Compositions and Methods for Editing Cytoplasmic DNA

Abstract

The present disclosure provides systems and methods for modifying target viral nucleic acids in the cytoplasm of eukaryotic cells.

Description

【Technical Field】 【0001】 Cross Reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 358,963, filed Jul. 7, 2022, the entire disclosure of which is hereby incorporated by reference. 【0002】 Incorporation by Reference of Electronically Filed Materials A Sequence Listing XML, generated on Jun. 26, 2023, having a size of 118,553 bytes, entitled “BERK-470WO_SEQ_LIST”, is provided with this specification as the Sequence Listing. The entire contents of the Sequence Listing XML are hereby incorporated by reference. 【Background Art】 【0003】 Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of viruses that harbor large (150 kbp to 1.2 Mbp) double-stranded DNA genomes and replicate in the cytoplasm of eukaryotic cells. NCLDVs include numerous viral families, including the Poxviridae, Asfaviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, Pithoviridae, and Mimiviridae, as well as Pandoraviruses, Molliviruses, and Faustoviruses. As a result of millions of years of natural evolution, NCLDVs have acquired the ability to deliver exogenous nucleic acids and proteins into cells. Certain virus species belonging to the NCLDV family have been adapted for use in recombinant vaccines, immunotherapy, adjuvants, gene therapy, expression systems for protein production, as pest management agents, and in other useful applications in biotechnology. However, natural evolution has not resulted in optimal qualities for many applications. Directed evolution is an iterative process that screens for genotypes that confer improved functions after generating diversity, and has been used as a powerful approach to engineer improved genetic code molecules, including systems with increased complexity such as gene delivery systems based on small viruses. However, NCLDVs have a large genome size, possess repetitive sequence regions, replicate in the cytoplasm, have limited homologous recombination efficiency, and rely on unique viral promoters and replication machinery, presenting barriers to applying directed evolution approaches to engineer NCLDVs using existing technologies. 【0004】 Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that are clustered contain a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. Such CRISPR-Cas systems can bind to a targeted nucleic acid and modify it. Due to the programmable nature of these CRISPR-Cas effector systems, they have become increasingly easy to use as versatile technologies for, for example, gene editing. 【0005】 There is a need in the art for compositions and methods for mutating large DNA viruses in the cytoplasm of eukaryotic cells. SUMMARY OF THE INVENTION 【0006】 The present disclosure provides systems and methods for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell. BRIEF DESCRIPTION OF THE DRAWINGS 【0007】 【Figure 1A】 Figures 1A-1F provide the amino acid sequences of DNA polymerase I (PolI) and mutants (SEQ ID NOs: 1-6, respectively). 【Figure 1B】 Figures 1A-1F provide the amino acid sequences of DNA polymerase I (PolI) and mutants (SEQ ID NOs: 1-6, respectively). 【Figure 1C】 Figures 1A-1F provide the amino acid sequences of DNA polymerase I (PolI) and mutants (SEQ ID NOs: 1-6, respectively). 【Figure 1D】 Figures 1A-1F provide the amino acid sequences of DNA polymerase I (PolI) and mutants (SEQ ID NOs: 1-6, respectively). 【Figure 1E】 Figures 1A-1F provide the amino acid sequences of DNA polymerase I (PolI) and mutants (SEQ ID NOs: 1-6, respectively). 【Figure 1F】 Figures 1A-1F provide the amino acid sequences of DNA polymerase I (PolI) and mutants (SEQ ID NOs: 1-6, respectively). 【0008】 【Figure 2】 Figure 2 provides the amino acid sequence of phi29 DNA polymerase (SEQ ID NO: 7). 【0009】 【Figure 3】 Figure 3 provides the amino acid sequence of T5 DNA polymerase (SEQ ID NO: 8). 【0010】 【Figure 4A】 Figures 4A-4B provide the amino acid sequences of T7 DNA polymerase and Sequenase (SEQ ID NOS: 9-10, respectively). 【Figure 4B】 Figures 4A-4B provide the amino acid sequences of T7 DNA polymerase and Sequenase (SEQ ID NOS: 9-10, respectively). 【0011】 【Figure 5】 Figure 5 provides the amino acid sequence of DNA polymerase iota (SEQ ID NO: 11). 【0012】 【Figure 6A】 Figures 6A-6B provide the amino acid sequence of DNA polymerase eta (SEQ ID NO: 12). 【Figure 6B】 Figures 6A-6B provide the amino acid sequence of DNA polymerase eta (SEQ ID NO: 12). 【0013】 【Figure 7A】 Figures 7A-7B provide the amino acid sequence of DNA polymerase kappa (SEQ ID NO: 13). 【Figure 7B】 Figures 7A-7B provide the amino acid sequence of DNA polymerase kappa (SEQ ID NO: 13). 【0014】 【Figure 8A】 Figures 8A-8B provide the amino acid sequence of DNA polymerase theta (SEQ ID NO: 14). 【Figure 8B】Figures 8A-8B provide the amino acid sequence of DNA polymerase θ (SEQ ID NO: 14). 【Figure 8C】 Figures 8A-8B provide the amino acid sequence of DNA polymerase θ (SEQ ID NO: 14). 【Figure 8D】 Figures 8A-8B provide the amino acid sequence of DNA polymerase θ (SEQ ID NO: 14). 【0015】 【Figure 9A】 Figures 9A-9B provide the amino acid sequence of DNA polymerase ν (SEQ ID NO: 15). 【Figure 9B】 Figures 9A-9B provide the amino acid sequence of DNA polymerase ν (SEQ ID NO: 15). 【0016】 【Figure 10A】 Figures 10A-10L provide the amino acid sequences of the CRISPR-Cas effector polypeptides (SEQ ID NOs: 16-27, respectively). 【Figure 10B】 Figures 10A-10L provide the amino acid sequences of the CRISPR-Cas effector polypeptides (SEQ ID NOs: 16-27, respectively). 【Figure 10C】 Figures 10A-10L provide the amino acid sequences of the CRISPR-Cas effector polypeptides (SEQ ID NOs: 16-27, respectively). 【Figure 10D】 Figures 10A-10L provide the amino acid sequences of the CRISPR-Cas effector polypeptides (SEQ ID NOs: 16-27, respectively). 【Figure 10E】 Figures 10A-10L provide the amino acid sequences of the CRISPR-Cas effector polypeptides (SEQ ID NOs: 16-27, respectively). 【Figure 10F】 Figures 10A-10L provide the amino acid sequences of the CRISPR-Cas effector polypeptides (SEQ ID NOs: 16-27, respectively). 【Figure 10G】 Figures 10A-10L provide the amino acid sequences of the CRISPR-Cas effector polypeptides (SEQ ID NOs: 16-27, respectively). 【Figure 10H】 Figures 10A - 10L provide the amino acid sequences of the CRISPR - Cas effector polypeptides (SEQ ID NOs: 16 - 27 respectively). 【Figure 10I】 Figures 10A - 10L provide the amino acid sequences of the CRISPR - Cas effector polypeptides (SEQ ID NOs: 16 - 27 respectively). 【Figure 10J】 Figures 10A - 10L provide the amino acid sequences of the CRISPR - Cas effector polypeptides (SEQ ID NOs: 16 - 27 respectively). 【Figure 10K】 Figures 10A - 10L provide the amino acid sequences of the CRISPR - Cas effector polypeptides (SEQ ID NOs: 16 - 27 respectively). 【Figure 10L】 Figures 10A - 10L provide the amino acid sequences of the CRISPR - Cas effector polypeptides (SEQ ID NOs: 16 - 27 respectively). 【0017】 【Figure 11A】 Figures 11A - 11E show a system for characterizing the diversification of user - defined loci in cytoplasmic DNA using the poxvirus vaccinia as a model (BFP amino acid and nucleic acid sequences: SEQ ID NOs: 28 - 29 respectively) (GFP amino acid and nucleic acid sequences: SEQ ID NOs: 30 - 31 respectively). 【Figure 11B】 Figures 11A - 11E show a system for characterizing the diversification of user - defined loci in cytoplasmic DNA using the poxvirus vaccinia as a model (BFP amino acid and nucleic acid sequences: SEQ ID NOs: 28 - 29 respectively) (GFP amino acid and nucleic acid sequences: SEQ ID NOs: 30 - 31 respectively). 【Figure 11C】 Figures 11A - 11E show a system for characterizing the diversification of user - defined loci in cytoplasmic DNA using the poxvirus vaccinia as a model (BFP amino acid and nucleic acid sequences: SEQ ID NOs: 28 - 29 respectively) (GFP amino acid and nucleic acid sequences: SEQ ID NOs: 30 - 31 respectively). 【Figure 11D】Figures 11A-11E show a system for characterizing diversification of a user-defined locus in cytoplasmic DNA using vaccinia virus as a model (BFP amino acid and nucleic acid sequences: SEQ ID NOs: 28-29, respectively) (GFP amino acid and nucleic acid sequences: SEQ ID NOs: 30-31, respectively). 【Figure 11E】 Figures 11A-11E show a system for characterizing diversification of a user-defined locus in cytoplasmic DNA using vaccinia virus as a model (BFP amino acid and nucleic acid sequences: SEQ ID NOs: 28-29, respectively) (GFP amino acid and nucleic acid sequences: SEQ ID NOs: 30-31, respectively). 【0018】 【Figure 12A】 Figures 12A-12C show data demonstrating that truncation of PAM-distal base pairs from the single-guide RNA (sgRNA) template-binding region increases nSpRy-PolI5M-mediated efficient single nucleotide polymorphism (SNP) generation. 【Figure 12B】 Figures 12A-12C show data demonstrating that truncation of PAM-distal base pairs from the single-guide RNA (sgRNA) template-binding region increases nSpRy-PolI5M-mediated efficient single nucleotide polymorphism (SNP) generation. 【Figure 12C】 Figures 12A-12C show data demonstrating that truncation of PAM-distal base pairs from the single-guide RNA (sgRNA) template-binding region increases nSpRy-PolI5M-mediated efficient single nucleotide polymorphism (SNP) generation. 【0019】 【Figure 13A】 Figures 13A-13B show data demonstrating that nSpRy-PolI5M guided by full-length (20 bp target site-binding) sgRNA and truncated (18 bp target site-binding) sgRNA, respectively, can target the AT-rich region of the A34R gene of vaccinia virus and generate site-specific diversity. 【Figure 13B】Figures 13A-13B show data indicating that nSpRy-PolI5M guided by full-length (20 bp target site-binding) sgRNA and excised (18 bp target site-binding) sgRNA can target the AT-rich region of the A34R gene of vaccinia virus and generate site-specific diversity. 【0020】 【Figure 14A】 Figures 14A-14D provide the amino acid sequences of the Cas9 mutants (SEQ ID NOs: 88-90 and 32, respectively). 【Figure 14B】 Figures 14A-14D provide the amino acid sequences of the Cas9 mutants (SEQ ID NOs: 88-90 and 32, respectively). 【Figure 14C】 Figures 14A-14D provide the amino acid sequences of the Cas9 mutants (SEQ ID NOs: 88-90 and 32, respectively). 【Figure 14D】 Figures 14A-14D provide the amino acid sequences of the Cas9 mutants (SEQ ID NOs: 88-90 and 32, respectively). 【0021】 【Figure 15】 Figure 15 shows data indicating that the RNA-guided nSpRY-PolI5M fusion complex confers on-target mutagenesis of VV-BFP with a low off-target effect. 【0022】 【Figure 16】 Figure 16 shows data indicating that the RNA-guided nSpRY-PolI5M fusion complex confers on-target mutagenesis in the genome of the distantly related poxvirus species, myxoma virus. 【0023】 【Figure 17】 Figure 17 shows data indicating that a miniaturized nuclease and polymerase fusion protein complexed with an excised (18 bp target site-binding) gRNA resulted in increased diversity at the targeted locus in VV-BFP. 【0024】 【Figure 18】 Figure 18 shows data indicating that the nSpRY-PolI5M fusion protein guided by a pool of 39 sgRNAs results in increased diversity across the endogenous gene of interest. 【0025】 【Figure 19】 Figure 19 provides the amino acid sequence (SEQ ID NO: 32) of the excised nSpRY-Cas9 mutant. 【0026】 【Figure 20】 Figure 20 provides the nucleic acid sequence (SEQ ID NO: 33) of the excised PolI5M mutant. 【DETAILED DESCRIPTION OF THE INVENTION】 【0027】 "Heterologous," as used herein in the context of a polypeptide, refers to an amino acid sequence not found in a native polypeptide. For example, a fusion CRISPR-Cas effector polypeptide comprises: a) a CRISPR-Cas effector polypeptide; and b) one or more heterologous polypeptides, where the heterologous polypeptides comprise amino acid sequences derived from proteins other than the CRISPR-Cas effector polypeptide. "Heterologous," as used herein in the context of a nucleic acid, refers to a nucleotide sequence not found in a native nucleic acid. As an example, in a guide nucleic acid, a heterologous guide nucleotide sequence (present in the targeting segment) capable of hybridizing to a target nucleotide sequence (target region) of a target nucleic acid is a nucleotide sequence not naturally found in the guide nucleic acid, together with a binding segment capable of binding to a CRISPR-Cas effector polypeptide. For example, in some cases, a heterologous target nucleotide sequence (present in a heterologous targeting segment) is from a different source than a binding nucleotide sequence (present in the binding segment) capable of binding to a CRISPR-Cas effector polypeptide of the present disclosure. The guide nucleic acids of the present disclosure may be generated by human intervention and may contain nucleotide sequences not found in naturally occurring guide nucleic acids. 【0028】 As used herein, when the term "naturally occurring" is applied to a nucleic acid, protein, cell, or organism, it refers to a nucleic acid, cell, protein, or organism that is found in nature. 【0029】 The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides or a combination thereof. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" are to be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides as applicable to the embodiments described. 【0030】 As used herein, terms such as "guide RNA" (gRNA) refer to an RNA that guides a CRISPR-Cas effector polypeptide (or a fusion protein comprising a CRISPR-Cas effector polypeptide) to a target sequence in a target nucleic acid. The term gRNA can also refer to a prime editing guide RNA (pegRNA), a nicking guide RNA (ngRNA), and a single guide RNA (sgRNA). In some cases, the term "gRNA molecule" refers to a nucleic acid encoding a gRNA. In some cases, the gRNA molecule is naturally occurring. In some cases, the gRNA molecule is non-naturally occurring. In some cases, the gRNA molecule is a synthetic gRNA molecule. 【0031】 The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. This term includes, but is not limited to, fusion proteins, including fusion proteins with heterologous amino acid sequences. 【0032】 Polypeptides as described herein also include polypeptides having various amino acid additions, deletions, or substitutions relative to the native amino acid sequence of the polypeptides of the present disclosure. In some embodiments, polypeptides that are homologs of the polypeptides of the present disclosure contain non-conservative changes of specific amino acids relative to the native sequence of the polypeptides of the present disclosure. In some embodiments, polypeptides that are homologs of the polypeptides of the present disclosure contain conservative changes of specific amino acids relative to the native sequence of the polypeptides of the present disclosure and can thus be referred to as conservative modified variants. Conservative modified variants can include individual substitutions, deletions, or additions to the polypeptide sequence that result in substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. Such conservative modified variants are added to polymorphic variants, interspecies homologs, and alleles of the present disclosure and do not exclude them. The following eight groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acid modifications that result in chemically similar amino acids can be referred to as similar amino acids. 【0033】 A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, which means that when aligned, that percentage of bases or amino acids are the same and in the same relative positions when the two sequences are compared. Sequence similarity can be determined in several different ways. To determine sequence identity, the sequences can be aligned using methods and computer programs including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST. See, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, a wholly owned subsidiary of Oxford Molecular Group, Inc., Madison, Wisconsin, USA. Other alignment techniques are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed., Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow gaps in the sequence. Smith-Waterman is one such algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the sequences may be aligned using the GAP program that uses the Needleman and Wunsch alignment method. See J. Mol. Biol. 48: 443-453 (1970). 【0034】 The terms "DNA control sequence", "regulatory element", and "control element" are used interchangeably herein and refer to transcriptional and translational regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., which provide and / or control the expression of a coding sequence and / or the production of the encoded polypeptide in a host cell. 【0035】 The term "transformation" is used interchangeably herein with "genetic modification" and refers to a permanent or transient genetic change induced in a cell after the introduction of a novel nucleic acid (e.g., DNA that is exogenous to the cell) into the cell. The genetic change ("modification") can be achieved either by the incorporation of the novel nucleic acid into the genome of the host cell or by the transient or stable maintenance of the novel nucleic acid as an episomal element. When the cell is a eukaryotic cell, a permanent genetic change is generally achieved by the introduction of novel DNA into the cell genome. 【0036】 "Linked so as to be functional" refers to proximal components that are in a relationship that permits the components so described to function in the manner in which they are intended. For example, a promoter is linked so as to be functional to a coding sequence if the promoter affects the transcription or expression of the coding sequence. As used herein, the terms "heterologous promoter" and "heterologous regulatory region" refer to promoters and other regulatory regions that are not normally associated in nature with a particular nucleic acid. For example, a "transcriptional regulatory region heterologous to a coding region" is a transcriptional regulatory region that is not normally associated in nature with that coding region. 【0037】 Before further describing the present invention, it is to be understood that the invention is not limited to the specific embodiments described, and as such, can of course be diverse. It is also to be understood that the terminology used herein is for the purpose of describing only particular embodiments and is not intended to be limiting, as the scope of the invention is limited only by the appended claims. 【0038】 When providing a range of values, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of the range, down to one-tenth of the unit of the lower limit, and any other recited or intervening values within the recited range are understood to be included within the present invention. The upper and lower limits of these smaller ranges may independently also be included within the smaller ranges, and this too is included within the present invention, depending on any specifically excluded limits within the recited range. Where one or both of the limits of the recited range are included, the range excluding either or both of these included limits is also included within the present invention. 【0039】 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the ones to which the publication is cited. 【0040】 As used in this specification and the appended claims, it should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a single-guide RNA" includes a plurality of such single-guide RNAs, and a reference to "the error-prone DNA polymerase" includes one or more error-prone DNA polymerases and their equivalents known to those of ordinary skill in the art, and the like. It is further noted that the claims may be drafted to exclude any optional element. As such, this reference is intended to serve as a precedent for the use of exclusionary terms such as "solely," "only," etc., or the use of "negative" limitations in connection with the recitation of claim elements. 【0041】 In the context of describing the present disclosure (particularly in the context of the following claims), the use of the terms "a", "an", and "the", and similar indicatives shall be considered to include both the singular and the plural, unless clearly indicated otherwise herein or clearly contradicted by the context. The terms "comprising", "having", "including", and "containing" shall be considered unrestricted terms (i.e., meaning "including but not limited to") unless otherwise stated. The recitation of a range of values herein is, unless otherwise indicated herein, merely intended to serve as a convenient method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if individually recited herein. For example, if a range of 10-15 is disclosed, 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or unless clearly contradicted by the context. The use of any examples, or exemplary language (e.g., "such as") provided herein is merely intended to better clarify embodiments of the present disclosure and does not impose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential for the practice of an embodiment of the present disclosure. 【0042】 As used herein, the term "about", when used in connection with an amount, indicates that the amount so referred to may vary by up to 10% of the recited amount. For example, "about 100" means an amount in the range of 90-110. When "about" is used in the context of a range, "about" used in reference to the lower amount of the range means that for the lower amount, an amount 10% lower than the lower amount of the range is included, and "about" used in reference to the higher amount of the range means that for the higher amount, an amount 10% higher than the higher amount of the range is included. For example, about 100-about 1000 means a range spanning from 90-1100. 【0043】 The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A alone; and B alone. Similarly, the term "and / or" as used herein in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone. 【0044】 It is understood that the aspects and embodiments of the present disclosure described herein include those that "comprise," "consist of," and "consist essentially of" the aspects and embodiments. 【0045】 It is recognized that, for clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments relevant to the present invention are specifically included by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Further, all sub-combinations of the various embodiments and their elements are also specifically included by the present invention and are disclosed herein as if each and every sub-combination were individually and explicitly disclosed. 【0046】 The publications discussed herein are provided only as regards their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates, which may need to be independently confirmed. 【0047】 Detailed Description The present disclosure provides systems and methods for modifying target viral nucleic acids in the cytoplasm of eukaryotic cells. 【0048】 The present disclosure provides a method for mutagenizing a region of cytoplasmic DNA defined by a user using a single guide RNA (sgRNA) or a combination of sgRNAs and a highly engineered fusion polypeptide, the fusion polypeptide comprising: a) an enzymatically active RNA-guided endonuclease that introduces a single-strand break in cytoplasmic DNA; and b) an error-prone DNA polymerase. In some cases, the fusion polypeptide comprises: a) an enzymatically active RNA-guided endonuclease that introduces a single-strand break in cytoplasmic DNA; b) a nuclear export sequence (NES); and c) an error-prone DNA polymerase. In some cases, the fusion polypeptide does not include a nuclear localization signal (NLS) polypeptide. 【0049】 A method for modifying a target nucleic acid in the cytoplasm of a eukaryotic cell The present disclosure provides a method for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell. In some cases, the method comprises contacting the target viral nucleic acid with: a) a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide that is a nickase (i.e., the CRISPR-Cas effector polypeptide introduces a single-strand break in the target viral nucleic acid), and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase and one of the two or more heterologous polypeptides comprises a nuclear export signal (NES) polypeptide; and b) one or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid, and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide. In some cases, the method comprises contacting the target viral nucleic acid with: a) a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide that is a nickase (i.e., the CRISPR-Cas effector polypeptide introduces a single-strand break in the target viral nucleic acid), and ii) one or more heterologous polypeptides, wherein one of the one or more heterologous polypeptides is an error-prone DNA polymerase; and b) one or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid, and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide. Contacting the target viral nucleic acid with the fusion polypeptide and the one or more guide nucleic acids provides modification of the target viral nucleic acid. In some cases, the fusion polypeptide does not comprise a nuclear localization signal (NLS) polypeptide. 【0050】 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -8 ~10 -2 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -8 A higher target mutation rate of mutations, e.g., 10 mutations per nucleotide per viral genome replication event -8 Greater than 10 -7 Greater than 10 -6 Greater than 10 -5 Greater than 10 -4 Greater than, or 10 -3 Greater than a target mutation rate of mutations. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -8 ~10 -7 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -7 ~10 -6 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -7 ~10 -5 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -5 ~10 -4 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -4 ~10 -3 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate of 10 mutations per nucleotide per viral genome replication event -3 ~10 -2Indicates the target mutation rate of the mutation. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it shows a target mutation rate of 1 mutation per nucleotide per viral genome replication event at 10 -2 ~10 -1 Indicates the target mutation rate of the mutation. 【0051】 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it shows a target mutation rate of 1 mutation per nucleotide per viral genome replication event. 【0052】 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it shows a ratio of target mutation rate to total mutation rate of at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 25:1, at least 50:1, at least 10 2 :1, at least 5x10 2 :1, at least 10 3 :1, at least 5x10 3 :1, at least 10 4 :1, or 10 4 :1 greater than, indicating the ratio of target mutation rate to total mutation rate. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it is about 1.5:1 to 10 4 :1, for example about 1.5:1 to 2:1, 2:1 to 5:1, 5:1 to 10:1, 10:1 to 25:1, 25:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1 to 5x10 2 :1, 5x10 2 :1 to 10 3 :1, 10 3 :1 to 5x10 3 :1, 5x10 3 :1 to 10 4 :1, or 10 4 :1 greater than, indicating the ratio of target mutation rate to total mutation rate. 【0053】 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate that is at least 2-fold higher than the target mutation rate shown by the error-prone DNA polymerase present in the fusion polypeptide when the error-prone DNA polymerase is not fused to the CRISPR-Cas effector polypeptide present in the fusion polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate that is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 10 2 -fold, at least 5x10 2 -fold, at least 10 3 -fold, at least 5x10 3 -fold, or at least 10 4 -fold higher than the target mutation rate shown by the error-prone DNA polymerase present in the fusion polypeptide when the error-prone DNA polymerase is not fused to the CRISPR-Cas effector polypeptide present in the fusion polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it exhibits a target mutation rate that is more than 10 4 -fold higher than the target mutation rate shown by the error-prone DNA polymerase present in the fusion polypeptide when the error-prone DNA polymerase is not fused to the CRISPR-Cas effector polypeptide present in the fusion polypeptide. 【0054】 In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 1 nucleotide to 10 4 nucleotides from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. For example, in some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 1 nucleotide (nt) to 10 nucleotides (nt), 10 nt to 50 nt, 50 nt to 100 nt, 100 nt to 500 nt, 50 nt to 10 3nt, 10 3 nt to 5x10 3 nt, or 5x10 3 nt to 10 4Introduce mutations at a distance of nt. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 1 nt to 10 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 1 nt to 25 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 10 nt to 25 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 1 nt to 50 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 10 nt to 50 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 25 nt to 50 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 1 nt to 100 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 10 nt to 100 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. In some cases, when the fusion polypeptide forms a complex with the guide RNA, it introduces mutations at a distance of 50 nt to 100 nt from the nick in the target DNA introduced by the CRISPR-Cas effector polypeptide. 【0055】 In some cases, the fusion polypeptide has a length of about 3000 amino acids or less. In some cases, the fusion polypeptide has a length of about 1000 amino acids to about 3000 amino acids. In some cases, the fusion polypeptide has a length of about 1000 amino acids to about 1250 amino acids, about 1250 amino acids to about 1500 amino acids, about 1500 amino acids to about 1750 amino acids, about 1750 amino acids to about 2000 amino acids, about 2000 amino acids to about 2250 amino acids, about 2250 amino acids to about 2500 amino acids, about 2500 amino acids to about 2750 amino acids, or about 2750 amino acids to about 3000 amino acids. 【0056】 Mutations that can be introduced into the target viral nucleic acid include insertions, deletions, substitutions, and the like. 【0057】 Target viral nucleic acid The viral nucleic acid that can be modified using the methods of the present disclosure is referred to as the "target viral nucleic acid." Suitable target viral nucleic acids are double-stranded DNA viruses having a genome length of about 50 kilobase pairs (kbp) to about 1.2 megabase pairs (mbp), where at least a portion of the replication cycle of the double-stranded DNA virus occurs in the cytoplasm of the cell. Such viruses are sometimes referred to as "nucleocytoplasmic large DNA viruses" or "NCLDVs." In some cases, suitable target viral nucleic acids are double-stranded DNA viruses having a genome length of about 50 kbp to 150 kbp, about 150 kbp to about 500 kbp, about 500 kbp to about 1000 kbp, or about 1000 kbp to about 1.2 mbp. 【0058】 NCLDVs include a number of viral families including the Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, Pithoviridae, Mimiviridae, Pandoraviridae, Mininucleoviridae, Mollivirus, and Faustovirus. 【0059】 In some cases, the target viral nucleic acid is a member of the family Poxviridae. The family Poxviridae includes the genera Avipoxvirus, Capripoxvirus, Centapoxvirus, Cervidpoxvirus, Crocodylidpoxvirus, Leporipoxvirus, Macroopoxvirus, Molluscipoxvirus, Mustelpoxvirus, Orthopoxvirus, Oryzopoxvirus, Parapoxvirus, Pteropopoxvirus, Scieuripoxvirus, Suipoxvirus, Vespertilionpoxvirus, Yatapoxvirus, Alphaentemopoxvirus, Betaentemopoxvirus, Deltaentomopoxvirus, Diachasmimorphaentemopoxvirus, and Gammaentomopoxvirus. The genus Orthopoxvirus includes the vaccinia virus, cowpox virus, monkey pox virus, and rabbit pox virus. In some cases, the target viral nucleic acid is the nucleic acid of a myxoma virus, a fibroma virus, or an ectromelia virus. In some cases, the target viral nucleic acid is the vaccinia virus.In some cases, the target viral nucleic acid is a vaccinia virus of any one of the following vaccinia virus strains: 1) Western Reserve; 2) Wyeth; 3) New York City Board of Health (NYCBH); 4) Paris; 5) Acambis 2000; 6) Bern; 7) Ankara; 8) IHD-J; 9) Copenhagen (Cop); 10) Temple of Heaven; 11) Dairen; 12) Lister; 13) Tian Tan; 14) Modified Vaccinia Ankara (MVA); 15) Lister clone 16m8 (LC16m8); and 16) Dairen I (DIs). In some cases, the target viral nucleic acid is a nucleic acid of a chimeric poxvirus strain or a recombinant virus strain encoding heterologous DNA. 【0060】 In some cases, the target nucleotide sequence in the target viral nucleic acid is a coding sequence, for example, the target nucleotide sequence encodes a polypeptide and / or RNA. The target nucleotide sequence in the target viral nucleic acid is a polypeptide involved in viral seeding, an antigenic polypeptide (e.g., a polypeptide in the viral capsid), a polypeptide providing oncolytic activity, a polypeptide functioning in the release of virus from cells, a polypeptide providing viral infectivity, a polypeptide modifying viral cell or host specificity, a polypeptide modifying the viral entry mechanism, a polypeptide modifying the intracellular transport of the virus, a polypeptide modifying the actin-based propulsion of the virus in the intracellular or extracellular environment, a polypeptide modifying the microtubule association or transport of the virus, a polypeptide involved in the formation and maturation of intracellular mature virions (IMV), a polypeptide involved in the formation of cell-associated enveloped virions (CEV), a polypeptide involved in the formation of extracellular enveloped virus (EEV), or a nucleotide sequence in a coding sequence encoding a polypeptide such as a polypeptide encoded by a virus that functions in antagonizing the host innate or acquired antiviral immune response. 【0061】 Any vaccinia virus gene may be the target nucleic acid. For example, vaccinia virus coding regions that may be of interest as target nucleotides include vaccinia virus genes selected from F13L, A36R, A34R, A53R, B5R, B7R, B13R, B15R, B22R, B28R, B29R, A33R, B8R, B18R, SPI-1, SPI-2, B15R, CUR, VGF, E3L, K2L, K3L, A41L, K7R, vC12L, vCKBP, and N1L. For example, A34R is a vaccinia virus glycoprotein required for cell release and infectivity of EEV. B5R, F13L, A36R, A34R, and A33R are examples of EEV-specific membrane proteins. 【0062】 In some cases, the methods of the disclosure provide for introducing one or more mutations into the target viral nucleic acid, thereby generating a mutant virus that exhibits increased oncolytic activity compared to a non-mutated virus (i.e., a control virus that does not contain one or more mutations but is otherwise identical to the mutant virus). In some cases, the methods of the disclosure provide for introducing one or more mutations into the target viral nucleic acid, thereby generating a mutant virus that exhibits at least 10%, at least 25%, at least 50%, at least 100% (or 2-fold), at least 5-fold, at least 10-fold, or more than 10-fold the oncolytic activity of a non-mutated virus (i.e., a control virus that does not contain one or more mutations but is otherwise identical to the mutant virus). 【0063】 In some cases, the methods of the present disclosure provide for introducing one or more mutations into a target viral nucleic acid, thereby generating a mutant virus that exhibits an increase in EEV production as compared to a non-mutated virus (i.e., a control virus that does not contain one or more mutations but is otherwise identical to the mutant virus). In some cases, the methods of the present disclosure provide for introducing one or more mutations into a target viral nucleic acid, thereby generating a mutant virus that exhibits at least 10%, at least 25%, at least 50%, at least 100% (or 2-fold), at least 5-fold, at least 10-fold, or more than 10-fold increase in EEV production as compared to a non-mutated virus (i.e., a control virus that does not contain one or more mutations but is otherwise identical to the mutant virus). 【0064】 In some cases, the methods of the present disclosure provide for introducing one or more mutations into a target viral nucleic acid, thereby generating a mutant virus that exhibits a decrease in neutralization by neutralizing antibodies in a mammalian host (e.g., human). In some cases, the methods of the present disclosure provide for introducing one or more mutations into a target viral nucleic acid, thereby generating a mutant virus that exhibits one or more of: 1) increased virion production in target cells; 2) increased CEV formation; 3) increased EEV formation; 4) modification of the cell entry mechanism to confer altered cell tropism; 5) increased intracellular trafficking; 6) increased kinetics of viral replication, maturation, and release; and 6) improved seeding among metastatic tumor cells in a human or non-human animal. 【0065】 In some cases, the methods of the present disclosure involve introducing into a eukaryotic cell in vitro: i) a fusion polypeptide (i.e., i) a CRISPR-Cas effector polypeptide and ii) two or more heterologous polypeptides, where one of the two or more heterologous polypeptides is an error-prone DNA polymerase and one of the two or more heterologous polypeptides comprises a NES polypeptide), and ii) one or more guide RNAs. In some cases, the guide RNA is a single guide RNA (“sgRNA”); i.e., the guide RNA is a single RNA molecule. In some cases, the nucleotide sequence encoding the fusion protein is operably linked to a promoter. In some cases, the promoter is a constitutive promoter. In some cases, the promoter is a controllable (e.g., inducible) promoter. 【0066】 In some cases, the methods of the present disclosure involve introducing into a eukaryotic cell in vitro a recombinant expression vector comprising: i) a first nucleotide sequence encoding one or more guide RNAs and ii) a second nucleotide sequence encoding a fusion polypeptide (i.e., i) a CRISPR-Cas effector polypeptide and ii) two or more heterologous polypeptides, where one of the two or more heterologous polypeptides is an error-prone DNA polymerase and one of the two or more heterologous polypeptides comprises a NES polypeptide). In some cases, the first nucleotide sequence is operably linked to a first promoter; the second nucleotide sequence is operably linked to a second promoter. 【0067】 A suitable promoter may be of viral origin and thus may be referred to as a viral promoter, or the promoter may be of any organism origin, including prokaryotes or eukaryotes. A suitable promoter may be used to drive expression by any RNA polymerase (e.g., polI, polII, polIII). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1;31(17)), the human H1 promoter (H1), etc. 【0068】 Suitable expression vectors include viral expression vectors (e.g., vaccinia virus; poliovirus; adenovirus; adeno-associated virus (AAV); SV40; herpes simplex virus; human immunodeficiency virus; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus)). In some cases, the recombinant expression vector is a recombinant adeno-associated virus (AAV) vector. In some cases, the recombinant expression vector is a recombinant lentiviral vector. In some cases, the recombinant expression vector is a recombinant retroviral vector. 【0069】 Suitable eukaryotic cells include in vitro cell lines, such as mammalian cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HEK 293T cells, HLHepG2 cells, and the like. 【0070】 Methods for introducing nucleic acids (e.g., recombinant expression vectors) into host cells are known in the art, and any suitable method may be used to introduce the nucleic acid into the cell. Suitable methods include, for example, viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. The fusion polypeptide and guide RNA may be present in a composition containing lipids. The fusion polypeptide and guide RNA may be present in lipid nanoparticles. Other suitable compositions are known in the art. 【0071】 In some cases, the methods of the disclosure for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell comprise: A) introducing into the eukaryotic cell a gene editing component, wherein the gene editing component comprises: a) a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase and one of the two or more heterologous polypeptides comprises a NES polypeptide; and b) one or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide, thereby producing a modified eukaryotic cell; and B) infecting the modified eukaryotic cell with a virus comprising the target viral nucleic acid, wherein the target viral nucleic acid is contacted with the gene editing component, and the contacting provides for modification of the target viral nucleic acid. In some cases, the infecting step (step B) is performed after the introducing step (step A). In some cases, step B is performed 2 to 96 hours after step A. For example, in some cases, step B is performed 2 to 4 hours, 4 to 8 hours, 8 to 12 hours, 12 to 18 hours, 18 to 24 hours, 24 to 36 hours, 36 to 48 hours, 48 to 72 hours, or 72 to 96 hours after step A. 【0072】 CRISPR-Cas effector polypeptide Suitable CRISPR-Cas effector polypeptides include type II CRISPR-Cas effector polypeptides, type III CRISPR-Cas effector polypeptides, type V CRISPR-Cas effector polypeptides, and type VI CRISPR-Cas effector polypeptides. 【0073】 In some cases, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide. In some cases, the type II CRISPR-Cas effector polypeptide is a Cas9 polypeptide, such as Streptococcus aureus Cas9, Streptococcus pyogenes Cas9 (SpCas9), etc. In some cases, the CRISPR-Cas effector polypeptide is a variant of wild-type SpCas9 and includes one or more of the following substitutions: A61R, L1111R, A1322R, D1135L, S1136W, G1218K, E1219Q, N1317R, R1333P, R1335A, and T1337R. In some cases, the CRISPR-Cas effector polypeptide is an SpG polypeptide or an SpRY polypeptide; see, for example, Walton et al. (2020) Science 368:290, and WO 2019 / 051097. SpRY is capable of targeting almost all protospacer adjacent motifs (PAMs) (NRN>NYN). In some cases, the CRISPR-Cas effector polypeptide is a miniaturized nSpRY-Cas9 with amino acids deleted. For example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide that includes D1135V, R1135Q, and T1137R substitutions compared to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide that includes D1135V, R1335Q, T1337R, and G1218R substitutions compared to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide that includes D1135L, S1136W, G1218K, E1219Q, R1335A, and T1337R substitutions compared to wild-type SpCas9.As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide comprising L1111R, A1322R, D1135L, S1136W, G1218K, E1219Q, R1335A, and T1337R substitutions compared to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide comprising A61R, L1111R, A1322R, D1135L, S1136W, G1218K, E1219Q, N1317R, R1333P, R1335A, and T1337R substitutions compared to wild-type SpCas9. The amino acid sequence of the wild-type SpCas9 polypeptide is provided in FIG. 10A. As another example, a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 19. 【0074】 In some cases, the CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide, such as a Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type VI CRISPR-Cas effector polypeptide, such as a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, or a Cas13d polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14a polypeptide, a Cas14b polypeptide, or a Cas14c polypeptide. For example, a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in any one of FIGS. 10A-10L. In some cases, a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in any one of FIGS. 14A-14D. 【0075】 In some cases, a CRISPR-Cas effector polypeptide suitable for use in the methods, systems, or compositions of the present disclosure is a nickase CRISPR-Cas effector polypeptide, i.e., a CRISPR-Cas effector polypeptide that binds to a target nucleic acid and cleaves only one strand of the target nucleic acid when complexed with a guide RNA. For example, in some cases, the CRISPR-Cas effector polypeptide is a Spy Cas9 polypeptide comprising a D10A substitution. 【0076】 NES and CPP In some cases, the heterologous polypeptide (fusion partner) provides intracellular localization, i.e., the heterologous polypeptide contains an intracellular localization sequence (e.g., a sequence that keeps the fusion protein outside the nucleus, such as a nuclear export sequence (NES), a sequence that retains the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an ER retention signal, etc.). In some cases, the CRISPR-Cas effector fusion polypeptide does not contain an NLS and thus the protein is not targeted to the nucleus (this may be suitable, for example, when the target nucleic acid is present in the cytoplasm). 【0077】 NESs are known in the art and any NES may be used in the fusion polypeptides of the present disclosure. See, for example, Xu et al. (2012) Mol. Biol. Cell 23:3677; and Fung et al. (2017) eLife 6:e23961. Examples of NESs include, for example, LPPLERLTL (SEQ ID NO: 34); LALKLAGLDL (SEQ ID NO: 35); MEELSQALASSFSV (SEQ ID NO: 36); EAETVSAMALLSVG (SEQ ID NO: 37); ELDELMASLSDFKF (SEQ ID NO: 38); VDQLRLERLQI (SEQ ID NO: 39); IDLSGLTLQ (SEQ ID NO: 40); LRALERLQID (SEQ ID NO: 41); LQKKLEELEL (SEQ ID NO: 42); MQELSNILNL (SEQ ID NO: 43); LCQAFSDVIL (SEQ ID NO: 44); RTFDMHSLESSLIDIMR (SEQ ID NO: 45); TNLEALQKKLEELELDE (SEQ ID NO: 46); RSFEMTEFNQALEEIKG (SEQ ID NO: 47); PLQLPPLERLTL (SEQ ID NO: 48); NELALKLAGLDI (SEQ ID NO: 49); ERFEMFRELNEALEL (SEQ ID NO: 50); DHAEKVAEKLEALSV (SEQ ID NO: 51); QLVEELLKIICAFQL (SEQ ID NO: 52); TNLEALQKKLEELEL (SEQ ID NO: 53); DVKEEMTSALATMRV (SEQ ID NO: 54); STNGSLAAEFRHLQL (SEQ ID NO: 55); PSVQELTEQIHRLLM (SEQ ID NO: 56); MNFKELKDFLKELNI (SEQ ID NO: 57); ENFEILMKLKESLEL (SEQ ID NO: 58); FETVYELTKMCTIRM (SEQ ID NO: 59); SGKASSSLGLQDFDL (SEQ ID NO: 60); PKYSDIDVDGLCSEL (SEQ ID NO: 61); and VDLACTPTDVRDVDI (SEQ ID NO: 62). NESs can have a length of 8 to 25 amino acids. In some cases, the NES has the following amino acid sequence: LPPLERLTL (SEQ ID NO: 34) and has a length of 9 amino acids. 【0078】 In some cases, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus: i) a CRISPR-Cas effector polypeptide; ii) one or more NESs; and iii) an error-prone DNA polymerase. In some cases, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus: i) a CRISPR-Cas effector polypeptide; ii) an error-prone DNA polymerase; and iii) one or more NESs. In some cases, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus: i) one or more NESs; ii) a CRISPR-Cas effector polypeptide; and iii) an error-prone DNA polymerase. In some cases, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus: i) a first NES; ii) a CRISPR-Cas effector polypeptide; iii) an error-prone DNA polymerase; and iv) a second NES. In some cases, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus: i) a first NES; ii) a CRISPR-Cas effector polypeptide; iii) a second NES; and iv) an error-prone DNA polymerase. A peptide linker may be disposed between any two polypeptides in the fusion protein, for example: i) between an NES and a CRISPR-Cas effector polypeptide; ii) between a first NES and a second NES; iii) between a CRISPR-Cas effector polypeptide and an error-prone DNA polymerase; iv) between an error-prone DNA polymerase and an NES, etc. 【0079】 In some cases, the heterologous polypeptide may provide a tag (i.e., the heterologous polypeptide may be a detectable label) to facilitate tracking and / or purification (e.g., a fluorescent protein, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, etc.; a histidine tag, such as a 6xHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag, etc.). 【0080】 In some cases, CRISPR-Cas effector polypeptides include a "protein transduction domain" or PTD (also known as a CPP - cell - penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule that may range from small polar molecules to macromolecules and / or nanoparticles facilitates the molecule's crossing of the membrane, for example, moving from the extracellular space into the intracellular space or from the cytosol into an organelle within the cell. In some embodiments, the PTD is covalently linked to the amino terminus of the fusion polypeptide. In some embodiments, the PTD is covalently linked to the carboxyl terminus of the fusion polypeptide. In some cases, the PTD is inserted internally within the fusion polypeptide at an appropriate insertion site. Examples of PTDs include, but are not limited to, the minimal undecapeptide protein transduction domain (corresponding to residues 47 - 57 of HIV - 1 TAT, including YGRKKRRQRRR; SEQ ID NO: 63); polyarginine sequences containing several arginines sufficient to enter cells directly (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10 - 50 arginines); the VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489 - 96); the Drosophila antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732 - 1737); truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248 - 1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003 - 13008); RRQRRTSKLMKR (SEQ ID NO: 64); transportan, GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 65); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 66); and RQIKIWFQNRRMKWKK (SEQ ID NO: 67).Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 63), RKKRRQRRR (SEQ ID NO: 68); arginine homopolymers of 3 arginine residues to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 63); RKKRRQRR (SEQ ID NO: 69); YARAAARQARA (SEQ ID NO: 70); THRLPRRRRRR (SEQ ID NO: 71); and GGRRARRRRRR (SEQ ID NO: 72). In some cases, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6): 371-381). The ACPP is a polycationic CPP (e.g., Arg9 or "R9") linked through a cleavable linker to a matching polyanion (e.g., Glu9 or "E9"), which reduces the net charge to near zero, thereby inhibiting adhesion and cellular uptake. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its native adhesiveness, thus "activating" the ACPP to cross the membrane. 【0081】 Error-prone DNA polymerase Several error-prone DNA polymerases are known in the art, and any of the known error-prone DNA polymerases are suitable for use in the fusion polypeptides of the present disclosure. Suitable error-prone DNA polymerases possess nick translation activity. 【0082】 Suitable error-prone DNA polymerases include, but are not limited to, Taq polymerase, Thermus flavus DNA polymerase I, Thermus thermophilus HB-8 DNA polymerase I, Thermophilus ruber DNA polymerase I, Thermophilus brokianus DNA polymerase I, Thermophilus caldophilus GK14 DNA polymerase I, Thermophilus filoformis DNA polymerase I, Bacillus stearothermophilus DNA polymerase I, Bacillus caldotonex YT-G DNA polymerase I, and Bacillus caldovelox YT-F DNA polymerase I. Suitable error-prone DNA polymerases include, but are not limited to, Niastella koreensis error-prone DNA polymerase, Mucilaginibacter paludis error-prone DNA polymerase, Methylobacterium extorquens error-prone DNA polymerase, and Stenotrophomonas maltophilia error-prone DNA polymerase. 【0083】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in any one of FIGS. 1A-1F. 【0084】 In some cases, a suitable error-prone DNA polymerase is Escherichia coli DNA polymerase I containing three fidelity-reducing mutations; this error-prone DNA polymerase is designated PolI3M. PolI3M contains the D424A, I709N, and A759R substitutions compared to wild-type Escherichia coli DNA polymerase I. In some cases, a suitable error-prone DNA polymerase contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in FIG. 1A; wherein the DNA polymerase has Ala at amino acid position 424, Asn at amino acid position 709, and Arg at amino acid position 759 of the amino acid sequence shown in FIG. 1A, or the corresponding amino acids in another DNA polymerase. 【0085】 In some cases, a suitable error-prone DNA polymerase is Escherichia coli DNA polymerase I containing five fidelity-reducing mutations; D424A, I709N, A759R, F742Y, and P796H. In some cases, a suitable error-prone DNA polymerase contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in FIG. 1A; wherein the DNA polymerase has Ala at amino acid position 424, Asn at amino acid position 709, Arg at amino acid position 759, Tyr at amino acid position 742, and His at amino acid position 796 of the amino acid sequence shown in FIG. 1A, or the corresponding amino acids in another DNA polymerase. 【0086】 In some cases, a suitable error-prone DNA polymerase contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in FIG. 1A; wherein the DNA polymerase has Ala at amino acid position 424 and Asn at amino acid position 709. 【0087】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in FIG. 2. 【0088】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in FIG. 3. 【0089】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in FIG. 4A or FIG. 4B. 【0090】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase iota amino acid sequence shown in FIG. 5. 【0091】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase eta amino acid sequence shown in FIGS. 6A-6B. 【0092】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase κ amino acid sequence shown in FIGS. 7A-7B. 【0093】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase θ amino acid sequence shown in FIGS. 8A-8D. 【0094】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase ν (nu) amino acid sequence shown in FIGS. 9A-9B. 【0095】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to DNA polymerase β having the following amino acid sequence: MSKRKAPQETLNGGITDMLTELANFEKNVSQAIHKYNAYRKAASVIAKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDTSSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNHHQRIGLKYFGDFEKRIPREEMLQMQDIVLNEVKKVDSEYIATVCGSFRRGAESSGDMDVLLTHPSFTSESTKQPKLLHQVVEQLQKVHFITDTLSKGETKFMGVCQLPSKNDEKEYPHRRIDIRLIPKDQYYCGVLYFTGSDIFNKNMRAHALEKGFTINEYTIRPLGVTGVAGEPLPVDSEKDIFDYIQWKYREPKDRSE (SEQ ID NO: 73). 【0096】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase iota having the following amino acid sequence: MEKLGVEPEEEGGGDDDEEDAEAWAMELADVGAAASSQGVHDQVLPTPNASSRVIVHVDLDCFYAQVEMISNPELKDKPLGVQQKYLVVTCNYEARKLGVKKLMNVRDAKEKCPQLVLVNGEDLTRYREMSYKVTELLEEFSPVVERLGFDENFVDLTEMVEKRLQQLQSDELSAVTVSGHVYNNQSINLLDVLHIRLLVGSQIAAEMREAMYNQLGLTGCAGVASNKLLAKLVSGVFKPNQQTVLLPESCQHLIHSLNHIKEIPGIGYKTAKCLEALGINSVRDLQTFSPKILEKELGISVAQRIQKLSFGEDNSPVILSGPPQSFSEEDSFKKCSSEVEAKNKIEELLASLLNRVCQDGRKPHTVRLIIRRYSSEKHYGRESRQCPIPSHVIQKLGTGNYDVMTPMVDILMKLFRNMVNVKMPFHLTLLSVCFCNLKALNTAKKGLIDYYLMPSLSTTSRSGKHSFKMKDTHMEDFPKDKETNRDFLPSGRIESTRTRESPLDTTNFSKEKDINEFPLCSLPEGVDQEVFKQLPVDIQEEILSGKSREKFQGKGSVSCPLHASRGVLSFFSKKQMQDIPINPRDHLSSSKQVSSVSPCEPGTSGFNSSSSSYMSSQKDYSYYLDNRLKDERISQGPKEPQGFHFTNSNPAVSAFHSFPNLQSEQLFSRNHTTDSHKQTVATDSHEGLTENREPDSVDEKITFPSDIDPQVFYELPEAVQKELLAEWKRAGSDFHIGHK(SEQ ID NO:11). In some cases, such DNA polymerases give rise to T→G substitutions. 【0097】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase iota having the following amino acid sequence (amino acids 1-445 of DNA polymerase iota): MEKLGVEPEEEGGGDDDEEDAEAWAMELADVGAAASSQGVHDQVLPTPNASSRVIVHVDLDCFYAQVEMISNPELKDKPLGVQQKYLVVTCNYEARKLGVKKLMNVRDAKEKCPQLVLVNGEDLTRYREMSYKVTELLEEFSPVVERLGFDENFVDLTEMVEKRLQQLQSDELSAVTVSGHVYNNQSINLLDVLHIRLLVGSQIAAEMREAMYNQLGLTGCAGVASNKLLAKLVSGVFKPNQQTVLLPESCQHLIHSLNHIKEIPGIGYKTAKCLEALGINSVRDLQTFSPKILEKELGISVAQRIQKLSFGEDNSPVILSGPPQSFSEEDSFKKCSSEVEAKNKIEELLASLLNRVCQDGRKPHTVRLIIRRYSSEKHYGRESRQCPIPSHVIQKLGTGNYDVMTPMVDILMKLFRNMVNVKMPFHLTLLSVCFCNLKALNTAK (SEQ ID NO: 74); having a length of 445 amino acids. In some cases, such DNA polymerases give rise to a T→G substitution. In some cases, such DNA polymerases have a T→G error rate approaching 1. 【0098】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase iota having the following amino acid sequence (amino acids 26 - 445 of DNA polymerase iota): ELADVGAAASSQGVHDQVLPTPNASSRVIVHVDLDCFYAQVEMISNPELKDKPLGVQQKYLVVTCNYEARKLGVKKLMNVRDAKEKCPQLVLVNGEDLTRYREMSYKVTELLEEFSPVVERLGFDENFVDLTEMVEKRLQQLQSDELSAVTVSGHVYNNQSINLLDVLHIRLLVGSQIAAEMREAMYNQLGLTGCAGVASNKLLAKLVSGVFKPNQQTVLLPESCQHLIHSLNHIKEIPGIGYKTAKCLEALGINSVRDLQTFSPKILEKELGISVAQRIQKLSFGEDNSPVILSGPPQSFSEEDSFKKCSSEVEAKNKIEELLASLLNRVCQDGRKPHTVRLIIRRYSSEKHYGRESRQCPIPSHVIQKLGTGNYDVMTPMVDILMKLFRNMVNVKMPFHLTLLSVCFCNLKALNTAK (SEQ ID NO: 75); having a length of 419 amino acids. In some cases, such DNA polymerases give rise to a T→G substitution. In some cases, such DNA polymerases have a T→G error rate approaching 1. 【0099】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA polymerase nu (ν) having the following amino acid sequence: ENYEALVGFDLCNTPLSSVAQKIMSAMHSGDLVDSKTWGKSTETMEVINKSSVKYSVQLEDRKTQSPEKKDLKSLRSQTSRGSAKLSPQSFSVRLTDQLSADQKQKSISSLTLSSCLIPQYNQEASVLQKKGHKRKHFLMENINNENKGSINLKRKHITYNNLSEKTSKQMALEEDTDDAEGYLNSGNSGALKKHFCDIRHLDDWAKSQLIEMLKQAAALVITVMYTDGSTQLGADQTPVSSVRGIVVLVKRQAEGGHGCPDAPACGPVLEGFVSDDPCIYIQIEHSAIWDQEQEAHQQFARNVLFQTMKCKCPVICFNAKDFVRIVLQFFGNDGSWKHVADFIGLDPRIAAWLIDPSDATPSFEDLVEKYCEKSITVKVNSTYGNSSRNIVNQNVRENLKTLYRLTMDLCSKLKDYGLWQLFRTLELPLIPILAVMESHAIQVNKEEMEKTSALLGARLKELEQEAHFVAGERFLITSNNQLREILFGKLKLHLLSQRNSLPRTGLQKYPSTSEAVLNALRDLHPLPKIILEYRQVHKIKSTFVDGLLACMKKGSISSTWNQTGTVTGRLSAKHPNIQGISKHPIQITTPKNFKGKEDKILTISPRAMFVSSKGHTFLAADFSQIELRILTHLSGDPELLKLFQESERDDVFSTLTSQWKDVPVEQVTHADREQTKKVVYAVVYGAGKERLAACLGVPIQEAAQFLESFLQKYKKIKDFARAAIAQCHQTGCVVSIMGRRRPLPRIHAHDQQLRAQAERQAVNFVVQGSAADLCKLAMIHVFTAVAASHTLTARLVAQIHDELLFEVEDPQIPECAALVRRTMESLEQVQALELQLQVPLKVSLSAGRSWGHLVPLQEAWGPPPGPCRTESPSNSLAAPGSPASTQPPPLHFSPSFCL(SEQ ID NO: 76). In some cases, such DNA polymerases result in G→T substitutions. 【0100】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to DNA polymerase eta (η) having the following amino acid sequence: MATGQDRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWKGGGIIAVSYEARAFGVTRSMWADDAKKLCPDLLLAQVRESRGKANLTKYREASVEVMEIMSRFAVIERASIDEAYVDLTSAVQERLQKLQGQPISADLLPSTYIEGLPQGPTTAEETVQKEGMRKQGLFQWLDSLQIDNLTSPDLQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVLAKLACGLNKPNRQTLVSHGSVPQLFSQMPIRKIRSLGGKLGASVIEILGIEYMGELTQFTESQLQSHFGEKNGSWLYAMCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQWWLLQLAQELEERLTKDRNDNDRVATQLVVSIRVQGDKRLSSLRRCCALTRYDAHKMSHDAFTVIKNCNTSGIQTEWSPPLTMLFLCATKFSASAPSSSTDITSFLSSDPSSLPKVPVTSSEAKTQGSGPAVTATKKATTSLESFFQKAAERQKVKEASLSSLTAPTQAPMSNSPSKPSLPFQTSQSTGTEPFFKQKSLLLKQKQLNNSSVSSPQQNPWSNCKALPNSLPTEYPGCVPVCEGVSKLEESSKATPAEMDLAHNSQSMHASSASKSVLEVTQKATPNPSLLAAEDQVPCEKCGSLVPVWDMPEHMDYHFALELQKSFLQPHSSNPQVVSAVSHQGKRNPKSPLACTNKRPRPEGMQTLESFFKPLTH (SEQ ID NO: 12). 【0101】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to DNA polymerase eta (η) having the following amino acid sequence: MATGQDRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWKGGGIIAVSYEARAFGVTRSMWADDAKKLCPDLLLAQVRESRGKANLTKYREASVEVMEIMSRFAVIERASIDEAYVDLTSAVQERLQKLQGQPISADLLPSTYIEGLPQGPTTAEETVQKEGMRKQGLFQWLDSLQIDNLTSPDLQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVLAKLACGLNKPNRQTLVSHGSVPQLFSQMPIRKIRSLGGKLGASVIEILGIEYMGELTQFTESQLQSHFGEKNGSWLYAMCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQWWLLQLAQELEERLTKDRNDNDRVATQLVVSIRVQGDKRLSSLRRCCALTRYDAHKMSHDAFTVIKNCNTSGIQTEWSPPLTMLFLCATKFSASAPSSSTDITSFLSSDPSSLPKVPVTSSEAKTQGSGPAVTATKKATTSLESFFQKAAERQKVKEASLSSLTAPTQAPMSN (SEQ ID NO: 77); having a length of 511 amino acids. 【0102】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to DNA polymerase kappa (κ) having the following amino acid sequence: MDSTKEKCDSYKDDLLLRMGLNDNKAGMEGLDKEKINKIIMEATKGSRFYGNELKKEKQVNQRIENMMQQKAQITSQQLRKAQLQVDRFAMELEQSRNLSNTIVHIDMDAFYAAVEMRDNPELKDKPIAVGSMSMLSTSNYHARRFGVRAAMPGFIAKRLCPQLIIVPPNFDKYRAVSKEVKEILADYDPNFMAMSLDEAYLNITKHLEERQNWPEDKRRYFIKMGSSVENDNPGKEVNKLSEHERSISPLLFEESPSDVQPPGDPFQVNFEEQNNPQILQNSVVFGTSAQEVVKEIRFRIEQKTTLTASAGIAPNTMLAKVCSDKNKPNGQYQILPNRQAVMDFIKDLPIRKVSGIGKVTEKMLKALGIITCTELYQQRALLSLLFSETSWHYFLHISLGLGSTHLTRDGERKSMSVERTFSEINKAEEQYSLCQELCSELAQDLQKERLKGRTVTIKLKNVNFEVKTRASTVSSVVSTAEEIFAIAKELLKTEIDADFPHPLRLRLMGVRISSFPNEEDRKHQQRSIIGFLQAGNQALSATECTLEKTDKDKFVKPLEMSHKKSFFDKKRSERKWSHQDTFKCEAVNKQSFQTSQPFQVLKKKMNENLEISENSDDCQILTCPVCFRAQGCISLEALNKHVDECLDGPSISENFKMFSCSHVSATKVNKKENVPASSLCEKQDYEAHPKIKEISSVDCIALVDTIDNSSKAESIDALSNKHSKEECSSLPSKSFNIEHCHQNSSSTVSLENEDVGSFRQEYRQPYLCEVKTGQALVCPVCNVEQKTSDLTLFNVHVDVCLNKSFIQELRKDKFNPVNQPKESSRSTGSSSGVQKAVTRTKRPGLMTKYSTSKKIKPNNPKHTLDIFFK (SEQ ID NO: 13). 【0103】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to DNA polymerase kappa (κ) having the following amino acid sequence: MDSTKEKCDSYKDDLLLRMGLNDNKAGMEGLDKEKINKIIMEATKGSRFYGNELKKEKQVNQRIENMMQQKAQITSQQLRKAQLQVDRFAMELEQSRNLSNTIVHIDMDAFYAAVEMRDNPELKDKPIAVGSMSMLSTSNYHARRFGVRAAMPGFIAKRLCPQLIIVPPNFDKYRAVSKEVKEILADYDPNFMAMSLDEAYLNITKHLEERQNWPEDKRRYFIKMGSSVENDNPGKEVNKLSEHERSISPLLFEESPSDVQPPGDPFQVNFEEQNNPQILQNSVVFGTSAQEVVKEIRFRIEQKTTLTASAGIAPNTMLAKVCSDKNKPNGQYQILPNRQAVMDFIKDLPIRKVSGIGKVTEKMLKALGIITCTELYQQRALLSLLFSETSWHYFLHISLGLGSTHLTRDGERKSMSVERTFSEINKAEEQYSLCQELCSELAQDLQKERLKGRTVTIKLKNVNFEVKTRASTVSSVVSTAEEIFAIAKELLKTEIDADFPHPLRLRLMGVRISSFPNEEDRKHQQRSIIGFLQAGNQALSATECTLEKTDKDKFVKPLE (SEQ ID NO: 78); having a length of 560 amino acids. 【0104】 In some cases, a suitable error-prone DNA polymerase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the excised PolI5M nucleic acid sequence shown in FIG. 20. 【0105】 Linker In some cases, the fusion polypeptides of the present disclosure include a linker disposed between a DNA polymerase and an RNA-guided endonuclease. 【0106】 In some embodiments, the subject fusion polypeptide may be fused to a fusion partner through a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. Proteins can generally be linked by flexible spacer peptides, although other chemical linkages are not excluded. Suitable linkers include polypeptides having from 4 to 40 amino acids in length, or from 4 to 25 amino acids in length. These linkers may be produced by coupling the proteins using oligonucleotides encoded by synthetic linkers, or may be encoded by nucleic acid sequences encoding fusion proteins. Peptide linkers having a degree of flexibility may be used. With the understanding that preferred linkers will generally have sequences that give rise to flexible peptides, the linking peptide may have substantially any amino acid sequence. The use of small amino acids such as glycine and alanine is what is used in generating flexible peptides. The generation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. 【0107】 Examples of linker polypeptides include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO:79), (GGSGGS) n (SEQ ID NO:80), and (GGGGS) n(SEQ ID NO: 81), wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers are included. Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 82), GGSGG (SEQ ID NO: 83), GSGSG (SEQ ID NO: 84), GSGGG (SEQ ID NO: 85), GGGSG (SEQ ID NO: 86), GSSSG (SEQ ID NO: 87), etc. One of ordinary skill in the art will recognize that for the design of a peptide conjugated to any desired element, a linker can include all or partially flexible linkers such that the linker can include a flexible linker as well as one or more moieties that provide a less flexible structure. 【0108】 CRISPR-Cas guide nucleic acid As described above, the guide nucleic acid includes: i) a binding region that binds to a CRISPR-Cas effector polypeptide, and ii) a targeting region that includes a nucleotide sequence complementary to the target sequence of the target nucleic acid. In some cases, the binding region is heterologous to the targeting region. In some cases, the nucleotide sequence complementary to the target sequence of the target nucleic acid is 15 to 19 nucleotides in length. In some cases, the nucleotide sequence complementary to the target sequence of the target nucleic acid is 18 nucleotides in length. The nucleotide sequence complementary to the target sequence of the target nucleic acid is 19 nucleotides or less in length, for example 18 nucleotides or less in length. 【0109】 A guide RNA comprising a nucleotide sequence complementary to the target sequence of a target nucleic acid provides an increased mutation rate when the nucleotide sequence is 18 nucleotides or less in length. For example, a guide RNA comprising an 18-nucleotide sequence that is complementary to the target sequence of a target nucleic acid, when complexed with the fusion polypeptide of the present disclosure (where the fusion polypeptide comprises an error-prone DNA polymerase and a CRISPR-Cas nickase polypeptide), provides a mutation rate that is 2- to 1000-fold higher (e.g., 2- to 5-fold, 5- to 10-fold, 10- to 25-fold, 25- to 50-fold, 50- to 100-fold, 100- to 250-fold, 250- to 500-fold, or 500- to 1000-fold higher) than the mutation rate obtained when the guide RNA comprises a 20-nucleotide sequence that is complementary to the target sequence of the target nucleic acid. 【0110】 In some cases, the CRISPR-Cas effector guide RNA has one or more modifications, such as one or more of a base modification, a backbone modification, and a sugar modification. 【0111】 Suitable modified backbones containing phosphorus atoms therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates including methyl and other alkyl phosphonates, phosphinates, 3'-aminophosphoramidates and phosphoramidates including aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates and boranophosphates having a normal 3'-5' linkage, their 2'-5' linkage analogs, and those having an inverted polarity in which one or more internucleotide linkages are 3'-3', 5'-5' or 2'-2' linkages. Suitable nucleic acids having an inverted polarity include a single 3'-3' linkage at the most 3'- internucleotide linkage, i.e., a single inverted nucleoside residue which may be basic (lacking a nucleobase or having a hydroxyl group instead). A variety of salts (e.g., potassium or sodium), mixed salts and free acid forms are also included. 【0112】 Suitable polynucleotides include sugar substituents selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. Particularly suitable are O((CH2) n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON((CH2) n CH3)2, where n and m are from 1 to about 10. Other suitable polynucleotides are C1-C 10Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and sugar substituents selected from other substituents having similar properties. Suitable modifications include 2'-methoxyethoxy (also known as 2'-O-CH2CH2OCH3, 2'-O-(2-methoxyethyl) or 2'-MOE) (the entire disclosure of which is incorporated herein by reference, Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. Further suitable modifications include 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, the O(CH2)2ON(CH3)2 group as described in the following examples herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2. 【0113】 The target nucleic acid may also include nucleic acid base (often simply referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleic acid bases include purine bases, adenine (A) and guanine (G), and pyrimidine bases, thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine may also be included. Further modified nucleic acid bases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp, such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indole-2-one), pyridoindole cytidine (H-pyrimido(3’,2’:4,5)pyrrolo(2,3-d)pyrimidine-2-one). 【0114】 System The present disclosure provides a system for implementing the methods of the present disclosure. 【0115】 In some cases, the system of the present disclosure comprises: a) a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide exhibiting nickase activity; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase lacking all or part of the 3'-to-5' exonuclease domain and / or lacking all or part of the 5'-to-3' exonuclease domain, one of the two or more heterologous polypeptides comprises a NES polypeptide, and the two or more heterologous polypeptides do not comprise a nuclear localization signal (NLS) polypeptide; and b) one or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in a target viral nucleic acid, the nucleotide sequence that binds to the target sequence having a length of 15 to 18 nucleotides; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide. In some cases, the error-prone DNA polymerase lacking all or part of the 3'-to-5' exonuclease domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 1F. 【0116】 In some cases, the systems of the present disclosure include: a) a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide exhibiting nickase activity; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase lacking all or a portion of the 3' to 5' exonuclease domain and / or lacking all or a portion of the 5' to 3' exonuclease domain, one of the two or more heterologous polypeptides comprises an NES polypeptide, and the two or more heterologous polypeptides do not comprise an NLS polypeptide; and b) one or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in a target viral nucleic acid, wherein the nucleotide sequence that binds to the target sequence has a length of 15 to 18 nucleotides; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide. 【0117】 In some cases, the systems of the present disclosure include: a) a nucleic acid (e.g., a recombinant expression vector) comprising: 1) a first nucleotide sequence encoding a fusion polypeptide, the fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide exhibiting nickase activity; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase lacking all or a portion of the 3'-to-5' exonuclease domain and / or lacking all or a portion of the 5'-to-3' exonuclease domain, and one of the two or more heterologous polypeptides comprises an NES polypeptide, and the two or more heterologous polypeptides do not comprise an NLS polypeptide; and b) a second nucleotide sequence encoding one or more guide nucleic acids, the one or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in a target viral nucleic acid, the nucleotide sequence that binds to the target sequence having a length of 15 to 18 nucleotides; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide. 【0118】 Examples of non-limiting aspects of the present disclosure Aspects including the above-described embodiments of the present subject matter may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure are provided below. As will be apparent to those skilled in the art upon reading the present disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects explicitly provided below: 【0119】 Aspect 1. A method for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell, the method comprising contacting the target viral nucleic acid with: a) a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase and one of the two or more heterologous polypeptides comprises a nuclear export signal (NES) polypeptide; and b) one or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide, wherein the contacting provides modification of the target viral nucleic acid. 【0120】 Aspect 2. The method of aspect 1, wherein the fusion polypeptide does not comprise a nuclear localization signal (NLS). 【0121】 Aspect 3. The method of aspect 1 or aspect 2, wherein the targeting region of the guide nucleic acid has a length of about 15 to 19 nucleobases. 【0122】 Aspect 4. The method of any one of aspects 1 to 3, wherein the error-prone DNA polymerase lacks 3'-5' exonuclease activity and / or lacks 5'-3' exonuclease activity. 【0123】 Aspect 5. The method of any one of aspects 1 to 4, wherein the fusion polypeptide has a length of about 3000 amino acids or less. 【0124】 Aspect 6. The method of any one of aspects 1 to 5, further comprising contacting the target viral nucleic acid with a donor nucleic acid. 【0125】 Aspect 7. The method according to any one of Aspects 1-6, wherein the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type III CRISPR-Cas effector polypeptide, a type IV CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide. 【0126】 Aspect 8. The method according to any one of Aspects 1-6, wherein the CRISPR-Cas effector polypeptide is a Cas9 polypeptide. 【0127】 Aspect 9. The method of Aspect 8, wherein the Cas9 polypeptide is a mutant Cas9 polypeptide having relaxed protospacer adjacent motif (PAM) requirements, and optionally, the mutant Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the amino acid sequence shown in any one of FIGS. 14A-14D. 【0128】 Aspect 10. The method according to any one of Aspects 1-9, wherein the fusion polypeptide exhibits a target mutation rate of 10 -8 ~10 -2 mutations per nucleotide per viral genome replication event. 【0129】 Aspect 11. The method according to any one of Aspects 1-9, wherein the fusion polypeptide exhibits a target mutation rate of 10 -6 ~10 -5 mutations per nucleotide per viral genome replication event. 【0130】 Aspect 12. The method according to any one of Aspects 1-9, wherein the fusion polypeptide exhibits a target mutation rate of 10 -5 ~10 -3 mutations per nucleotide per viral genome replication event. 【0131】 Aspect 13. When the fusion polypeptide forms a complex with the guide RNA, it has a target mutation rate of 10 nucleotides per viral genome replication event -3 ~10 -2 to 10 mutations, and the method according to any one of Aspects 1 to 9. 【0132】 Aspect 14. The method according to any one of Aspects 1 to 13, wherein the CRISPR-Cas effector polypeptide is a nickase. 【0133】 Aspect 15. The method according to any one of Aspects 1 to 13, wherein the CRISPR-Cas effector polypeptide lacks catalytic activity but retains binding to the target viral nucleic acid. 【0134】 Aspect 16. The method according to any one of Aspects 1 to 15, wherein the target viral nucleic acid is a nucleic acid of a double-stranded DNA virus having a genome length of about 50 kbp to about 1.2 Mbp, or about 150 kbp to 1.2 Mbp, and at least a part of the replication cycle of the double-stranded DNA virus occurs in the cytoplasm of the cell. 【0135】 Aspect 17. The method of Aspect 16, wherein the double-stranded DNA virus is a virus of a family selected from the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, Pithoviridae, Mimiviridae, Pandoraviridae, Mollivirus, and Faustovirus. 【0136】 Aspect 18. The method according to any one of Aspects 1 to 17, wherein the DNA polymerase comprises an amino acid sequence having at least 85% amino acid sequence identity to the DNA polymerase I amino acid sequence shown in FIG. 1A or FIG. 1F, where the DNA polymerase has one or more of Ala at amino acid position 424, Asn at amino acid position 709, Tyr at amino acid position 742, Arg at amino acid position 759, and His at amino acid position 796, and optionally, the DNA polymerase lacks all or part of the 3' to 5' exonuclease domain and / or lacks all or part of the 5' to 3' exonuclease domain. 【0137】 Aspect 19. The method according to any one of Aspects 1 to 17, wherein the DNA polymerase is DNA polymerase beta, DNA polymerase iota, DNA polymerase nu, DNA polymerase eta, or DNA polymerase kappa. 【0138】 Aspect 20. The method according to any one of Aspects 1 to 19, wherein the fusion polypeptide exhibits a target mutation rate of one mutation per nucleotide per viral genome replication event when complexed with the guide RNA. 【0139】 Aspect 21. The method according to any one of Aspects 1 to 20, wherein the method comprises introducing into a eukaryotic cell a recombinant expression construct comprising a nucleotide sequence encoding the fusion polypeptide. 【0140】 Aspect 22. The method of Aspect 21, wherein the recombinant expression construct comprises a nucleotide sequence encoding the guide RNA. 【0141】 Aspect 23. A method for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell, comprising: A) introducing a gene editing component into the eukaryotic cell, wherein the gene editing component comprises: a) a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase and one of the two or more heterologous polypeptides comprises a nuclear export signal (NES) polypeptide; and b) one or more guide nucleic acids, comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide, thereby generating a modified eukaryotic cell; B) infecting the modified eukaryotic cell with a virus comprising the target viral nucleic acid, such that the target viral nucleic acid is contacted with the gene editing component, wherein the contact provides for modification of the target viral nucleic acid. 【0142】 Aspect 24. A system for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell, comprising: 【0143】 a1) a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide having nickase activity; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides lacks all or part of the 3' to 5' exonuclease domain and / or lacks all or part of the 5' to 3' exonuclease domain, and is an error-prone DNA polymerase, one of the two or more heterologous polypeptides comprises a nuclear export signal (NES) polypeptide, and the two or more heterologous polypeptides do not comprise a nuclear localization signal (NLS) polypeptide; 【0144】 b1) One or more guide nucleic acids, comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in a target viral nucleic acid, wherein the nucleotide sequence that binds to the target sequence has a length of 15 to 18 nucleotides, and ii) a protein-binding region that binds to a CRISPR-Cas effector polypeptide; or 【0145】 a2) A nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises: i) a CRISPR-Cas effector polypeptide exhibiting nickase activity, and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase lacking all or part of the 3'-to-5' exonuclease domain and / or lacking all or part of the 5'-to-3' exonuclease domain, and one of the two or more heterologous polypeptides comprises an NES polypeptide, and the two or more heterologous polypeptides do not comprise an NLS polypeptide; 【0146】 b2) One or more guide nucleic acids, comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in a target viral nucleic acid, wherein the nucleotide sequence that binds to the target sequence has a length of 15 to 19 nucleotides, and ii) a protein-binding region that binds to a CRISPR-Cas effector polypeptide; or 【0147】 a3) A nucleic acid, comprising: 【0148】 1) A first nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises: i) a CRISPR-Cas effector polypeptide exhibiting nickase activity; and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase lacking all or a part of the 3'-to-5' exonuclease domain and / or lacking all or a part of the 5'-to-3' exonuclease domain, one of the two or more heterologous polypeptides comprises a NES polypeptide, and the two or more heterologous polypeptides do not comprise an NLS polypeptide, and the first nucleotide sequence as described above. 【0149】 2) A second nucleotide sequence encoding one or more guide nucleic acids, wherein the one or more guide nucleic acids comprise: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in a target viral nucleic acid, and the nucleotide sequence that binds to the target sequence has a length of 15 to 18 nucleotides; and ii) a protein-binding region that binds to a CRISPR-Cas effector polypeptide, and the second nucleotide sequence as described above. The nucleic acid as described above comprising The system as described above comprising. 【Examples】 【0150】 The following examples are set forth to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention. Nor are the following experiments intended to represent all or the only experiments that are performed. Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, for example, bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular; i.p., intraperitoneal; s.c., subcutaneous, etc. 【0151】 Example 1 As a first proof of concept to exemplify the idea and estimate the mutation rate that causes random mutagenesis within a user-defined locus in the NCLDV genome, we adapted the conversion assay from blue fluorescent protein to green fluorescent protein (GFP) (Glaser et al. (2016) Molecular Therapy - Nucleic Acids 5:e334); this assay enables visualization and quantification of the amino acid substitution from histidine to tyrosine (H68Y) resulting from a single nucleotide substitution from cytosine to thymine (Figure 11A). We knocked out both copies of the gene products J2R (encoding thymidine kinase (TK), which is essential for nucleotide precursor processing) and C11R (encoding vaccinia growth factor (VGF), which promotes the progression of neighboring cells into the S phase) to restrict replication in non-cancerous cells and thus ensure safety, and we incorporated the bfp gene sequence into the J2R locus of the Western Reserve strain of vaccinia virus (VV-BFP). We generated a plasmid that drives the expression of an sgRNA targeted to the bfp region of VV-BFP and a plasmid that encodes an mCherry-tagged fusion protein consisting of an RNA-guided nickase fused to an error-prone DNA polymerase. 【0152】 To achieve useful activity levels of such fusion proteins in cytoplasmic DNA virus genomes, including poxviruses and vaccinia, three engineering improvements were essential. 1. We engineered a D10A mutation in the SpRY Cas9 variant so that the A-T rich genome of poxviruses is targetable at all loci. 2. We used a nuclear export sequence (NES) instead of a nuclear localization sequence (NLS) to localize the complex to the cytoplasmic site of NCLDV genome replication. 3. We excised the template-binding region of each sgRNA from the standard 20 bp to 18 bp, significantly increasing the on-target observed mutation rate. 【0153】 To test the highly engineered complex, each plasmid was transfected into human embryonic kidney (HEK293-T) cells. Next, cells expressing the editing system were infected with VV-BFP 24 hours post-transfection (HPT). Subsequently, crude lysates were harvested, subjected to three freeze-thaw cycles, and rescued by infecting HEK293-Ad cells with the crude lysates at an MOI of 0.1. After rescue, the cells were fixed with 4% PFA, and the GFP reversion rate was quantified by flow cytometry using an Attune flow cytometer, and the data were analyzed using FlowJo software (Figure 11B). The initial results showed nuclease- and sgRNA-dependent GFP reversion efficiencies in the range of three orders of magnitude higher than background (0.108%), near the background reversion rate of the virus passaged without the editing system (Figure 11C). The use of S. pyogenes Cas9 with enhanced nicking fused to PolI5M (rather than nicking by SpRy Cas9) showed low GFP reversion rates. Optimizing the system by using a nickase with flexible PAM usage, tagging the molecule with two strategically placed NES sequences, and excising the template-binding region of the sgRNA from 20 bp to 18 bp resulted in all tested sgRNAs yielding GFP reversion rates exceeding 0.1%, and the highest-performing sgRNA yielded 1.18% GFP reversion after a single passage of the virus on HEK293-T cells expressing the editing system (Figure 11D). 【0154】 Figures 11A - 11E. System for in vivo diversification of user - defined loci in cytoplasmic DNA using the poxvirus vaccinia as a model. A) Through the generation of the H67Y (BFP→GFP) mutation, a measurable shift in emission is observable, which is caused by a single C→T nucleic acid substitution. B) Map showing the plasmid expression system of an RNA - guided fusion protein used for site - specific diversification of the NCLDV genome in mammalian cells. C) Method for a proof - of - concept experiment to evaluate the diversification of the bfp gene (VV - BFP) incorporated within the J2R locus of the vaccinia genome. The GFP reversion rate corresponds to only one of many possible nucleotide substitutions and thus provides an estimate of the diversification efficiency. D) The GFP reversion rates evaluated using 12 different sgRNAs and two different nickases demonstrate both the utility and the advancement of the present invention. Each sample corresponds to four independently transfected, infected, and rescued biological replicates. The statistical significance of individual samples relative to the empty vector control was calculated by Student's t - test. E) Representative dot plots for a positive gate control (VV - GFP = vaccinia stably expressing the corresponding gfp from the J2R locus) and two negative controls (VV - BFP virus not passaged on cells and VV - BFP passaged on cells expressing only the mCherry empty vector). 【0155】 Figures 12A - 12C. Excision of PAM - distal base pairs from the sgRNA template - binding region increases nSpRY - PolI5M - mediated efficient SNP generation. A) Direct (head - to - head) quantification of GFP reversion rates in the VV - BFP population. Four independent transfect / infect biological replicates show that excision of 2 base pairs from the PAM - distal side of the sgRNA binding region increases GFP reversion rates for all sgRNAs, regardless of initial performance. B) Representative dot - plot of individual biological replicates shown in Figure 12A. C) Genomic DNA of the VV - BFP population shown in Figure 12A was harvested, amplified by PCR, and subjected to Illumina sequencing. Analysis of rare mutants, subtracting the mutant frequency of the empty vector condition from each condition, showed an increase in the mutations observed in the viral population passaged on the excised (18bp template - binding region - containing) sgRNA samples relative to the full - length (20bp template - binding region - containing) sgRNA condition. 【0156】 To further illustrate the proof-of-concept of the editing system, the inventors designed a second functional assay with the conserved vaccinia gene A34R. A34R encodes a type II transmembrane glycoprotein with functions essential for the cell release and infectivity of EEV. Some previous studies provided proof-of-concept that point mutations in A34R could improve oncolytic vaccinia by increasing EEV production. First, the vaccinia IHD-J strain naturally produces up to 30% virions in EEV type 16, in contrast to <1% produced by other vaccinia strains, including WR, due to differences in only two residues. When tested in an oncolytic background, vaccinia WR expressing the IHD-J A34 protein variant instead of endogenous A34 showed enhanced dissemination in vivo, from subcutaneous tumors to lung tumors. Furthermore, a single rationally designed K151E point mutation in A34R of the WR vaccinia increased EEV production and resulted in improved dissemination and replication of the oncolytic vaccinia backbone in a peritoneal carcinomatosis model of MC38 colon cancer. Two sgRNAs were designed with 20bp template-binding regions targeting the locus encoding the lysine at the 151st amino acid position of A34R, and the sequences were subjected to NGS. The initial results showed that the non-optimized version of the editing system generated observable diversity within the targeting window. Next, the optimized excision sgRNA was used to guide nSpRY-PolI5M to the locus. After three cycles of diversification, selection was performed with the IMV neutralizing antibody 7D11. NGS and functional analysis of these experimental results are currently underway. Collectively, these data represent the first evidence of user-defined targeting diversification of cytoplasmic DNA in mammalian cells. Several NCLDV species harbor AT-rich genomes. To enable targeting diversification at any locus, the potential of a technique targeting the AT-rich region of the vaccinia virus (containing C11R and J2R gene deletions) was tested. 【0157】 Figures 13A - 13B. The nSpRY - PolI5M fusion complex guided by full - length (non - excised) sgRNA or excised (18bp) sgRNA induced on - target mutagenesis at an AT - rich endogenous locus in the vaccinia virus genome. A) nSpRY - PolI5M guided by full - length (20bp target - site binding) sgRNA generates site - specific diversity detectable at 100,000 reads. B) To enable targeting diversification at any locus, the potential of a technique targeting the AT - rich region of the vaccinia virus A34R gene when using an excised (18bp target - site binding) gRNA was tested. Two passages of VV - GFP on HEK293 - T cells transiently transfected with nSpRY - PolI5M and on - target sgRNA showed increased diversity at the endogenous A34R locus when analyzed by Illumina amplicon sequencing (compared to VV - GFP passaged on HEK293 - T transfected with empty vector). Collectively, these data provide further evidence demonstrating targeting diversification of user - defined loci within cytoplasmic DNA in mammalian cells. 【0158】 Example 2 A direct comparison of GFP conversion rates (quantified by flow cytometry) was performed to assess the off - target effects of the CRISPR fusion protein editing complex. VV - BFP passaged once on HEK293T cells expressing an sgRNA targeting the codons encoding HIS56 of nSpRY and BFP but lacking the fused polymerase contained no increased mutations compared to un - passaged VV - BFP or VV - BFP passaged on cells transfected with empty vector. 【0159】 Figure 15 The RNA-guided nSpRY-PolI5M fusion complex induced on-target mutagenesis of VV-BFP with low off-target effects. Conditions containing the nSpRY-PolI5M fusion protein and sgRNAs targeting off-target or non-target sites resulted in a slight increase in GFP conversion compared to the background, which was independent of the distance of the sgRNA target site from the site encoding HIS67 of BFP. On the other hand, only the condition containing the nSpRY-PolI5M fusion protein, together with on-target sgRNA alone or on-target sgRNA co-transfected with a pool of three off-target gRNAs, resulted in a GFP conversion rate that was more than three orders of magnitude higher than that of the background rate of GFP conversion of VV-BFP. All conditions shown in Figure 15 utilized n = 4 independent biological replicates, and all sgRNAs utilized an 18bp template-binding region. The statistical significance of each group shown was calculated by one-way ANOVA. These data indicate that both on-target gRNA and the nickase-polymerase fusion complex are required to achieve increased diversification at the loci targeted in the model NCLDV. 【0160】 To show that this technique works broadly among NCLDVs, we tested the ability of nSpRY-PolI5M to target a GFP-encoding gene stably integrated within the genome of a distantly related poxvirus known as myxoma virus (MYXV). Myxoma virus is a member of the genus Leporipoxvirus, which is a poxvirus genus with a host range limited to lagomorphs (rabbits and hares). Vaccinia, on the other hand, is a member of the genus Orthopoxvirus and contains numerous gene deletions, additions, and mutations that are responsible for its expanded host range. Both viruses have been considered as anti-cancer agents, and MYXV has historically also been used as a biological control agent against the invasive European rabbit in the Australian continent. 【0161】 Figure 16 The RNA-guided nSpRY-PolI5M fusion complex induced on-target mutagenesis in the genome of the distantly related poxvirus species, myxoma virus. Single-passage of MYXV on HEK293 cells transiently transfected with sgRNA targeting the incorporated genes encoding nSpRY-PolI5M and GFP showed increased diversity at the target locus when analyzed by Illumina amplicon sequencing. These data indicate that this technology works in a broad range of NCLDVs. 【0162】 A potential use of this technology is to generate targeted continuous diversity of cytoplasmic DNA in vivo. However, major gene delivery systems, such as adenovirus or lentivirus, require stable gene expression of RNA-guided fusion proteins in vivo and have the drawback of limited coding capacity with respect to heterologous gene cargo. Therefore, we tested whether the nickase and polymerase excision types could diversify the codons encoding HIS67 of BFP in VV-BFP. 【0163】 Figure 17 The excision nuclease and polymerase complex generated increased diversity at the targeted locus in VV-BFP. Single-passage of MYXV-GFP on HEK293 cells transiently transfected with a plasmid encoding a fusion protein composed of on-target sgRNA and excision nSpRY and excision PolI5M showed increased GFP conversion when analyzed by flow cytometry. These data indicate that the excision type of the technology, within the packaging limits of the standard lentiviral vector, retains functional activity. 【0164】 To evaluate multiplexed on-target mutagenesis, both copies of the gene products J2R (encoding thymidine kinase (TK), which is essential for nucleotide precursor processing) and C11R (encoding vaccinia growth factor (VGF), which promotes the progression of neighboring cells into the S phase) were knocked out to restrict replication in non-cancerous cells, and thus VV-BFP with ensured safety was passaged on HEK293-T cells expressing nSpRY-PolI5M and a pool of 39 sgRNAs whose target sites were tiled across the entire A34R gene. After two rounds of passage on cells expressing the editing machinery, viral genomic DNA was harvested, the A34R locus in a ~200 bp window was PCR amplified, and analyzed by next-generation sequencing. 【0165】 Figure 18 The nSpRY-PolI5M fusion protein guided by a pool of 39 sgRNAs generates increased diversity across the endogenous gene of interest. VV-BFP was passaged on HEK293-T cells where a pool of 39 sgRNAs was tiled across the A34R locus. Analysis of rare variants shows an increase in the number of unique mutations in the selected region of the A34R locus for VV-BFP passaged on HEK293-T cells expressing nSpRY-PolI5M and a pool of sgRNAs compared to VV-BFP passaged on HEK293-T cells expressing an empty vector. These data indicate that this technique is multiplexable across endogenous loci of the model NCLDV genome. 【0166】 The present invention has been described with reference to specific embodiments, but those skilled in the art must understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. Furthermore, many modifications can be made to adapt a particular situation, material, composition, process, or singular or plural process steps to the objectives, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the appended claims herein.

Claims

[Claim 1] A method for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell, wherein the target viral nucleic acid is: a) A fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide, and ii) two or more heterologous polypeptides, wherein one of the two or more heterologous polypeptides is an error-prone DNA polymerase, and one of the two or more heterologous polypeptides comprises a nuclear export signal (NES) polypeptide, b) one or more guide nucleic acids, comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid, and ii) a protein-binding region that binds to a CRISPR-Cas effector polypeptide, and contacting the one or more guide nucleic acids, The contact provides modification of the target viral nucleic acid. The aforementioned method. [Claim 2] The method of claim 1, wherein a) the viral nucleic acid is derived from a poxvirus, and / or b) the CRISPR-Cas effector polypeptide is a Spy Cas9 polypeptide containing a D10A substitution. [Claim 3] The method according to claim 1 or claim 2, wherein the targeting region of the guide nucleic acid has a length of about 15 to 19 nucleic acid bases. [Claim 4] The method according to claim 1 or 2, wherein the error-prone DNA polymerase has reduced 3'-5' exonuclease activity or lacks 3'-5' exonuclease activity. [Claim 5] The method according to claim 1 or claim 2, wherein the fusion polypeptide has a length of about 3000 amino acids or less. [Claim 6] The method according to claim 1 or 2, further comprising contacting the target viral nucleic acid with a donor nucleic acid. [Claim 7] The method according to claim 1 or 2, wherein the CRISPR-Cas effector polypeptide is a mutant Cas9 polypeptide having a relaxed protospacer adjacent motif (PAM) requirement, and optionally the mutant Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity with respect to the amino acid sequence shown in any one of SEQ ID NOs. 88-90 and 32. [Claim 8] The method according to claim 1 or 2, wherein the CRISPR-Cas effector polypeptide lacks catalytic activity but retains binding to the target viral nucleic acid. [Claim 9] The method according to claim 1 or 2, wherein the target viral nucleic acid is the nucleic acid of a double-stranded DNA virus having a genome length of about 50 kbp to about 1.2 mbp, or about 150 kbp to 1.2 mbp, at least a portion of the replication cycle of the double-stranded DNA virus occurs in the cytoplasm of the cell, and optionally the double-stranded DNA virus is a virus from a family selected from the Poxviridae, Asphaviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, Pitoviridae, Mimiviridae, Pandoraviridae, Morivirus, and Faustovirus. [Claim 10] The method of claim 1 or 2, wherein the DNA polymerase comprises an amino acid sequence having at least 85% amino acid sequence identity with respect to the DNA polymerase I amino acid sequence of SEQ ID NO: 1, wherein the DNA polymerase has one or more Ala at amino acid position 424, Asn at amino acid position 709, Tyr at amino acid position 742, Arg at amino acid position 759, and His at amino acid position 796, or the DNA polymerase is DNA polymerase beta, DNA polymerase iota, DNA polymerase neo, DNA polymerase eta, or DNA polymerase kappa. [Claim 11] When the aforementioned fusion polypeptide forms a complex with the guide RNA, 10 nucleotides per viral genome replication event -8 ~10 -2 The method of claim 1 or claim 2, which provides a target mutation rate of 10⁻⁶ to 10⁻⁵, 10⁻⁵ to 10⁻⁧, or 10⁻⁧ to 10⁻⁰ mutations, or 1 mutation. [Claim 12] The method according to claim 1 or 2, comprising introducing a recombinant expression construct comprising a nucleotide sequence encoding the fusion polypeptide into the eukaryotic cell, wherein the recombinant expression construct optionally comprises a nucleotide sequence encoding the guide RNA. [Claim 13] The method according to claim 1 or 2, wherein prior to the contact, the eukaryotic cell is infected with a virus containing the target viral nucleic acid. [Claim 14] A system for modifying target viral nucleic acids in the cytoplasm of eukaryotic cells: a1) A fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide exhibiting nickas activity; and ii) two or more heterologous polypeptides, one of which is an error-prone DNA polymerase lacking all or part of the 3' to 5' exonuclease domain and / or all or part of the 5' to 3' exonuclease domain, and one of which comprises a nuclear export signal (NES) polypeptide, and the two or more heterologous polypeptides not comprising a nuclear localization signal (NLS) polypeptide, the fusion polypeptide comprising: b1) One or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid, wherein the nucleotide sequence that binds to the target sequence has a length of 15 to 18 nucleotides; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide; or a2) A nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises: i) a CRISPR-Cas effector polypeptide exhibiting nickase activity, and ii) two or more heterologous polypeptides, one of which is an error-prone DNA polymerase lacking all or part of the 3' to 5' exonuclease domain and / or all or part of the 5' to 3' exonuclease domain, and one of which comprises an NES polypeptide and the other two or more heterologous polypeptides not comprising an NLS polypeptide, and the nucleic acid comprising: b2) One or more guide nucleic acids comprising: i) a targeting region comprising a nucleotide sequence that binds to a target sequence in the target viral nucleic acid, wherein the nucleotide sequence that binds to the target sequence has a length of 15 to 18 nucleotides; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide; or a3) Nucleic acids: 1) A first nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises: i) a CRISPR-Cas effector polypeptide exhibiting nickase activity, and ii) two or more heterologous polypeptides, one of which is an error-prone DNA polymerase lacking all or part of the 3' to 5' exonuclease domain and / or all or part of the 5' to 3' exonuclease domain, and one of which comprises an NES polypeptide and the other two or more heterologous polypeptides not comprising an NLS polypeptide, the first nucleotide sequence, 2) A second nucleotide sequence encoding one or more guide nucleic acids, wherein the one or more guide nucleic acids include: i) a nucleotide sequence that binds to a target sequence in the target viral nucleic acid, wherein the nucleotide sequence that binds to the target sequence has a targeting region having a length of 15 to 18 nucleotides; and ii) a protein-binding region that binds to the CRISPR-Cas effector polypeptide. The nucleic acid, including The system including the above. [Claim 15] The system of claim 14, wherein a) the viral nucleic acid is derived from a poxvirus, and / or b) the CRISPR-Cas effector polypeptide is a Spy Cas9 polypeptide containing a D10A substitution.

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