplex Editing at Three Loci

[00145] T cells will be electroporated with TdTSmRNA and three Cas9 systems (either RNPs or Cas9mRNA + sgRNA) targeted three different genomic loci. The percent of chromosomal translocations, the indel patterns, and the editing efficiency will be analyzed as described above. 

Claims

38 CLAIMS What is claimed is:

1. A method for gene editing at multiple genomic loci, the method comprising delivering to a cell:

(a) two or more CRISPR-Cas nuclease systems or two or more nucleic acids encoding two or more CRISPR-Cas nuclease systems, wherein each CRISPR-Cas nuclease system is targeted to a different genomic locus; and

(b) an X-family DNA polymerase or a nucleic acid encoding an X family DNA polymerase; wherein the two or more CRISPR-Cas nuclease systems generate cleaved genomic loci and the X-family DNA polymerase catalyzes addition of nucleotides to 3’ termini of the cleaved genomic loci, such that chromosomal translocations are reduced between the cleaved genomic loci, thereby facilitating editing at multiple genomic loci.

2. The method of claim 1 , wherein addition of nucleotides increases nucleotide insertion frequencies, increases insertion lengths, and decreases nucleotide deletion frequencies as compared to a comparable cell in which the X-family DNA polymerase is not delivered.

3. The method of claims 1 or 2, wherein chromosomal translocations are reduced by at least 5% when two genomic loci are edited as compared to a comparable cell in which the X-family DNA polymerase is not delivered.

4. The method of any one of claims 1 to 3, wherein each CRISPR-Cas nuclease system comprises a CRISPR-Cas nuclease and a gRNA.

5. The method of claim 4, wherein the CRISPR-Cas nuclease is a type II CRISPR-Cas nuclease or a type V CRISPR-Cas nuclease. 39 The method of claims 4 or 5, wherein the CRISPR-Cas nuclease is SpCas9, SluCas9, SaCas9, or a variant thereof with increased fidelity. The method of any one of claims 4 to 6, wherein the gRNA is a single molecule gRNA (sgRNA). The method of any one of claims 1 to 7, wherein each of the CRISPR-Cas nuclease systems is delivered as a ribonucleoprotein (RNP) complex. The method of any one of claims 1 to 7, wherein each of the CRISPR-Cas nuclease systems is delivered as a CRISPR-Cas nuclease and a gRNA. The method of any one of claims 1 to 7, wherein each of the CRISPR-Cas nuclease systems is delivered as mRNA encoding the CRISPR-Cas nuclease and a gRNA. The method of any one of claims 1 to 7, wherein each of the CRISPR-Cas nuclease systems is delivered as DNA encoding the CRISPR-Cas nuclease and DNA encoding the gRNA. The method of any one of claims 1 to 11 , wherein the X-family DNA polymerase is terminal deoxyribonucleotidyl transferase (TdT), polymerase lambda (Pol l), or polymerase mu (Pol m). The method of any one of claims 1 to 12, wherein the X-family DNA polymerase is a short isoform of TdT (TdtS). The method of claim 13, wherein TdTS has an amino acid sequence of SEQ ID NO: 34. The method of any one of claims 1 to 14, wherein the X-family DNA polymerase is delivered as mRNA. 40 The method of any one of claims 1 to 14, wherein the X-family DNA polymerase is delivered as protein. The method of any one of claims 1 to 14, wherein the X-family DNA polymerase is delivered as DNA. The method of any one of claims 1 to 17, wherein two CRISPR-Cas nuclease systems are delivered to the cell. The method of any one of claims 1 to 17, wherein three CRISPR-Cas nuclease systems are delivered to the cell. The method of any one of claims 1 to 17, wherein four CRISPR-Cas nuclease systems are delivered to the cell. The method of any one of claims 1 to 17, wherein five CRISPR-Cas nuclease systems are delivered to the cell. The method of any one of claims 1 to 21 , wherein delivering comprises electroporation. The method of any one of claims 1 to 22, wherein the cell is a mammalian cell. The method of any one of claims 1 to 23, wherein the cell is other than an immature, pre-B or pre-T lymphoid cell, a leukemia cell, a lymphoma cell, or an immortalized cell derived therefrom. The method of any one of claims 1 to 24, wherein the cell is a primary T cell or a NK cell. The method of any one of claims 1 to 25, wherein the cell is in vitro, ex vivo, in situ, or in vivo. 41 A cell comprising (a) two or more CRISPR-Cas nuclease systems and (b) a short isoform of terminal deoxyribonucleotidyl transferase (TdTS), wherein the CRISPR-Cas nuclease systems and the TdTS are exogenous to the cell. The cell of claim 27, wherein each CRISPR-Cas nuclease systems of (a) is a ribonucleoprotein (RNP) complex. The cell of claim 27, wherein each CRISPR-Cas nuclease system of (a) comprises CRISPR-Cas mRNA and gRNA. The cell of claim 28 or 29, wherein (b) comprises mRNA encoding TdTS The cell of claim 28 or 29, wherein (b) comprises TdTS protein. The cell of claim 27, wherein each CRISPR-Cas nuclease system of (a) comprises DNA encoding CRISPR-Cas nuclease and DNA encoding gRNA and (b) comprises DNA encoding TdTS. A kit for multiplex gene editing, the kit comprising (a) two or more CRISPR- Cas nuclease systems, and (b) a short isoform of terminal deoxyribonucleotidyl transferase (TdTS). The kit of claim 33, wherein each CRISPR-Cas nuclease systems of (a) is a RNP complex. The kit of claim 33, wherein each CRISPR-Cas nuclease system of (a) comprises CRISPR-Cas mRNA and gRNA. The kit of claim 34 or 35, wherein (b) comprises mRNA encoding TdTS

The kit of claim 34 or 35, wherein (b) comprises TdTS protein. 42 The kit of claim 33, wherein each CRISPR-Cas nuclease system of (a) comprises DNA encoding CRISPR-Cas nuclease and DNA encoding gRNA and (b) comprises DNA encoding TdTS. The kit of any one of claims 34 to 40, further comprising at least one reagent for gene editing.