WO2021183783A1

WO2021183783A1 - Chimeric crispr/cas effector polypeptides and methods of use thereof

Info

Publication number: WO2021183783A1
Application number: PCT/US2021/021942
Authority: WO
Inventors: Jennifer A. Doudna; Audrone LAPINAITE; Enrique LIN SHIAO; David Frank Savage
Original assignee: The Regents Of The University Of California
Priority date: 2020-03-12
Filing date: 2021-03-11
Publication date: 2021-09-16

Abstract

The present disclosure provides a modified type II CRISPR/Cas effector polypeptide comprising a heterologous polypeptide in place of the HNH domain. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the modified type II CRISPR/Cas effector polypeptide, as well as cells comprising the modified type II CRISPR/Cas effector polypeptide and/or nucleic acid. A modified type II CRISPR/Cas effector polypeptide, or nucleic acid encoding such modified type II CRISPR/Cas effector polypeptide, of the present disclosure finds use in methods of modifying a target nucleic acid or a polypeptide associated with a target nucleic acid, which methods are also provided.

Description

CHIMERIC CRISPR/CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/988,556, filed March 12, 2020, which application is incorporated herein by reference in its entirety.

INTRODUCTION

[0002] CRISPR-Cas systems include Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a guide RNA(s), which includes a segment that binds Cas proteins and a segment that binds to a target nucleic acid. For example, Class 2 CRISPR-Cas systems comprise a single Cas protein bound to a guide RNA (gRNA), where the Cas protein binds to and cleaves a targeted nucleic acid. The programmable nature of these systems has facilitated their use as a versatile technology for use in modification of target nucleic acid.

[0003] There is a need in the art for CRISPR/Cas effector polypeptides comprising heterologous functionalities that are allosterically regulated by domains within the CRISPR/Cas effector polypeptide.

SUMMARY

[0004] The present disclosure provides a modified type II CRISPR/Cas effector polypeptide comprising a heterologous polypeptide in place of the HNH domain. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the modified type II CRISPR/Cas effector polypeptide, as well as cells comprising the modified type II CRISPR/Cas effector polypeptide and/or nucleic acid. A modified type II CRISPR/Cas effector polypeptide, or nucleic acid encoding such modified type II CRISPR/Cas effector polypeptide, of the present disclosure finds use in methods of modifying a target nucleic acid or a polypeptide associated with a target nucleic acid, which methods are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1A-1D depict the dynamics of Streptococcus pyogenes Cas9 HNH domain.

[0006] FIG. 2 depicts the domain organization of the DNA adenine base editor ABE 7.10 and a scheme illustrating the flexibility of the deaminase domain fused to Cas9 through a long and flexible linker. [0007] FIG. 3A-3C depict a strategy of HNH domain replacement with a DNA deaminase domain.

[0008] FIGs. 4-8 provide amino acid sequence of various Cas9 polypeptides.

[0009] FIGs. 9-11 provide schematic depictions of examples of modified CRISPR/Cas effector polypeptides of the present disclosure.

DEFINITIONS

[0010] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0011] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non- covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a guanine (G) (e.g., of dsRNA duplex of a guide RNA molecule; of a guide RNA base pairing with a target nucleic acid, etc.) is considered complementary to both a uracil (U) and to an adenine (A). For example, when a G/U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0012] Flybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. [0013] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0014] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489), and the like.

[0015] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0016] "Binding" as used herein (e.g. with reference to an RNA-binding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a modified CRISPR/Cas effector polypeptide/guide RNA complex and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. Binding interactions are generally characterized by a dissociation constant (K_D) of less than 10⁶ M, less than 10⁷ M, less than 10 ^s M, less than 10⁹ M, less than 10¹⁰ M, less than 10¹¹ M, less than 10¹² M, less than 10¹³ M, less than 10¹⁴ M, or less than 10¹⁵ M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower K_D.

[0017] By "binding domain" it is meant a protein domain that is able to bind non-covalently to another molecule. A binding domain can bind to, for example, a DNA molecule (a DNA-binding domain), an RNA molecule (an RNA-binding domain) and/or a protein molecule (a proteinbinding domain). In the case of a protein having a protein-binding domain, it can in some cases bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more regions of a different protein or proteins.

[0018] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic -hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine-glycine, and asparagine- glutamine.

[0019] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0020] A DNA sequence that "encodes" a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a “non-coding” RNA (ncRNA), a guide RNA, etc.).

[0021] A "protein coding sequence" or a sequence that encodes a particular protein or polypeptide, is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.

[0022] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence and/or regulate translation of an encoded polypeptide.

[0023] As used herein, a “promoter” or a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. For purposes of the present disclosure, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive expression by the various vectors of the present disclosure.

[0024] The term "naturally-occurring" or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature is naturally occurring. [0025] The term “fusion” as used herein as applied to a nucleic acid or polypeptide refers to two components that are defined by structures derived from different sources. For example, where "fusion" is used in the context of a fusion polypeptide (e.g., a fusion polypeptide comprising a CRISPR/Cas effector polypeptide and a fusion partner(s)), the fusion polypeptide includes amino acid sequences that are derived from different polypeptides. A fusion polypeptide may comprise either modified or naturally-occurring polypeptide sequences (e.g., a first amino acid sequence from a CRISPR/Cas effector polypeptide; and a second amino acid sequence from a protein other than a CRISPR/Cas effector polypeptide, etc.).

[0026] The term “fusion polypeptide” refers to a polypeptide which is made by the combination (i.e., “fusion”) of two otherwise separated segments of amino acid sequence, usually through human intervention.

[0027] “Heterologous,” as used herein, means a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. For example, in some cases, in a modified CRISPR/Cas effector polypeptide of the present disclosure, a portion of a naturally-occurring CRISPR/Cas effector polypeptide (or a variant thereof) may be fused to a heterologous polypeptide (i.e. an amino acid sequence from a protein other than a CRISPR/Cas effector polypeptide; or an amino acid sequence from another organism). As another example, a modified CRISPR/Cas effector polypeptide of the present disclosure comprises a portion of a naturally- occurring CRISPR/Cas effector (or variant thereof) fused to a heterologous polypeptide, i.e., a polypeptide from a protein other than CRISPR/Cas effector, or a polypeptide from another organism. The heterologous polypeptide may exhibit an activity (e.g., enzymatic activity) that will also be exhibited by the modified CRISPR/Cas effector polypeptide.

[0028] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences"). Alternatively, DNA sequences encoding RNA (e.g., guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence. An example of such a case is a DNA (a recombinant) encoding a wild-type protein where the DNA sequence is codon optimized for expression of the protein in a cell (e.g., a eukaryotic cell) in which the protein is not naturally found (e.g., expression of a modified CRISPR/Cas effector polypeptide of the present disclosure in a eukaryotic cell). A codon-optimized DNA can therefore be recombinant and non-naturahy occurring while the protein encoded by the DNA may have a wild type amino acid sequence.

[0029] Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose amino acid sequence does not naturally occur. Instead, a “recombinant” polypeptide is encoded by a recombinant non-naturahy occurring DNA sequence, but the amino acid sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant, etc.). Thus, a "recombinant" polypeptide is the result of human intervention, but may have a naturally occurring amino acid sequence.

[0030] A "vector" or “expression vector” is a replicon, such as plasmid, phage, virus, artificial chromosome, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell.

[0031] An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence (or the coding sequence can also be said to be operably linked to the promoter) if the promoter affects its transcription or expression.

[0032] The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and an insert. Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0033] A cell has been “genetically modified” or "transformed" or "transfected" by exogenous DNA or exogenous RNA, e.g. a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0034] Suitable methods of genetic modification (also referred to as “transformation”) include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) -mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283- 9. doi: 10.1016/j.addr.2012.09.023 ), and the like.

[0035] The choice of method of genetic modification is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0036] A “target nucleic acid” as used herein is a polynucleotide (e.g., DNA such as genomic DNA) that includes a site ("target site" or "target sequence") targeted by a modified CRISPR/Cas effector polypeptide of the present disclosure. The target sequence is the sequence to which the guide sequence of a guide nucleic acid (e.g., guide RNA; e.g., a dual guide RNA or a singlemolecule guide RNA) will hybridize. For example, the target site (or target sequence) 5'- GAGCAUAUC-3' within a target nucleic acid is targeted by (or is bound by, or hybridizes with, or is complementary to) the sequence 5’-GAUAUGCUC-3’. Suitable hybridization conditions include physiological conditions normally present in a cell. For a double stranded target nucleic acid, the strand of the target nucleic acid that is complementary to and hybridizes with the guide RNA is referred to as the “complementary strand” or “target strand”; while the strand of the target nucleic acid that is complementary to the “target strand” (and is therefore not complementary to the guide RNA) is referred to as the “non-target strand” or “noncomplementary strand.”

[0037] By “cleavage” it is meant the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events.

[0038] “Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[0039] By "cleavage domain" or “active domain” or “nuclease domain” of a nuclease it is meant the polypeptide sequence or domain within the nuclease which possesses the catalytic activity for nucleic acid cleavage. A cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.

[0040] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0041] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. [0042] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0043] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0044] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0045] 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 now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0046] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a type II CRISPR/Cas effector polypeptide” includes a plurality of such polypeptides and reference to “the heterologous polypeptide” includes reference to one or more heterologous polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. [0047] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0048] The publications discussed herein are provided solely for 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 publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0049] The present disclosure provides a modified type II CRISPR/Cas effector polypeptide comprising a heterologous polypeptide in place of the HNH domain. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the modified type II CRISPR/Cas effector polypeptide, as well as cells comprising the modified type II CRISPR/Cas effector polypeptide and/or nucleic acid. A modified type II CRISPR/Cas effector polypeptide or nucleic acid encoding such modified type II CRISPR/Cas effector polypeptide of the present disclosure finds use in methods of modifying a target nucleic acid or a polypeptide associated with a target nucleic acid, which methods are also provided.

[0050] Streptococcus pyogenes Cas9 (Spy Cas9) is an example of a type II CRISPR/Cas enzyme. Spy Cas9 is a multi-domain polypeptide that includes two catalytically active nuclease domains: RuvC and HNH. The HNH domain is dynamic when the Cas9 protein is in the gRNA bound state. It undergoes a large conformational rearrangement upon interaction of a Cas9/gRNA complex with a fully complementary target double-stranded DNA (dsDNA), and docks onto the non-target strand of the DNA. HNH’s activity and conformational switch are allosterically regulated via direct interactions of REC lobe domains REC2 and REC3 with gRNA/target DNA heteroduplexes. If the dsDNA is not fully complementary to the target-binding sequence present in the gRNA, the HNH domain becomes trapped in a catalytically inactive state. This is illustrated schematically in FIG. 1A-1B. [0051] Table 1. Table 1 lists 4 motifs that are present in Cas9 sequences from various species. The amino acids listed in Table 1 are from the Cas9 from S. pyogenes (FIG. 4).

[0052] FIG. 2 depicts an example of a chimeric SpyCas9 protein in which an adenosine deaminase (e.g. TagA) is fused via a flexible linker to the C-terminus of a SpyCas9 protein. In such a chimeric polypeptide, the adenosine is not allosterically controlled by the REC lobe. FIG. 2 depicts the numerous potential conformations that the chimeric protein may assume where the adenosine deaminase may be found in many different locations with respect to the Cas9 polypeptide.

[0053] The present disclosure provides a modified type II CRISPR/Cas effector polypeptide comprising a heterologous polypeptide in place of the HNH domain. Just as the HNH domain in an unmodified type II CRISPR/Cas effector polypeptide is allosterically regulated via REC2 and REC3 (collectively referred to as the “REC lobe”), the heterologous polypeptide is allosterically regulated by REC2 and REC3 in the modified type II CRISPR/Cas effector polypeptide. MODIFIED TYPE II CRISPR/CAS EFFECTOR POLYPEPTIDES

[0054] The present disclosure provides a modified type II CRISPR/Cas effector polypeptide comprising a heterologous polypeptide in place of the HNH domain, such that all, or substantially all (e.g., from 50% to 55%, from 55% to 60%, from 60% to 65%, from 65% to 70%, from 70% to 75%, from 75% to 80%, from 80% to 85%, from 85% to 90%, from 90% to 95%, or from 95% to 100%; or, e.g., from 50 amino acids to 100 amino acids, or from 100 amino acids to the entire length of the HNH domain) of the HNH domain of a corresponding unmodified type II CRISPR/Cas effector polypeptide is replaced with the heterologous polypeptide, such that the modified type II CRISPR/Cas effector polypeptide lacks HNH function, and such that the heterologous polypeptide is allosterically regulated by the REC lobe.

[0055] The HNH domain cleaves the DNA strand complementary to the guide RNA sequence; i.e., the HNH domain cleaves the target strand. The HNH domain includes a conserved Tyr823 residue and an Arg864 residue, as well as conserved residues Asp839, His840, and Asp861 (based on the Spy Cas9 amino acid sequence depicted in FIG. 4, where the conserved residues are highlighted). [0056] As depicted schematically in FIG. 1A, amino acids 775-909 of Spy Cas9 constitute the HNH domain. A type II CRISPR/Cas polypeptide comprises an HNH domain, where the HNH domain is amino acids 775-909 of a Spy Cas9 (e.g., a Cas9 polypeptide comprising the amino acid sequence depicted in FIG. 4 where the HNH domain is shown in bold underlined text), or corresponding amino acids of another Cas9 polypeptide; see, e.g., Nishimasu et al. (2014) Cell 156:935. For example, the HNH domain of the Staphylococcus aureus Cas9 is amino acids 520- 629 of the amino acid sequence depicted in FIG. 5 where the HNH domain is shown in bold underlined text; see, e.g., Nishimasu et al. (2015) Cell 162:1113. As another example, the HNH domain of the Corynebacterium diphtheriae Cas9 is amino acids 498-664 of the amino acid sequence depicted in FIG. 6 where the HNH domain is shown in bold underlined text; see, e.g., Hirano et al. (2019) Nat. Commun. 10:1968. As another example, the HNH domain of the Campylobacter jejuni Cas9 is amino acids 481-641 of the amino acid sequence depicted in FIG. 7 where the HNH domain is shown in bold underlined text; see, e.g., Yamada et al. (2017)

Molec. Cell 65:1109. As another example, the HNH domain of the Francisella novicida Cas9 is amino acids 932-1070 of the amino acid sequence depicted in FIG. 8 where the HNH domain is shown in bold underlined text; see, e.g., Hirano et al. (2016) Cell 164:950. See also, Jinek et al. (2014) Science 343:1247997.

[0057] A modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises a heterologous polypeptide, such that the heterologous polypeptide replaces all or a portion of the HNH domain of a corresponding unmodified type II CRISPR/Cas effector polypeptide. For example, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises a heterologous polypeptide, such that the heterologous polypeptide replaces from 50% to 100% of the HNH domain of a corresponding unmodified type II CRISPR/Cas effector polypeptide. For example, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises a heterologous polypeptide, such that the heterologous polypeptide replaces from 50% to 55%, from 55% to 60%, from 60% to 65%, from 65% to 70%, from 70% to 75%, from 75% to 80%, from 80% to 85%, from 85% to 90%, from 90% to 95%, or from 95% to 100%, of the HNH domain of a corresponding unmodified type II CRISPR/Cas effector polypeptide. A modified type II CRISPR/Cas effector polypeptide of the present disclosure lacks HNH function.

[0058] In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 775-845, amino acids 775-850, 775- 860, 775-870, 775-880, 775-890, 775-900, or 775-909, of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 780-850, 780-860, 780-870, 780-880, 780-890, 780- 900, or 780-909, of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 790-860, 790-870, 790-880, 790-890, 790-900, or 790-909, of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 800-870, 800-880, 800-890, 800-900, or 800-909, of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 810-880, 810-890, 810-900, or 810-909, of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 820-890, 820-900, or 820-909, of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 830-900, or 830-909, of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 840-909 of the SpyCas9 amino acid sequence depicted in FIG. 4, or the corresponding amino acids in another Cas9 polypeptide.

[0059] In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 520-570, 520-580, 520-590, 520-600, 520-610, 520-620, or 520-629, of the SauCas9 amino acid sequence depicted in FIG. 5, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 530-580, 530-590, 530-600, 530-610, 520-620, or 520-629, of the SauCas9 amino acid sequence depicted in FIG. 5, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 540-590, 540-600, 540-610, 540-620, or 540-629, of the SauCas9 amino acid sequence depicted in FIG. 5, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 550-600, 550-610, 550-620, or 550-629, of the SauCas9 amino acid sequence depicted in FIG. 5, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 560-610, 560-620, or 560-629, of the SauCas9 amino acid sequence depicted in FIG. 5, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 570-620, or 570-629, of the SauCas9 amino acid sequence depicted in FIG. 5, or the corresponding amino acids in another Cas9 polypeptide.

[0060] In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 498-550, 498-560, 498-570, 498-580, 498-590, 498-600, 498-610, 498-620, 498-630, 498-640, 498-650, or 498-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 510-560, 510-570, 510-580, 510-590, 510-600, 510-610, 510-620, 510-630, 510-640, 510-650, or 510- 664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 520-570, 520-580, 520-590, 520-600, 520-610, 520-620, 520-630, 520-640, 520-650, or 520-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 530-580, 530-590, 530-600, 530-610, 530-620, 530-630, 530- 640, 530-650, or 530-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 540-590, 540-600, 540-610, 540-620, 540-630, 540-640, 540- 650, or 540-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 550-600, 550-610, 550-620, 550-630, 550-640, 550-650, or 550- 664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 560-610, 560-620, 560-630, 560-640, 560-650, or 560-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 570-620, 570- 630, 570-640, 570-650, or 570-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 580-630, 580-640, 580-650, or 580-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 590-640, 590-650, or 590-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 600-650, or 600-664, of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6, or the corresponding amino acids in another Cas9 polypeptide.

[0061] In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 481-530, 481-540, 481-550, 481-560, 481-570, 481-580, 481-590, 481-600, 481-610, 481-620, 481-630, or 481-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 490-540, 490- 550, 490-560, 490-570, 490-580, 490-590, 490-600, 490-610, 490-620, 490-630, or 490-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 500-550, 500-560, 500-570, 500-580, 500-590, 500-600, 500-610, 500-620, 500-630, or 500-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 510-560, 510-570, 510-580, 510-590, 510-600, 510-610, 510-620, 510-630, or 510-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 520-570, 520- 580, 520-590, 520-600, 520-610, 520-620, 520-630, or 520-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 530-580, 530-590, 530-600, 530-610, 530-620, 530-630, or 530-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 540-590, 540-600, 540-610, 540-620, 540-630, or 540-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 550-600, 550-610, 550-620, 550-630, or 550-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 560-610, 560-620, 560-630, or 560-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 570-620, 570- 630, or 570-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 580-630, or 580-641, of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7, or the corresponding amino acids in another Cas9 polypeptide.

[0062] In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 932-980, 932-990, 932-1000, 932- 1010, 932-1020, 932-1030, 932-1040, 932-1050, 932-1050, 932-1060, or 932-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 940-990, 940-1000, 940-1010, 940-1020, 940-1030, 940-1040, 940-1050, 940-1050, 940-1060, or 940- 1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 950-1000, 950-1010, 950-1020, 950-1030, 950-1040, 950-1050, 950-1050, 950-1060, or 950-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 960-1010, 960-1020, 960-1030, 960-1040, 960-1050, 960-1050, 960-1060, or 960-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 970- 1020, 970-1030, 970-1040, 970-1050, 970-1050, 970-1060, or 970-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 980-1030, 980- 1040, 980-1050, 980-1050, 980-1060, or 980-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 990-1040, 990-1050, 990-1050, 990-1060, or 990- 1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 1000-1050, 1000-1050, 1000-1060, or 1000-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 1010-1050, 1010-1060, or 1010-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide. In some cases, the heterologous polypeptide present in a modified type II CRISPR/Cas effector polypeptide of the present disclosure replaces amino acids 1020-1060, or 1020-1070, of the F. novicida Cas9 amino acid sequence depicted in FIG. 8, or the corresponding amino acids in another Cas9 polypeptide.

[0063] In some cases, as depicted schematically in FIG. 9, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain; 2) a bridge helix (BH) domain; 3) a recognition (REC) lobe; 4) a RuvC-II domain; 5) a heterologous polypeptide; 6) a RuvC-III domain; and 7) a PAM-interacting (PI) domain.

[0064] In some cases, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1-60 of the S. pyogenes Cas9 amino acid sequence depicted in FIG. 5; 2) a BH domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 61-94 of the S. pyogenes Cas9 amino acid sequence depicted in FIG. 5; 3) a REC lobe comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 95-718 of the S. pyogenes Cas9 amino acid sequence depicted in FIG. 5; 4) a RuvC-II domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 719-775 of the S. pyogenes Cas9 amino acid sequence depicted in FIG. 5; 5) a heterologous polypeptide; 6) a RuvC-III domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 910-1099 of the S. pyogenes Cas9 amino acid sequence depicted in FIG. 5; and 7) a PI domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1100-1368 of the S. pyogenes Cas9 amino acid sequence depicted in FIG. 5.

[0065] In some cases, as depicted schematically in FIG. 10, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain; 2) a BH domain; 3) a REC lobe; 4) a RuvC-II domain; 5) a heterologous polypeptide; 6) a RuvC-III domain; 7) a Wedge (WED) domain; 8) a topoisomerase-homology (TOPO) domain; and 9) a carboxyl-terminal domain (CTD) domain.

[0066] In some cases, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1-41 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5; 2) a BH domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 42-74 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5; 3) a REC lobe comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 75-426 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5; 4) a RuvC-II domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 436-481 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5; 5) a heterologous polypeptide; 6) a RuvC- III domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 650-775 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5; 7) a WED domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 788-910 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5; 8) a TOPO domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 911-968 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5; and 9) a CTD domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 969-1053 of the S. aureus Cas9 amino acid sequence depicted in FIG. 5. In some cases, the polypeptide comprises a peptide linker of from about 10 amino acids to about 40 amino acids (e.g., from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, or from about 35 amino acids to about 40 amino acids) between one or more of: i) the REC lobe and the RuvC-II domain; ii) the heterologous polypeptide and the RuvC-III domain; and iii) the RuvC-III domain and the WED domain.

[0067] In some cases, as depicted schematically in FIG. 11, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain; 2) a BF1 domain; 3) a REC lobe; 4) a RuvC-II domain; 5) a heterologous polypeptide; 6) a RuVC-III domain; 7) a WED domain; and 8) a PI domain.

[0068] In some cases, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1-52 of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6; 2) a BF1 domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 53-86 of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6; 3) a REC lobe comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 87-449 of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6; 4) a RuvC-II domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 450-498 of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6; 5) a heterologous polypeptide; 6) a RuvC-III domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 665-808 of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6; 7) a WED domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 821-905 of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6; and 8) a PI domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 906-1084 of the C. diphtheriae Cas9 amino acid sequence depicted in FIG. 6. In some cases, the polypeptide comprises a peptide linker of from about 10 amino acids to about 40 amino acids (e.g., from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, or from about 35 amino acids to about 40 amino acids) between the RuvC- III domain and the WED domain.

[0069] In some cases, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1-45 of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7; 2) a BH domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 46-77 of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7; 3) a REC lobe comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 78-427 of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7; 4) a RuvC-II domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 428-481 of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7; 5) a heterologous polypeptide; 6) a RuvC- III domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 641-778 of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7; 7) a WED domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 792-828 of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7 ; and 8) a PI domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 829-984 of the C. jejuni Cas9 amino acid sequence depicted in FIG. 7. In some cases, the polypeptide comprises a peptide linker of from about 10 amino acids to about 40 amino acids (e.g., from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, or from about 35 amino acids to about 40 amino acids) between the RuvC-III domain and the WED domain.

[0070] In some cases, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: 1) a RuvC-I domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1-51 of the F. novicida Cas9 amino acid sequence depicted in FIG. 8; 2) a BFi domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 52-83 of the F. novicida Cas9 amino acid sequence depicted in FIG. 8; 3) a REC lobe comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 84-858 of the F. novicida Cas9 amino acid sequence depicted in FIG. 8; 4) a RuvC-II domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 859-899 of the F. novicida Cas9 amino acid sequence depicted in FIG. 8; 5) a heterologous polypeptide; 6) a RuvC-III domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1088-1224 of the F. novicida Cas9 amino acid sequence depicted in FIG. 8; 7) a WED domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1244-1469 of the F. novicida Cas9 amino acid sequence depicted in FIG. 8; and 8) a PI domain comprising 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%, at least 99%, or 100%, amino acid sequence identity to amino acids 1479-1629 of the F. novicida Cas9 amino acid sequence depicted in FIG. 8. In some cases, the polypeptide comprises a peptide linker of from about 10 amino acids to about 40 amino acids (e.g., from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, or from about 35 amino acids to about 40 amino acids) between one or more of: i) the RuvC-II domain and the heterologous polypeptide; ii) the heterologous polypeptide and the RuvC-III domain; iii) the RuvC-III domain and the WED domain; and iv) the WED domain and the PI domain.

[0071] In some cases, a modified CRISPR/Cas polypeptide of the present disclosure comprises a substitution of Asp-10 (D10) of the Streptococcus pyogenes amino acid sequence depicted in FIG. 4, or a corresponding amino acid of another Cas9 polypeptide. A modified CRISPR/Cas polypeptide of the present disclosure that comprises a D10 substitution can cleave the complementary strand of a double stranded target nucleic acid but has reduced ability to cleave the non-complementary strand of a double stranded target nucleic acid (thus resulting in a single strand break (SSB) instead of a double strand break (DSB) when the variant Cas9 protein cleaves a double stranded target nucleic acid) (see, for example, Jinek et al. (2012) Science 17;337(6096):816-21. For example, in some cases, a modified CRISPR/Cas polypeptide of the present disclosure comprises a D10A substitution, i.e., a substitution of D10 of the Streptococcus pyogenes amino acid sequence depicted in FIG. 4, or a corresponding amino acid of another Cas9 polypeptide, with an Ala.

Heterologous polypeptides

[0072] As noted above, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises a heterologous polypeptide in place of the HNH domain, such that all, or substantially all (e.g., from 50% to 55%, from 55% to 60%, from 60% to 65%, from 65% to 70%, from 70% to 75%, from 75% to 80%, from 80% to 85%, from 85% to 90%, from 90% to 95%, or from 95% to 100%; or, e.g., from 50 amino acids to 100 amino acids, or from 100 amino acids to the entire length of the HNH domain) of the HNH domain of a corresponding unmodified type II CRISPR/Cas effector polypeptide is replaced with the heterologous polypeptide, such that the modified type II CRISPR/Cas effector polypeptide lacks HNH function, and such that the heterologous polypeptide is allosterically regulated by the REC lobe.

[0073] Suitable heterologous polypeptides include, e.g., a polypeptide that exhibits one or more enzymatic activities. For example, a suitable heterologous polypeptides include, e.g., a polypeptide that exhibits an enzymatic activity selected from: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0074] A suitable heterologous polypeptide includes a heterologous polypeptide that exhibits histone modification activity. A suitable heterologous polypeptide includes a polypeptide that exhibits one or more enzymatic activities selected from: methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity, and deglycosylation activity.

[0075] In some cases, the heterologous polypeptide has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).

[0076] In some cases, the heterologous polypeptide has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the heterologous polypeptide include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT 1C and EHMT2), SUV39H2, ESET/SETDB 1 , and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARIDlB/PLU-1, JARID1C/SMCX, JARID1D/SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HB01/MYST2, FiMOF/MYSTl, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., FiDACl, FIDAC2, FIDAC3, FIDAC8, FIDAC4, FIDAC5, FIDAC7, FIDAC9, SIRT1, SIRT2, FiDACl 1, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[0077] In some cases, the heterologous polypeptide has enzymatic activity that remodels chromatin and alters nucleosome positioning. Non-limiting examples of such polypeptides include SMARCA5 (also referred to as SNF2FI or ISWI).

[0078] In some cases, the heterologous polypeptide is a metabolic enzyme. In some cases, a suitable metabolic enzyme is an acetyl-CoA synthetase 2 (ACSS2) that generates acetyl coenzyme A (acetyl-CoA) in the nucleus to regulate local histone acetylation (Mews et al. (2017) Nature 546:381). In some cases, a suitable metabolic enzyme is a methionine adenosyltransferase that catalyzes the reaction of methionine and adenosine triphosphate to produce S- adenosylmethionine (SAM).

[0079] In some cases, the heterologous polypeptide has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the heterologous polypeptide include but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., Fokl nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Fihal DNA m5c-methyltransferase (M.Fihal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like) , DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinFI106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity)

[0080] In some cases, the heterologous polypeptide is a reverse transcriptase. In some cases, the heterologous polypeptide is a deaminase. For example, in some cases, the heterologous polypeptide is an adenosine deaminase or a cytosine deaminase.

[0081] Non-limiting examples of heterologous polypeptides for use when targeting ssRNA target nucleic acids include, but are not limited to: splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain).

[0082] In some cases, the heterologous polypeptide is a nuclease. Suitable nucleases include, but are not limited to, a homing nuclease polypeptide; a Fokl polypeptide; a transcription activator-like effector nuclease (TALEN) polypeptide; a MegaTAL polypeptide; a meganuclease polypeptide; a zinc finger nuclease (ZFN); an ARCUS nuclease; and the like. The meganuclease can be engineered from an LADLIDADG homing endonuclease (LF1E). A megaTAL polypeptide can comprise a TALE DNA binding domain and an engineered meganuclease. See, e.g., WO 2004/067736 (homing endonuclease); Urnov et al. (2005) Nature 435:646 (ZFN); Mussolino et al. (2011) Nucle. Acids Res. 39:9283 (TALE nuclease); Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTAL).

[0083] In some cases, the heterologous polypeptide is a reverse transcriptase polypeptide. In some cases, the modified CRISPR/Cas effector polypeptide is catalytically inactive. Suitable reverse transcriptases include, e.g., a murine leukemia virus reverse transcriptase; a Rous sarcoma virus reverse transcriptase; a human immunodeficiency virus type I reverse transcriptase; a Moloney murine leukemia virus reverse transcriptase; and the like.

[0084] In some cases, the heterologous polypeptide is a base editor. Suitable base editors include, e.g., an adenosine deaminase; a cytidine deaminase (e.g., an activation-induced cytidine deaminase (AID)); APOBEC3G; and the like); and the like.

[0085] A suitable adenosine deaminase is any enzyme that is capable of deaminating adenosine in DNA. In some cases, the deaminase is a TadA deaminase.

[0086] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid

[0087] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

[0088] In some cases, a modified CRISPR/Cas effector polypeptide comprises, as the heterologous polypeptide, one deaminase domain. In some cases, a modified CRISPR/Cas effector polypeptide comprises, as the heterologous polypeptide, two deaminase domains.

[0089] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

[0090] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Staphylococcus aureus TadA amino acid sequence:

[0091] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Bacillus subtilis TadA amino acid sequence:

[0092] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Salmonella typhimurium TadA:

[0093] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Shewanella putrefaciens TadA amino acid sequence:

[0094] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Haemophilus influenzae F3031 TadA amino acid sequence:

[0095] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Caulobacter crescentus TadA amino acid sequence:

[0096] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Geobacter sulfurreducens TadA amino acid sequence:

[0097] Suitable cytidine deaminases include any enzyme that is capable of deaminating cytidine in

DNA. In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA- editing complex (APOBEC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC 1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is an activation induced deaminase (AID).

[0098] In some cases, a suitable cytidine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

[0099]

[00100] In some cases, a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSEEMNRRK

[00101] In some cases, a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK

[00102] In some cases, the heterologous polypeptide is a recombinase. Suitable recombinases include, e.g., a Cre recombinase; a Hin recombinase; a Tre recombinase; a FLP recombinase; and the like.

[00103] In some cases, the heterologous polypeptide is a polypeptide that confers an inducible conformational change on the modified CRISPR/Cas effector polypeptide (e.g., a ligand binding domain of an estrogen receptor, an EF domain from calmodulin which specifically binds Ca²⁺, and the like). In some cases, the heterologous polypeptide is a receptor, e.g., a hormone receptor, metabolite receptor, GPCR, or any other suitable receptor, or a suitable portion thereof. In some cases, the heterologous polypeptide includes a hormone receptor, or a portion thereof. In some cases, the heterologous polypeptide includes a ligand-binding domain of the hormone receptor. The hormone receptor may be any suitable hormone receptor, including, but not limited to an estrogen receptor, estrogen-related receptor, androgen receptor, glucocorticoid receptor, mineralocorticoid receptor, progesterone receptor, retinoic acid receptor, vitamin D receptor, thyroid hormone receptor, peroxisome proliferator-activated receptor (PPAR), Rev-ErbA receptor, RAR-related orphan receptor, liver X receptor, farnesoid X receptor, pregnane X receptor, constitutive androstane receptor, hepatocyte nuclear factor-4, retinoid receptor, testicular receptor, nerve growth factor IB, nuclear receptor related 1, neuron-derived orphan receptor, steroidogenic factor 1, liver receptor homolog- 1, germ cell nuclear factor, or any other hormone receptor . In some cases, the hormone receptor is an estrogen receptor. In certain embodiments, the heterologous polypeptide contains the ligand binding domain of an estrogen receptor, e.g., estrogen receptor alpha.

[00104] For example, in some cases, a heterologous polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following estrogen receptor ligand binding domain polypeptide:

Additional polypeptides

[00105] In some cases, a modified type II CRISPR/Cas effector polypeptide of the present disclosure comprises one or more additional heterologous polypeptides (“fusion partners”). The one or more additional polypeptides can be fused to the N-terminus of the modified type II CRISPR/Cas effector polypeptide. The one or more additional polypeptides can be fused to the C-terminus of the modified type II CRISPR/Cas effector polypeptide. The one or more additional polypeptides can be fused to the N-terminus and the C-terminus of the modified type II CRISPR/Cas effector polypeptide. The one or more additional polypeptides can be fused to the modified type II CRISPR/Cas effector polypeptide directly or via a peptide linker.

[00106] Suitable additional polypeptides include an endosomal escape peptide, a chloroplast transit peptide (CTP), a plastid transit peptide, a nuclear localization signal (NLS), a nuclear export sequence (NES), a mitochondrial localization signal for targeting to the mitochondria and the like. Other suitable additional polypeptides include: a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FFAG tag; a Myc tag; and the like).

[00107] A suitable endosomal escape polypeptide comprises the amino acid sequence

, wherein each X is independently selected from lysine, histidine, and arginine. In some cases, an endosomal escape polypeptide comprises the amino acid sequence

[00108] In some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure comprises (is fused to) a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus.

[00109] In some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure includes (is fused to) from 1 to 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NLSs). In some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure includes (is fused to) from 2 to 5 NLSs (e.g., 2-4, or 2-3 NLSs).

[00110] Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the

SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:20); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence

[00111] In some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure includes 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 traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino terminus of a modified CRISPR/Cas effector polypeptide of the present disclosure, to generate a fusion protein. In some embodiments, a PTD is covalently linked to the carboxyl terminus of a modified CRISPR/Cas effector polypeptide of the present disclosure to generate a fusion protein. In some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure includes (is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, a PTD includes a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:36); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256);

embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol ( Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane.

Linkers (e.g., for fusion partners )

[00112] In some cases, modified CRISPR/Cas effector polypeptide of the present disclosure is fused to a fusion partner via 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 be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation 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.

[00113] Examples of linker polypeptides include glycine polymers (G)_n, glycine-serine polymers

(including, for example,

(GS)„, GSGGS_n (S Q O 6), GGSGGS_n (S Q O ), and where n is an integer of at least one), glycine-alanine polymers,

alanine-serine polymers. Exemplary linkers can comprise amino acid sequences including, but not limited to

and the like.

The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. NUCLEIC ACIDS, EXPRESSION VECTORS, AND HOST CELLS

[00114] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides one or more nucleic acids comprising one or more of: a donor polynucleotide, a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure, a guide nucleic acid (e.g., a guide RNA), and a nucleotide sequence encoding a guide nucleic acid (e.g., a guide RNA). The present disclosure provides a recombinant expression vector that comprises a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides a recombinant expression vector that comprises: a) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and b) a nucleotide sequence encoding a guide RNA(s). In some cases, the nucleotide sequence encoding the modified CRISPR/Cas effector polypeptide of the present disclosure and/or the nucleotide sequence encoding the guide RNA is operably linked to a promoter that is operable in a cell type of choice (e.g., a eukaryotic cell; such as a plant cell, an animal cell, a mammalian cell, a non-human primate cell, a rodent cell, a human cell, an insect cell, an arachnid cell, a yeast cell, etc.).

[00115] In some cases, a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure is codon optimized. This type of optimization can entail a mutation of a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence could be used. As another nonlimiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a plant cell, then a plant codon- optimized modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were an insect cell, then an insect codon-optimized modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence could be generated.

[00116] Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www[dot]kazusa[dot]or[dot]jp[forwardslash]codon. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a eukaryotic cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide- encoding nucleotide sequence that is codon optimized for expression in an animal cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a fungus cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a plant cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a monocotyledonous plant species. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a dicotyledonous plant species. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a gymnosperm plant species. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in an angiosperm plant species. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a corn cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a soybean cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptideencoding nucleotide sequence that is codon optimized for expression in a rice cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a wheat cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a cotton cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a sorghum cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in an alfalfa cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a sugar cane cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in an Arabidopsis cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a tomato cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptideencoding nucleotide sequence that is codon optimized for expression in a cucumber cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in a potato cell. In some cases, a nucleic acid of the present disclosure comprises a modified CRISPR/Cas effector polypeptide-encoding nucleotide sequence that is codon optimized for expression in an algae cell.

[00117] The present disclosure provides one or more recombinant expression vectors that include

(in different recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence of a donor template nucleic acid (where the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome)); (ii) a nucleotide sequence that encodes a guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (iii) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors that include (in separate recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence of a donor template nucleic acid (where the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome)); and (ii) a nucleotide sequence that encodes a guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors that include (in separate recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence that encodes a guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (ii) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell).

[00118] Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV)

(see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[00119] For plant applications, viral vectors based on Tobamoviruses, Potexviruses, Potyviruses,

Tobraviruses, Tombusviruses, Geminiviruses, Bromoviruses, Carmoviruses, Alfamoviruses, or Cucumoviruses can be used. See, e.g., Peyret and Lomonossoff (2015) Plant Biotechnol. J. 13:1121. Suitable Tobamovirus vectors include, for example, a tomato mosaic virus (ToMV) vector, a tobacco mosaic virus (TMV) vector, a tobacco mild green mosaic virus (TMGMV) vector, a pepper mild mottle virus (PMMoV) vector, a paprika mild mottle virus (PaMMV) vector, a cucumber green mottle mosaic virus (CGMMV) vector, a kyuri green mottle mosaic virus (KGMMV) vector, a hibiscus latent fort pierce virus (HLFPV) vector, an odontoglossum ringspot virus (ORSV) vector, a rehmannia mosaic vims (ReMV) vector, a Sammon's opuntia vims (SOV) vector, a wasabi mottle vims (WMoV) vector, a youcai mosaic virus (YoMV) vector, a sunn-hemp mosaic virus (SHMV) vector, and the like. Suitable Potexvims vectors include, for example, a potato virus X (PVX) vector, a potato aucubamosaicvirus (PAMV) vector, an Alstroemeria vims X (AlsVX) vector, a cactus virus X (CVX) vector, a Cymbidium mosaic vims (CymMV) vector, a hosta virus X (HVX) vector, a lily vims X (LVX) vector, a Narcissus mosaic vims (NMV) vector, a Nerine virus X (NVX) vector, a Plantago asiatica mosaic vims (P1AMV) vector, a strawberry mild yellow edge vims (SMYEV) vector, a tulip vims X (TVX) vector, a white clover mosaic virus (WC1MV) vector, a bamboo mosaic virus (BaMV) vector, and the like. Suitable Potyvirus vectors include, for example, a potato vims Y (PVY) vector, a bean common mosaic vims (BCMV) vector, a clover yellow vein vims (C1YVV) vector, an East Asian Passiflora virus (EAPV) vector, a Freesia mosaic virus (FreMV) vector, a Japanese yam mosaic virus (JYMV) vector, a lettuce mosaic virus (LMV) vector, a Maize dwarf mosaic virus (MDMV) vector, an onion yellow dwarf virus (OYDV) vector, a papaya ringspot virus (PRSV) vector, a pepper mottle virus (PepMoV) vector, a Perilla mottle virus (PerMoV) vector, a plum pox virus (PPV) vector, a potato virus A (PVA) vector, a sorghum mosaic virus (SrMV) vector, a soybean mosaic virus (SMV) vector, a sugarcane mosaic virus (SCMV) vector, a tulip mosaic virus (TulMV) vector, a turnip mosaic virus (TuMV) vector, a watermelon mosaic virus (WMV) vector, a zucchini yellow mosaic virus (ZYMV) vector, a tobacco etch virus (TEV) vector, and the like. Suitable Tobravirus vectors include, for example, a tobacco rattle virus (TRV) vector and the like. Suitable Tombusvirus vectors include, for example, a tomato bushy stunt virus (TBSV) vector, an eggplant mottled crinkle virus (EMCV) vector, a grapevine Algerian latent virus (GALV) vector, and the like. Suitable Cucumovirus vectors include, for example, a cucumber mosaic virus (CMV) vector, a peanut stunt virus (PSV) vector, a tomato aspermy virus (TAV) vector, and the like. Suitable Bromovirus vectors include, for example, a brome mosaic virus (BMV) vector, a cowpea chlorotic mottle virus (CCMV) vector, and the like. Suitable Carmovirus vectors include, for example, a carnation mottle virus (CarMV) vector, a melon necrotic spot virus (MNSV) vector, a pea stem necrotic virus (PSNV) vector, a turnip crinkle virus (TCV) vector, and the like. Suitable Alfamovirus vectors include, for example, an alfalfa mosaic virus (AMV) vector, and the like.

[00120] Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[00121] In some cases, a nucleotide sequence encoding a guide RNA is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In some embodiments, a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.

[00122] The transcriptional control element can be a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, etc.). [00123] Non-limiting examples of eukaryotic promoters (promoters functional in a eukaryotic cell) include EF1α, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused a modified CRISPR/Cas effector polypeptide of the present disclosure.

[00124] In some cases, a nucleotide sequence encoding a guide RNA and/or a modified

CRISPR/Cas effector polypeptide of the present disclosure is operably linked to an inducible promoter. In some embodiments, a nucleotide sequence encoding a guide RNA and/or a modified CRISPR/Cas effector polypeptide of the present disclosure is operably linked to a constitutive promoter.

[00125] A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/”ON” state), it may be an inducible promoter (i.e., a promoter whose state, active/”ON” or inactive/^' OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

[00126] Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (FTR) promoter; adenovirus major late promoter (Ad MFP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1 ;31(17)), a human HI promoter (H1), and the like. [00127] In some cases, a nucleotide sequence encoding a guide RNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an HI promoter, and the like). As would be understood by one of ordinary skill in the art, when expressing an RNA (e.g., a guide RNA) from a nucleic acid (e.g., an expression vector) using a U6 promoter (e.g., in a eukaryotic cell), or another PolIII promoter, the RNA may need to be mutated if there are several Ts in a row (coding for Us in the RNA). This is because a string of Ts (e.g., 5 Ts) in DNA can act as a terminator for polymerase III (PolIII). Thus, in order to ensure transcription of a guide RNA in a eukaryotic cell it may sometimes be necessary to modify the sequence encoding the guide RNA to eliminate runs of Ts. In some cases, a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EF1 a promoter, an estrogen receptor-regulated promoter, and the like).

[00128] Methods of introducing a nucleic acid (e.g., a nucleic acid comprising a donor polynucleotide sequence, one or more nucleic acids encoding a modified CRISPR/Cas effector polypeptide of the present disclosure and/or a guide RNA, and the like) into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., 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.

[00129] Introducing the recombinant expression vector into cells can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant expression vector into a target cell can be carried out in vivo or ex vivo. Introducing the recombinant expression vector into a target cell can be carried out in vitro.

Introducing components into a target cell

[00130] A guide RNA (or a nucleic acid comprising a nucleotide sequence encoding same) and/or a modified CRISPR/Cas effector polypeptide of the present disclosure (or a nucleic acid comprising a nucleotide sequence encoding same) and/or a donor polynucleotide (donor template) can be introduced into a host cell by any of a variety of well-known methods. Any of a variety of compounds and methods can be used to deliver to a target cell a system of the present disclosure (e.g., where a system of the present disclosure comprises: a) a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide nucleic acid; b) a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide nucleic acid, and a DNA donor template; c) a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide RNA; d) a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide RNA, and a DNA donor template; e) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide nucleic acid; f) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide nucleic acid, and a DNA donor template; g) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide RNA; h) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide RNA, and a DNA donor template; i) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and ii) a nucleotide sequence encoding a CRISPR/Cas guide nucleic acid; j) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; ii) a nucleotide sequence encoding a CRISPR/Cas guide nucleic acid; and iii) a DNA donor template; k) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and ii) a nucleotide sequence encoding a CRISPR/Cas guide RNA; or 1) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; ii) a nucleotide sequence encoding a CRISPR/Cas guide RNA; and a DNA donor template; or some variation of one of (a) through (1). As a non-limiting example, a system of the present disclosure can be combined with a lipid. As another non-limiting example, a system of the present disclosure can be combined with a particle, or formulated into a particle.

[00131] Methods of introducing a nucleic acid into a host cell are known in the art, and any convenient method can be used to introduce a subject nucleic acid (e.g., an expression construct/vector) into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) -mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023 ), and the like.

[00132] In some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes the modified CRISPR/Cas effector polypeptide. In some cases, a modified CRISPR/Cas effector polypeptide of the present disclosure is provided directly as a protein (e.g., without an associated guide RNA or with an associate guide RNA, i.e., as a ribonucleoprotein complex). A modified CRISPR/Cas effector polypeptide of the present disclosure can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, a modified CRISPR/Cas effector polypeptide of the present disclosure can be injected directly into a cell (e.g., with or without a guide RNA or nucleic acid encoding a guide RNA, and with or without a donor polynucleotide). As another example, a preformed complex of a modified CRISPR/Cas effector polypeptide of the present disclosure and a guide RNA (an RNP) can be introduced into a cell (e.g., a eukaryotic cell) (e.g., via injection, via nucleofection; via a protein transduction domain (PTD) conjugated to one or more components, e.g., conjugated to the modified CRISPR/Cas effector polypeptide of the present disclosure, conjugated to a guide RNA, conjugated to a modified CRISPR/Cas effector polypeptide of the present disclosure and a guide RNA; etc.).

[00133] In some cases, a nucleic acid (e.g., a guide RNA; a nucleic acid comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; etc.) is delivered to a cell (e.g., a target host cell) and/or a polypeptide (e.g., a modified CRISPR/Cas effector polypeptide of the present disclosure) in a particle, or associated with a particle. In some cases, a system of the present disclosure is delivered to a cell in a particle, or associated with a particle. The terms “particle” and “nanoparticle” can be used interchangeably, as appropriate. A recombinant expression vector comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure and/or a guide RNA, an mRNA comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure, and guide RNA may be delivered simultaneously using particles or lipid envelopes; for instance, a modified CRISPR/Cas effector polypeptide of the present disclosure and a guide RNA, e.g., as a complex (e.g., a ribonucleoprotein (RNP) complex), can be delivered via a particle, e.g., a delivery particle comprising lipid or lipidoid and hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP) or l,2-ditetradecanoyl-sn-glycero-3- phosphocholine (DMPC) and/or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and/or wherein the particle further comprises cholesterol (e.g., particle from formulation l=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5). For example, a particle can be formed using a multistep process in which a modified CRISPR/Cas effector polypeptide of the present disclosure and a guide RNA are mixed together, e.g., at a 1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g., in sterile, nuclease free 1 x phosphate-buffered saline (PBS); and separately, DOTAP, DMPC,

PEG, and cholesterol as applicable for the formulation are dissolved in alcohol, e.g., 100% ethanol; and, the two solutions are mixed together to form particles containing the complexes).

[00134] A modified CRISPR/Cas effector polypeptide of the present disclosure (or an mRNA comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; or a recombinant expression vector comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure) and/or guide RNA (or a nucleic acid such as one or more expression vectors encoding the guide RNA) may be delivered simultaneously using particles or lipid envelopes. For example, a biodegradable coreshell structured nanoparticle with a poly (b-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used. In some cases, particles/nanoparticles based on self assembling bioadhesive polymers are used; such particles/nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. A molecular envelope technology, which involves an engineered polymer envelope which is protected and delivered to the site of the disease, can be used. Doses of about 5 mg/kg can be used, with single or multiple doses, depending on various factors, e.g., the target tissue.

[00135] Lipidoid compounds (e.g., as described in US patent application 20110293703) are also useful in the administration of polynucleotides, and can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure In one aspect, the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles. The aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[00136] A poly(beta-amino alcohol) (PBAA) can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. US Patent Publication No. 20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that has been prepared using combinatorial polymerization. [00137] Sugar-based particles may be used, for example GalNAc, as described with reference to WO2014118272 (incorporated herein by reference) and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961) can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell.

[00138] In some cases, lipid nanoparticles (LNPs) are used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely l,2-diIineoyI-3-dimethyIammonium-propane (DLinDAP), l,2-diIinoIeyIoxy-3-N,N-dimethyIaminopropane (DLinDMA), 1 ,2-dilinolcyloxy- keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1 ,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2-DMA). Preparation of LNPs and is described in, e.g., Rosin et al. (2011) Molecular Therapy 19:1286-2200). The cationic lipids 1 ,2-dilineoyl- 3-dimethylammonium-propane (DLinDAP), 1 ,2-diIinoIeyIoxy-3-N,N-dimethyIaminopropane (DLinDMA), l,2-diIinoIeyk>xyketo-N,N-dimethyI-3-aminopropane (DLinK-DMA), 1,2- dilinoleyl-4-(2-dimethylaminoethyl)- [ 1 ,3] -dioxolane (DLinKC2-DMA) , (3-o- [2"- (methoxypolyethyleneglycol 2000) succinoyI]-l,2-dimyristoyI-sn-gIycoI (PEG-S-DMG), and R- 3-[(.omega.-methoxy-poly(ethylene glycol)2000) carbamoyl] -l,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be used. A nucleic acid (e.g., a guide RNA; a nucleic acid of the present disclosure; etc.) may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic Iipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios). In some cases, 0.2% SP-DiOC18 is incorporated.

[00139] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell.. See, e.g., Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962- 6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 20127:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19): 7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ral52 (2013) and Mirkin, et al., Small, 10:186- 192.

[00140] Self-assembling nanoparticles with RNA may be constructed with polyethyleneimine

(PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG).

[00141] In general, a "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. In some cases, nanoparticles suitable for use in delivering a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell have a diameter of from 25 nm to 200 nm. In some cases, nanoparticles suitable for use in delivering a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell have a diameter of 100 nm or less In some cases, nanoparticles suitable for use in delivering a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell have a diameter of from 35 nm to 60 nm.

[00142] Nanoparticles suitable for use in delivering a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically below 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure.

[00143] Semi-solid and soft nanoparticles are also suitable for use in delivering a modified

CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. A prototype nanoparticle of semi-solid nature is the liposome. [00144] In some cases, an exosome is used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs.

[00145] In some cases, a liposome is used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties. Lor instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. A liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1 ,2- distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.

[00146] A stable nucleic-acid-lipid particle (SNALP) can be used to deliver a modified

CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. The SNALP formulation may contain the lipids 3 -N-[(methoxypoly (ethylene glycol) 2000) carbamoyl] - 1 ,2-dimyristyloxy-propylamine (PEG-C-DMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar percent ratio. The SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DM A/DSPC7PLG-C-DM A. The resulting SNALP liposomes can be about 80-100 nm in size. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis,

Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N- [(w-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyrestyloxypropylamine, and cationic 1 ,2-dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprise synthetic cholesterol (Sigma- Aldrich), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and l,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

[00147] Other cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA) can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. A preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl- 1- (methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of approximately 0.05 (w/w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11.+-.0.04 (n=56), the particles may be extruded up to three times through 80 nm membranes prior to adding the guide RNA. Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further optimized to enhance in vivo activity.

[00148] Lipids may be formulated with a system of the present disclosure or component(s) thereof or nucleic acids encoding the same to form lipid nanoparticles (LNPs). Suitable lipids include, but are not limited to, DLin-KC2-DMA4, Cl 2-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated with a system, or component thereof, of the present disclosure, using a spontaneous vesicle formation procedure. The component molar ratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG) .

[00149] A system of the present disclosure, or a component thereof, may be delivered encapsulated in PLGA microspheres such as that further described in US published applications 20130252281 and 20130245107 and 20130244279.

[00150] Supercharged proteins can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. [00151] Cell Penetrating Peptides (CPPs) can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.

[00152] An implantable device can be used to deliver a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure (e.g., a guide RNA, a nucleic acid encoding a guide RNA, a nucleic acid encoding a modified CRISPR/Cas effector polypeptide of the present disclosure, a donor template, and the like), or a system of the present disclosure, to a target cell (e.g., a target cell in vivo, where the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.). An implantable device suitable for use in delivering a modified CRISPR/Cas effector polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a system of the present disclosure, to a target cell (e.g., a target cell in vivo, where the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.) can include a container (e.g., a reservoir, a matrix, etc.) that comprises the modified CRISPR/Cas effector polypeptide of the present disclosure, the RNP, or the system (or component thereof, e.g., a nucleic acid of the present disclosure).

[00153] A suitable implantable device can comprise a polymeric substrate, such as a matrix for example, that is used as the device body, and in some cases additional scaffolding materials, such as metals or additional polymers, and materials to enhance visibility and imaging. An implantable delivery device can be advantageous in providing release locally and over a prolonged period, where the polypeptide and/or nucleic acid to be delivered is released directly to a target site, e.g., the extracellular matrix (ECM), the vasculature surrounding a tumor, a diseased tissue, etc. Suitable implantable delivery devices include devices suitable for use in delivering to a cavity such as the abdominal cavity and/or any other type of administration in which the drug delivery system is not anchored or attached, comprising a biostable and/or degradable and/or bioabsorbable polymeric substrate, which may for example optionally be a matrix. In some cases, a suitable implantable drug delivery device comprises degradable polymers, wherein the main release mechanism is bulk erosion. In some cases, a suitable implantable drug delivery device comprises non degradable, or slowly degraded polymers, wherein the main release mechanism is diffusion rather than bulk erosion, so that the outer part functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months). Combinations of different polymers with different release mechanisms may also optionally be used. The concentration gradient at the can be maintained effectively constant during a significant period of the total releasing period, and therefore the diffusion rate is effectively constant (termed "zero mode" diffusion). By the term "constant" it is meant a diffusion rate that is maintained above the lower threshold of therapeutic effectiveness, but which may still optionally feature an initial burst and/or may fluctuate, for example increasing and decreasing to a certain degree. The diffusion rate can be so maintained for a prolonged period, and it can be considered constant to a certain level to optimize the therapeutically effective period, for example the effective silencing period.

[00154] In some cases, the implantable delivery system is designed to shield the nucleotide based therapeutic agent from degradation, whether chemical in nature or due to attack from enzymes and other factors in the body of the subject.

[00155] The site for implantation of the device, or target site, can be selected for maximum therapeutic efficacy. For example, a delivery device can be implanted within or in the proximity of a tumor environment, or the blood supply associated with a tumor. The target location can be, e.g.: 1) the brain at degenerative sites like in Parkinson or Alzheimer disease at the basal ganglia, white and gray matter; 2) the spine, as in the case of amyotrophic lateral sclerosis (ALS); 3) uterine cervix; 4) active and chronic inflammatory joints; 5) dermis as in the case of psoriasis; 7) sympathetic and sensoric nervous sites for analgesic effect; 7) a bone; 8) a site of acute or chronic infection; 9) Intra vaginal; 10) Inner ear— auditory system, labyrinth of the inner ear, vestibular system; 11) Intra tracheal; 12) Intra-cardiac; coronary, epicardiac; 13) urinary tract or bladder; 14) biliary system; 15) parenchymal tissue including and not limited to the kidney, liver, spleen; 16) lymph nodes; 17) salivary glands; 18) dental gums; 19) Intra-articular (into joints); 20) Intra-ocular; 21) Brain tissue; 22) Brain ventricles; 23) Cavities, including abdominal cavity (for example but without limitation, for ovary cancer); 24) Intra esophageal; and 25) Intra rectal; and 26) into the vasculature.

[00156] The method of insertion, such as implantation, may optionally already be used for other types of tissue implantation and/or for insertions and/or for sampling tissues, optionally without modifications, or alternatively optionally only with non-major modifications in such methods. Such methods optionally include but are not limited to brachytherapy methods, biopsy, endoscopy with and/or without ultrasound, such as stereotactic methods into the brain tissue, laparoscopy, including implantation with a laparoscope into joints, abdominal organs, the bladder wall and body cavities. CELLS

[00157] The present disclosure provides a cell (a “modified cell”) comprising a modified

CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides a cell (a “modified cell”) comprising a nucleic acid of the present disclosure (e.g., a nucleic acid comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure).

[00158] The present disclosure provides a modified cell comprising a modified CRISPR/Cas effector polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides a modified cell comprising a modified CRISPR/Cas effector polypeptide of the present disclosure, where the modified cell is a cell that does not normally comprise a modified CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with an mRNA comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and b) a nucleotide sequence encoding a guide RNA of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; b) a nucleotide sequence encoding a guide RNA of the present disclosure; and c) a nucleotide sequence encoding a donor template.

[00159] A cell that serves as a recipient for a modified CRISPR/Cas effector polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure and/or a guide RNA and/or a donor template, can be any of a variety of eukaryotic cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; etc. A cell that serves as a recipient for a modified CRISPR/Cas effector polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure and/or a guide RNA is referred to as a “host cell” or a “target cell.” A host cell or a target cell can be a recipient of a system of the present disclosure, or a component of a system of the present disclosure. A host cell or a target cell can be a recipient of an RNP of the present disclosure. A host cell or a target cell can be a recipient of a single component of a system of the present disclosure.

[00160] Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[00161] A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). A cell can be and in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.

[00162] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc. [00163] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogenic cells, and post-natal stem cells.

[00164] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[00165] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[00166] Adult stem cells are resident in differentiated tissue, but retain the properties of selfrenewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[00167] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[00168] Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7,

LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[00169] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34⁺ and CD3 . HSCs can repopulate the erythroid, neutrophil- macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[00170] In other instances, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[00171] In other instances, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

[00172] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon.

[00173] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non- Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (Chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (russian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled/ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like.

[00174] In some cases, the plant cell is a cell of a plant component such as a leaf, a stem, a root, a seed, a flower, pollen, an anther, an ovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod, an embryo, endosperm, an explant, a callus, or a shoot.

[00175] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a suborder, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola , Hymenoptera , Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera , Mecoptera , Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[00176] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.

COMPOSITIONS

[00177] The present disclosure provides a composition comprising a modified CRISPR/Cas effector polypeptide of the present disclosure. The composition may comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, “Remington: The Science and Practice of Pharmacy”, 19^th Ed. (1995), or latest edition, Mack Publishing Co; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et ak, eds., 3^rd ed. Amer. Pharmaceutical Assoc.

[00178] A composition of the present disclosure can include: a) one or more of: i) a modified

CRISPR/Cas effector polypeptide of the present disclosure; ii) a guide nucleic acid (e.g., a guide RNA); and iii) a donor template; and b) one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative. Suitable buffers include, but are not limited to, (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2- hydroxyethyl)piperazine-N'3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2- hydroxyehtylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate, 3-(N- tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl- glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), etc.). Suitable salts include, e.g., NaCl, MgCE, KC1, MgSCh, etc.

[00179] A composition of the present disclosure can include: a) a system of the present disclosure; and b) one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative. Suitable buffers include, but are not limited to, (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyehtylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethane- sulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2- hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), etc.). Suitable salts include, e.g., NaCl, MgCT, KC1, MgSCh, etc.

[00180] In some cases, the composition is sterile. In some cases, the composition is suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and/or other toxins.

[00181] A composition of the present disclosure may include other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride, sulfate salts, solvates (e.g., mixed ionic salts, water, organics), hydrates (e.g., water), and the like. In some cases, a composition of the present disclosure comprises saline. SYSTEMS

[00182] The present disclosure provides a system comprising a modified CRISPR/Cas effector polypeptide of the present disclosure.

[00183] The present disclosure provides a system comprising one of: a) a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide nucleic acid; b) a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide nucleic acid, and a DNA donor template; c) a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide RNA; d) a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide RNA, and a DNA donor template; e) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide nucleic acid; f) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide nucleic acid, and a DNA donor template; g) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and a CRISPR/Cas guide RNA; h) an mRNA encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; a CRISPR/Cas guide RNA, and a DNA donor template; i) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and ii) a nucleotide sequence encoding a CRISPR/Cas guide nucleic acid; j) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; ii) a nucleotide sequence encoding a CRISPR/Cas guide nucleic acid; and iii) a DNA donor template; k) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; and ii) a nucleotide sequence encoding a CRISPR/Cas guide RNA; and 1) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a modified CRISPR/Cas effector polypeptide of the present disclosure; ii) a nucleotide sequence encoding a CRISPR/Cas guide RNA; and a DNA donor template; or some variation of one or (a) through (1).

Guide nucleic acid

[00184] A guide nucleic acid suitable for inclusion in a system of the present disclosure can include: i) a first segment (referred to herein as a “targeting segment”); and ii) a second segment (referred to herein as a “protein-binding segment”). By “segment” it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule. A segment can also mean a region/section of a complex such that a segment may comprise regions of more than one molecule. The “targeting segment” is also referred to herein as a “variable region” of a guide RNA. The “protein-binding segment” is also referred to herein as a “constant region” of a guide RNA. The first segment (targeting segment) of a guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within a target nucleic acid (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.). The protein-binding segment (or “protein-binding sequence”) interacts with (binds to) a CRISPR/Cas effector polypeptide. The protein-binding segment of a guide RNA includes two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex). Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the guide RNA (the guide sequence of the guide RNA) and the target nucleic acid.

[00185] A guide RNA and a CRISPR/Cas effector polypeptide form a complex (e.g., bind via non- covalent interactions). The guide RNA provides target specificity to the complex by including a targeting segment, which includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid). The CRISPR/Cas effector polypeptide of the complex provides the site-specific activity (e.g., cleavage activity or an activity provided by the CRISPR/Cas effector polypeptide when the CRISPR/Cas effector polypeptide is a CRISPR/Cas effector polypeptide is a modified CRISPR/Cas effector polypeptide of the present disclosure). In other words, the CRISPR/Cas effector polypeptide is guided to a target nucleic acid sequence (e.g. a target sequence in a chromosomal nucleic acid, e.g., a chromosome; a target sequence in an extrachromosomal nucleic acid, e.g. an episomal nucleic acid, a minicircle, an ssRNA, an ssDNA, etc. ; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid; a target sequence in a viral nucleic acid; etc.) by virtue of its association with the guide RNA.

[00186] The “guide sequence” also referred to as the “targeting sequence” of a guide RNA can be modified so that the guide RNA can target a CRISPR/Cas effector polypeptide to any desired sequence of any desired target nucleic acid, with the exception that the protospacer adjacent motif (PAM) sequence can be taken into account. Thus, for example, a guide RNA can have a targeting segment with a sequence (a guide sequence) that has complementarity with (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like. [00187] In some cases, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, or a “two-molecule guide RNA” a “dual guide RNA”, or a “dgRNA.” In some embodiments, the activator and targeter are covalently linked to one another (e.g., via intervening nucleotides) and the guide RNA is referred to as a “single guide RNA”, a “Cas9 single guide RNA”, a “singlemolecule Cas9 guide RNA,” or a “one-molecule Cas9 guide RNA”, or simply “sgRNA.”

[00188] Examples of various CRISPR/Cas effector polypeptides and guide RNAs (as well as information regarding requirements related to protospacer adjacent motif (PAM) sequences present in targeted nucleic acids) can be found in the art, for example, see Jinek et al., Science.

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[00189] In some cases, a guide nucleic acid comprises ribonucleotides only, deoxyribonucleotides only, or a mixture of ribonucleotides and deoxyribonucleotides. In some cases, a guide nucleic acid comprises ribonucleotides only, and is referred to herein as a “guide RNA.” In some cases, a guide nucleic acid comprises deoxyribonucleotides only, and is referred to herein as a “guide DNA.” In some cases, a guide nucleic acid comprises both ribonucleotides and deoxyribonucleotides. A guide nucleic acid can comprise combinations of ribonucleotide bases, deoxyribonucleotide bases, nucleotide analogs, modified nucleotides, and the like; and may further include naturally-occurring backbone residues and/or linkages and/or non-naturally- occurring backbone residues and/or linkages.

Donor DNA template

[00190] In some cases, a system of the present disclosure comprises a donor nucleic acid. By a

“donor nucleic acid” or “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site cleaved by a CRISPR/Cas effector protein (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) can support homology-directed repair. Donor polynucleotides can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc. Donor polynucleotides can be from 25 nucleotides to 50 nucleotides, from 50 nucleotides to 100 nucleotides, from 100 nucleotides to 500 nucleotides, from 500 nucleotides to 1000 nucleotides, from 1000 nucleotides to 5000 nucleotides, or from 5000 nucleotides to 10,000 nucleotides, or more than 10,000 nucleotides, in length.

[00191] The donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease-causing base pair or a non disease-causing base pair). In some embodiments, the donor sequence comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region. Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.

[00192] The donor sequence may comprise certain sequence differences as compared to the genomic sequence, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor sequence at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). In some cases, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). Alternatively, these sequences differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence.

[00193] In some cases, the donor sequence is provided to the cell as single-stranded DNA. In some cases, the donor sequence is provided to the cell as double-stranded DNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and/or selfcomplementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the termini of a linear donor sequence, additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination. A donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.

METHODS

[00194] The present disclosure provides a method of modifying a target nucleic acid, or a polypeptide associated with the target nucleic acid, the method comprising contacting the target nucleic acid with: a) the modified CRISPR/Cas effector polypeptide of the present disclosure; and b) a guide nucleic acid that comprises: i) a guide sequence that hybridizes to a target sequence of the target nucleic acid; and ii) an activator sequence that binds to the modified CRISPR/Cas effector polypeptide.

[00195] Modifications include any modification that can be effected by the heterologous polypeptide present in a modified CRISPR/Cas effector polypeptide of the present disclosure.

[00196] For example, where the heterologous polypeptide is a deaminase, a method of the present disclosure provides for deamination of a target nucleotide sequence in a target nucleic acid, wherein the target nucleic acid is determined by the target-binding nucleotide sequence present in the guide nucleic acid (e.g., guide RNA). For example, in some cases, a modified CRISPR/Cas effector polypeptide functions as a base editor.

[00197] A heterologous polypeptide present in a modified CRISPR/Cas effector polypeptide of the present disclosure is allosterically regulated by the REC lobe present in the modified CRISPR/Cas effector polypeptide. Whether the heterologous polypeptide present in a modified CRISPR/Cas effector polypeptide of the present disclosure is allosterically regulated by the REC lobe present in the modified CRISPR/Cas effector polypeptide can be determined using any known method.

[00198] Whether a heterologous polypeptide present in a modified CRISPR/Cas effector polypeptide of the present disclosure is allosterically regulated by the REC lobe upon binding to a target nucleic acid can be determined using various methods. For example, a modified CRISPR/Cas effector polypeptide of the present disclosure can be complexed with a guide RNA that comprises a nucleotide sequence that binds to a target nucleic acid, where the target nucleic acid comprises a nucleotide sequence that encodes a green fluorescent protein (GFP), and where the modified CRISPR/Cas effector polypeptide/guide RNA complex represses transcription of the GFP-encoding nucleotide sequence. Where the heterologous polypeptide is allosterically controlled by the REC lobe upon binding to the target nucleic acid, production of GFP will be reduced. Where the heterologous polypeptide is not allosterically controlled by the REC lobe upon binding to the target nucleic acid, production of GFP will not be reduced. A two-color assay such as that described in Oakes et al. ((2014) Methods Enzymol. 546:491) can also be used. See also Reynolds et al. (2011) Cell 147:1564; and Nadler et al. (2016) Nat. Commun. 7:12266.

[00199] A modified CRISPR/Cas effector polypeptide of the present disclosure, when bound to

(complexed with) a guide RNA, can bind to a target nucleic acid, and in some cases, can bind to and modify a target nucleic acid. A target nucleic acid can be any nucleic acid (e.g., DNA,

RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted).

[00200] A target nucleic acid can be DNA or RNA. A target nucleic acid can be double stranded

(e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is single stranded. In some cases, a target nucleic acid is a single stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and microRNA (miRNA). In some cases, a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some cases, a target nucleic acid is single stranded.

[00201] A target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo. Suitable target cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell from a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell from a mammal (e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).

Examples of Non-Limiting Aspects of the Disclosure

[00202] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this 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 combinations of aspects explicitly provided below:

[00203] Aspect 1. A modified type II CRISPR/Cas effector polypeptide, wherein the modified

CRISPR/Cas effector polypeptide comprises a heterologous polypeptide in place of an HNH domain present in a corresponding unmodified CRISPR/Cas effector polypeptide, wherein the heterologous polypeptide is an enzyme, and wherein, when complexed with a guide nucleic acid, the modified CRISPR/Cas effector polypeptide binds a target nucleic acid.

[00204] Aspect 2. The modified CRISPR/Cas effector polypeptide of aspect 1, wherein the activity of the heterologous enzyme is allosterically regulated by a REC lobe present in the modified CRISPR/Cas effector polypeptide.

[00205] Aspect 3. The modified CRISPR/Cas effector polypeptide of aspect 1 or aspect 2, wherein the heterologous polypeptide exhibits one or more enzymatic activities selected from: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[00206] Aspect 4. The modified CRISPR/Cas effector polypeptide of aspect 1 or aspect 2, wherein the heterologous polypeptide exhibits histone modification activity.

[00207] Aspect 5. The modified CRISPR/Cas effector polypeptide of aspect 4, wherein the heterologous polypeptide exhibits one or more enzymatic activities selected from: methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity, and deglycosylation activity. [00208] Aspect 6. The modified CRISPR/Cas effector polypeptide of aspect 1, wherein the heterologous polypeptide is a deaminase.

[00209] Aspect 7. The modified CRISPR/Cas effector polypeptide of aspect 6, wherein the deaminase is an adenosine deaminase or a cytosine deaminase.

[00210] Aspect 8. The modified CRISPR/Cas effector polypeptide of any one of aspects 1-7, wherein the modified CRISPR/Cas effector polypeptide comprises a nucleotide localization signal.

[00211] Aspect 9. The modified CRISPR/Cas effector polypeptide of any one of aspects 1-8, wherein the modified type II CRISPR/Cas effector polypeptide is a Cas9 polypeptide.

[00212] Aspect 10. The modified CRISPR/Cas effector polypeptide of any one of aspects 1-9, wherein the HNH domain is amino acids 775-909 of the Cas9 amino acid sequence depicted in FIG. 4 or a corresponding region of another Cas9 polypeptide.

[00213] Aspect 11. The modified CRISPR/Cas effector polypeptide of any one of aspects 1-10, wherein the modified CRISPR/Cas effector polypeptide comprises, in order from N-terminus to C-terminus:

[00214] a) a RuvC-I domain;

[00215] b) a BH domain;

[00216] c) a REC domain;

[00217] d) a RuvC-II domain;

[00218] e) the heterologous polypeptide;

[00219] f) a RuvC-III domain; and

[00220] g) a PI domain.

[00221] Aspect 12. The modified CRISPR/Cas effector polypeptide of any one of aspects 1-11, wherein the CRISPR/Cas effector polypeptide comprises a mutation of Asp at position 10 of Streptococcus pyogenes Cas9, or a corresponding position in another type II CRISPR/Cas effector polypeptide, such that the modified CRISPR/Cas effector polypeptide does not cleave a non-target strand of a target nucleic acid.

[00222] Aspect 13. A nucleic acid comprising a nucleotide sequence encoding the modified

CRISPR/Cas effector polypeptide of any one of aspects 1-12.

[00223] Aspect 14. The nucleic acid of aspect 13, wherein the nucleotide sequence is operably linked to one or more transcriptional control elements.

[00224] Aspect 15. The nucleic acid of aspect 14, wherein the one or more transcriptional control elements is a promoter. [00225] Aspect 16. The nucleic acid of aspect 15, wherein the promoter is a constitutive promoter, an inducible promoter, a cell type-specific promoter, or a tissue-specific promoter.

[00226] Aspect 17. A recombinant expression vector comprising the nucleic acid of any one of aspects 13-16.

[00227] Aspect 18. The recombinant expression vector of aspect 17, wherein the recombinant expression vector is a recombinant adenoassociated viral vector, a recombinant retroviral vector, or a recombinant lentiviral vector.

[00228] Aspect 19. A cell comprising the nucleic acid of any one of aspects 13-16 or the recombinant expression vector of aspect 17 or aspect 18.

[00229] Aspect 20. A composition comprising:

[00230] a) the modified CRISPR/Cas effector polypeptide of any one of aspects 1-12; and

[00231] b) a buffer.

[00232] Aspect 21. A composition comprising:

[00233] a) the modified CRISPR/Cas effector polypeptide of any one of aspects 1-12; and

[00234] b) a CRISPR/Cas guide nucleic acid, or one or more DNA molecules comprising a nucleotide sequence(s) encoding the CRISPR/Cas guide nucleic acid.

[00235] Aspect 22. The composition of aspect 21, wherein the CRISPR/Cas guide nucleic acid is an RNA, a DNA, or an RNA/DNA hybrid.

[00236] Aspect 23. A method of modifying a target nucleic acid or a polypeptide associated with the target nucleic acid, the method comprising contacting the target nucleic acid with:

[00237] a) the modified CRISPR/Cas effector polypeptide of any one of aspects 1-12; and

[00238] b) a guide nucleic acid that comprises: i) a guide sequence that hybridizes to a target sequence of the target nucleic acid; and ii) an activator sequence that binds to the modified CRISPR/Cas effector polypeptide.

[00239] Aspect 24. The method of aspect 23, wherein said modification comprises deamination of one or more nucleotides in the target nucleic acid.

[00240] Aspect 25. The method of aspect 23 or aspect 24, wherein said modification takes place inside of a cell in vitro.

[00241] Aspect 26. The method of aspect 23 or aspect 24, wherein said modification takes place inside of a cell in vivo.

[00242] Aspect 27. The method of aspect 25 or aspect 26, wherein the cell is a prokaryotic cell.

[00243] Aspect 28. The method of aspect 25 or aspect 26, wherein the cell is a eukaryotic cell.

[00244] Aspect 29. The method of aspect 28, wherein the cell is selected from: a plant cell, a fungal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

EXAMPLES

[00245] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description 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 they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, 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(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1

[00246] A library is constructed by PCR using primers covering tolerant HNH domain deletions.

A deaminase domain (e.g., a Tad deaminase domain) is inserted into the deletion, creating a library of base editors with a deaminase domain inserted into sites of HNH deletion. Functional candidates are selected from the library by a coupled on-target/off-target screen which allows simultaneous assay of base editors that are allosterically regulated to only edit at the correct (“on-target”) site (a nucleotide sequence in a target nucleic acid that has complementarity to a target-binding nucleotide sequence in a guide RNA) and do not edit at off-target sites.

[00247] The method of screening identifies candidates that are active under the proper conditions while counter-screening against candidates that are improperly active under a ‘decoy’ condition, a known method for engineering proteins to possess allosteric regulation of enzymatic activity (Nadler et al. (2016) Nat. Commun. 7:12266). In brief, on-target base editing activity is identified following reversion of an inserted stop codon within a T7 polymerase sequence. A double reporter containing two fluorescent proteins (e.g., green fluorescent protein and red fluorescent protein) under a T7 promoter is expressed if the functional T7 is produced, which is dependent on the on-target base editing activity. At the same time, the off-target activity is identified using mismatched gRNAs that target one of the fluorescent proteins to cause a switching off of fluorescence (i.e., reduced or no expression of one of the fluorescent proteins). These gRNAs are designed to only have partial complementarity to the target DNA and are known to be unable to drive HNH conformational changes in the native SpCas9 context (Sternberg et al. (2015) Nature 527:110; Chen et al. (2017) Nature 550:407). Editing activity associated with these off-target gRNAs will turn off the fluorescence of only one of the fluorescent proteins (i.e., reduced or no expression of one of the fluorescent proteins), keeping the other one active, allowing identification of on-target activity while identifying any library variant possessing spurious activity. The coupled screen is used to select only functional Cas9- base editor insertion constructs that can be allosterically regulated. The base editor library is screened using the coupled screen and base editors are identified with only on-target activity.

[00248] FIG. 3A-3B depict a strategy of HNH domain replacement with a DNA deaminase domain.

[00249] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A modified type II CRISPR/Cas effector polypeptide, wherein the modified CRISPR/Cas effector polypeptide comprises a heterologous polypeptide in place of an HNH domain present in a corresponding unmodified CRISPR/Cas effector polypeptide, wherein the heterologous polypeptide is an enzyme, and wherein, when complexed with a guide nucleic acid, the modified CRISPR/Cas effector polypeptide binds a target nucleic acid.

2. The modified CRISPR/Cas effector polypeptide of claim 1, wherein the activity of the heterologous enzyme is allosterically regulated by a REC lobe present in the modified CRISPR/Cas effector polypeptide.

3. The modified CRISPR/Cas effector polypeptide of claim 1 or claim 2, wherein the heterologous polypeptide exhibits one or more enzymatic activities selected from: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

4. The modified CRISPR/Cas effector polypeptide of claim 1 or claim 2, wherein the heterologous polypeptide exhibits histone modification activity.

5. The modified CRISPR/Cas effector polypeptide of claim 4, wherein the heterologous polypeptide exhibits one or more enzymatic activities selected from: methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity, and deglycosylation activity.

6. The modified CRISPR/Cas effector polypeptide of claim 1, wherein the heterologous polypeptide is a deaminase.

7. The modified CRISPR/Cas effector polypeptide of claim 6, wherein the deaminase is an adenosine deaminase or a cytosine deaminase.

8. The modified CRISPR/Cas effector polypeptide of any one of claim 1-7, wherein the modified CRISPR/Cas effector polypeptide comprises a nucleotide localization signal.

9. The modified CRISPR/Cas effector polypeptide of any one of claims 1-8, wherein the modified type II CRISPR/Cas effector polypeptide is a Cas9 polypeptide.

10. The modified CRISPR/Cas effector polypeptide of any one of claims 1-9, wherein the HNH domain is amino acids 775-909 of the Cas9 amino acid sequence depicted in FIG. 4 or a corresponding region of another Cas9 polypeptide.

11. The modified CRISPR/Cas effector polypeptide of any one of claims 1-10, wherein the modified CRISPR/Cas effector polypeptide comprises, in order from N-terminus to C-terminus: a) a RuvC-I domain; b) a BH domain; c) a REC domain; d) a RuvC-II domain; e) the heterologous polypeptide; f) a RuvC-III domain; and g) a PI domain.

12. The modified CRISPR/Cas effector polypeptide of any one of claims 1-11, wherein the CRISPR/Cas effector polypeptide comprises a mutation of Asp at position 10 of Streptococcus pyogenes Cas9, or a corresponding position in another type II CRISPR/Cas effector polypeptide, such that the modified CRISPR/Cas effector polypeptide does not cleave a non-target strand of a target nucleic acid.

13. A nucleic acid comprising a nucleotide sequence encoding the modified CRISPR/Cas effector polypeptide of any one of claims 1-12.

14. The nucleic acid of claim 13, wherein the nucleotide sequence is operably linked to one or more transcriptional control elements.

15. The nucleic acid of claim 14, wherein the one or more transcriptional control elements is a promoter.

16. The nucleic acid of claim 15, wherein the promoter is a constitutive promoter, an inducible promoter, a cell type-specific promoter, or a tissue-specific promoter.

17. A recombinant expression vector comprising the nucleic acid of any one of claims 13-

16.

18. The recombinant expression vector of claim 17, wherein the recombinant expression vector is a recombinant adenoassociated viral vector, a recombinant retroviral vector, or a recombinant lentiviral vector.

19. A cell comprising the nucleic acid of any one of claims 13-16 or the recombinant expression vector of claim 17 or claim 18.

20. A composition comprising: a) the modified CRISPR/Cas effector polypeptide of any one of claims 1-12; and b) a buffer.

21. A composition comprising : a) the modified CRISPR/Cas effector polypeptide of any one of claims 1-12; and b) a CRISPR/Cas guide nucleic acid, or one or more DNA molecules comprising a nucleotide sequence(s) encoding the CRISPR/Cas guide nucleic acid.

22. The composition of claim 21, wherein the CRISPR/Cas guide nucleic acid is an RNA, a DNA, or an RNA/DNA hybrid.

23. A method of modifying a target nucleic acid or a polypeptide associated with the target nucleic acid, the method comprising contacting the target nucleic acid with: a) the modified CRISPR/Cas effector polypeptide of any one of claims 1-12; and b) a guide nucleic acid that comprises: i) a guide sequence that hybridizes to a target sequence of the target nucleic acid; and ii) an activator sequence that binds to the modified CRISPR/Cas effector polypeptide.

24. The method of claim 23, wherein said modification comprises deamination of one or more nucleotides in the target nucleic acid.

25. The method of claim 23 or claim 24, wherein said modification takes place inside of a cell in vitro.

26. The method of claim 23 or claim 24, wherein said modification takes place inside of a cell in vivo.

27. The method of claim 25 or claim 26, wherein the cell is a prokaryotic cell.

28. The method of claim 25 or claim 26, wherein the cell is a eukaryotic cell.

29. The method of claim 28, wherein the cell is selected from: a plant cell, a fungal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a nonhuman primate cell, and a human cell.