CRISPR-CAS effector polypeptides and methods of use thereof

Abstract

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising the RNA-guided CRISPR-Cas effector proteins of the present disclosure and a guide RNA. The present disclosure provides methods for modifying target nucleic acids using the RNA-guided CRISPR-Cas effector proteins and guide RNAs of the present disclosure.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 815,179, filed March 7, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Introduction CRISPR-Cas systems include a Cas protein, which is responsible for acquiring, targeting, and cleaving foreign DNA or RNA, and a guide RNA(s) that includes a segment that binds to the Cas protein and a segment that binds to the target nucleic acid. For example, a Class 2 CRISPR-Cas system contains a single Cas protein bound to a guide RNA, and the Cas protein binds to and cleaves the targeted nucleic acid. The programmable nature of these systems facilitates their use as a versatile technology for modifying target nucleic acids. Summary of the Invention

[0003] overview The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising the RNA-guided CRISPR-Cas effector proteins of the present disclosure and a guide RNA. The present disclosure provides methods for modifying target nucleic acids using the RNA-guided CRISPR-Cas effector proteins and guide RNAs of the present disclosure. [Brief explanation of the drawings]

[0004] [Figure 1A]Size distributions are shown for complete bacteriophage genomes from this study, the recently reported Lak phage from a subset of the same samples, and reference sources (all dsDNA genomes from RefSeq v92 and non-artifact assemblies >200 kb from (Paez-Espino et al. (2016) Nature 536:425)). [Figure 1B] 1 shows a histogram of genome size distribution of phages with genomes larger than 200 kb from this study, Lak, and the reference genome. FIG. 1 shows a boxplot of tRNA counts per genome as a function of genome size. [Figure 2] Figure 1 shows a phylogenetic tree constructed using terminase sequences from the large phage genomes of this study and associated database sequences. Colored regions of the tree indicate large clades of phages, all of which have large genomes. [Figure 3] This model shows how phage-encoded capabilities can function to redirect the host's translation system to produce phage proteins. While no large phages possess all of these genes, many possess tRNAs (cloverleaf-shaped) and tRNA synthetases (aaRSs). Phage proteins with up to six ribosomal protein S1 domains occur in some genomes. S1 binds to mRNA and delivers it to the site on the ribosome where it is decoded. Ribosomal protein S21 (S21) can selectively initiate translation of phage mRNA, and many sequences possess N-terminal extensions (dashed lines in the ribosome inset, based on PDB codes 6bu8 and pmid:29247757 for the ribosome and S1 structural model) that may be involved in RNA binding. Some phages possess initiation factors (IFs) and elongation factor G (EFG), and some possess rpL7 / L12, which may mediate efficient ribosome binding. Abbreviations: RNA pol, RNA polymerase. [Figure 4A] Figure 1 shows bacteria-phage interactions (cell diagram) involving CRISPR targeting. [Figure 4B]1 shows an interaction network illustrating the targeting of bacterial (top to bottom: SEQ ID NOs: 49-50) and phage-encoded (top to bottom: SEQ ID NOs: 49-50) CRISPR spacers. [Figure 5-1] Phages with genomes >200 kbp and ecosystems with several plasmids are shown, grouped by sampling location type. Each box represents a phage genome; boxes are arranged in order of decreasing genome size, with size ranges for each location type listed to the right. Color indicates inferred host phylum based on genome phylogenetic profiles with confirmation by CRISPR targeting (X) or information system gene phylogenetic analysis (T). [Figure 5-2] See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. [Figure 5-4] See description of Figure 5-1. [Figure 6-1] Figures 6A-6BB provide the amino acid sequences of example CRISPR-Cas effector polypeptides of the present disclosure (from top to bottom: SEQ ID NOS: 51-97). [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 6-4] See description of Figure 6-1. [Figure 6-5] See description of Figure 6-1. [Figure 6-6] See description of Figure 6-1. [Figure 6-7] See description of Figure 6-1. [Figure 6-8] See description of Figure 6-1. [Figure 6-9] See description of Figure 6-1. [Figure 7-1] Figures 7A-7D provide the nucleotide sequences of the constant region portions of CRISPR-Cas effector guide RNAs (from top to bottom: SEQ ID NOs: 98-116). [Figure 7-2] See description of Figure 7-1. [Figure 8-1]1 provides an alignment of the amino acid sequences of CRISPR-Cas effector polypeptides (top to bottom: SEQ ID NOs: 117-187). [Figure 8-2] See description of Figure 8-1. [Figure 8-3] See description of Figure 8-1. [Figure 8-4] See description of Figure 8-1. [Figure 8-5] See description of Figure 8-1. [Figure 8-6] See description of Figure 8-1. [Figure 9] 1 provides the amino acid sequence of a CRISPR-Cas effector polypeptide (SEQ ID NO: 188). [Figure 10] A phylogenetic tree showing the various CRISPR-Cas effector protein families is provided. DETAILED DESCRIPTION OF THE INVENTION

[0005] definition The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0006] "Hybridizable," "complementary," or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) contains a sequence of nucleotides that allows it to non-covalently bind, i.e., form Watson-Crick and / or G / U base pairs, "anneal," or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base pairings include adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, guanine (G) can also base pair with uracil (U) 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 nucleobase is paired with a guide RNA). For example, G / U base pairing, in conjunction with tRNA anticodon base pairing with codons in mRNA, is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code. Thus, in the context of the present disclosure, guanine (G) (e.g., G of the guide RNA base that pairs with the target nucleic acid in the dsRNA duplex of the guide RNA molecule) is considered complementary to both uracil (U) and adenine (A). For example, if a G / U base pair can be made at a given nucleotide position in the dsRNA duplex of the guide RNA molecule, that position is not considered non-complementary, but instead is considered complementary.

[0007] Hybridization and washing conditions are well known and are 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). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0008] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases are possible. Suitable conditions 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 melting temperature (Tm) value for hybrids of nucleic acids having those sequences. For hybridization 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 location of mismatches can be important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of a hybridizable nucleic acid is 8 or more nucleotides (e.g., 10 or more, 12 or more, 15 or more, 20 or more, 22 or more, 25 or more, or 30 or more nucleotides). Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary, depending on factors such as the length of the regions of complementarity and the degree of complementarity.

[0009] It is understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. Furthermore, a polynucleotide may hybridize across one or more segments, such that intervening or adjacent segments are not involved in the hybridization event (for example, a bulge, a loop structure, or a hairpin structure). A polynucleotide may 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 with the target region within the target nucleic acid sequence to which the polynucleotide hybridizes. For example, an antisense nucleic acid in which 18 of the 20 nucleotides of an antisense compound are complementary to the target region and therefore specifically hybridize represents 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be adjacent to each other or to complementary nucleotides. The percent complementarity between specific stretches of nucleic acid sequences within a nucleic acid can be determined using any convenient method. Exemplary methods include the BLAST program (basic local alignment search tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), and the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), which uses, for example, the Smith-Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489) using default settings.

[0010] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0011] As used herein, "binding" (e.g., with respect to an RNA-binding domain of a polypeptide, binding to a target nucleic acid, etc.) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid, between a CRISPR-Cas effector polypeptide / guide RNA complex and a target nucleic acid, etc.). While in a state of non-covalent interaction, the macromolecules are said to be "associated" or "interacting" or "bound" (e.g., when molecule X is said to interact with molecule Y, it means that 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), although some portions of a binding interaction may be sequence-specific. Binding interactions generally occur in a variety of ways, including: -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M or 10 -15 A dissociation constant (K D "Affinity" refers to the strength of binding, and increased binding affinity is characterized by a lower K D correlates with.

[0012] By "binding domain" is meant a protein domain that can non-covalently bind to another molecule. A binding domain can bind, for example, to a DNA molecule (DNA-binding domain), an RNA molecule (RNA-binding domain), and / or a protein molecule (protein-binding domain). In the case of a protein having a protein-binding domain, the protein can optionally bind to itself (to form a homodimer, homotrimer, etc.) and / or the protein can bind to one or more regions of a different protein(s).

[0013] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins that have similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; the group of amino acids with acidic side chains consists of glutamate and aspartate; and the group of amino acids with 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.

[0014] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, the percentage of bases or amino acids are the same and in the same relative positions when the two sequences are compared. Sequence identity can be determined in several different ways. To determine sequence identity, sequences can be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the World Wide Web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.

[0015] A DNA sequence that "encodes" a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide can encode an RNA (mRNA) that is translated into a protein (thus, both DNA and mRNA encode proteins), or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, microRNA (miRNA), "non-coding" RNA (ncRNA), guide RNA, etc.).

[0016] 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 translated into a polypeptide (in the case of mRNA), in vitro or in vivo, when placed under the control of appropriate regulatory sequences.

[0017] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, e.g., promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a guide RNA) or a coding sequence (e.g., an RNA-guided endonuclease, a GeoCas9 polypeptide, a GeoCas9 fusion polypeptide, etc.) and / or regulate the translation of an encoded polypeptide.

[0018] As used herein, a "promoter" or "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 this disclosure, a promoter sequence is flanked at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence, a transcription initiation site is found, as well as protein binding domains responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive expression by the various vectors of this disclosure.

[0019] As used herein, the terms "native" or "unmodified" or "wild-type," when applied to a nucleic acid, polypeptide, cell, or organism, refer to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence present in an organism that can be isolated from a natural source is native.

[0020] As used herein, the term "fusion," when applied to a nucleic acid or polypeptide, refers to two components defined by their structure derived from different sources. For example, when "fusion" is used in the context of a fusion polypeptide (e.g., a fusion CRISPR-Cas effector protein), the fusion polypeptide comprises amino acid sequences derived from different polypeptides. A fusion polypeptide can comprise either a modified or native polypeptide sequence (e.g., a first amino acid sequence from a modified or unmodified CRISPR-Cas effector protein and a second amino acid sequence from a modified or unmodified protein other than a CRISPR-Cas effector protein, etc.). Similarly, a "fusion" in the context of a polynucleotide encoding a fusion polypeptide comprises nucleotide sequences derived from different coding regions (e.g., a first nucleotide sequence encoding a modified or unmodified CRISPR-Cas effector protein and a second nucleotide sequence encoding a polypeptide other than a CRISPR-Cas effector protein).

[0021] The term "fusion polypeptide" refers to a polypeptide made by the combination (ie, "fusion") of two otherwise separated segments of amino acid sequence, usually through human intervention.

[0022] As used herein, "heterologous" refers to a nucleotide or polypeptide sequence not found in the native nucleic acid or protein, respectively. For example, in some cases, in the variant CRISPR-Cas effector proteins of the present disclosure, a portion of a native 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 the CRISPR-Cas effector polypeptide or an amino acid sequence from another organism). As another example, a fused CRISPR-Cas effector polypeptide may comprise all or a portion of a native CRISPR-Cas effector polypeptide (or a variant thereof) fused with a heterologous polypeptide, i.e., a polypeptide from a protein other than the CRISPR-Cas effector polypeptide or a polypeptide from another organism. The heterologous polypeptide may exhibit an activity (e.g., an enzymatic activity) that is also exhibited by the variant CRISPR-Cas effector protein or fusion CRISPR-Cas effector protein (e.g., biotin ligase activity, nuclear localization, etc.). A heterologous nucleic acid sequence can be linked (eg, by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to create a nucleotide sequence that encodes a fusion polypeptide (fusion protein).

[0023] As used herein, "recombinant" 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 that result in a construct with structural coding or non-coding sequences distinguishable from endogenous nucleic acids found in nature. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide synthetic nucleic acids capable of expression from recombinant transcription units contained in cells or cell-free transcription and translation systems. Genomic DNA containing relevant sequences can also be used in forming recombinant genes or transcription units. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with the manipulation or expression of the coding region, or may actually act to regulate production of a desired product by various mechanisms (see "DNA regulatory sequences"). Alternatively, DNA sequences encoding non-translated RNA (e.g., guide RNA) may also be considered recombinant. Thus, for example, the term "recombinant" nucleic acid refers to one that is not naturally occurring, e.g., created by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often achieved either by chemical synthesis means or by the artificial manipulation of isolated segments of nucleic acid, e.g., genetic engineering techniques. This is usually done to replace codons with codons that encode the same amino acid, a conserved amino acid, or a non-conserved amino acid. Alternatively, it is performed to join nucleic acid segments of desired functions together to generate a desired combination of functions. This artificial combination is often achieved either by chemical synthesis means or by the artificial manipulation of isolated segments of nucleic acid, e.g., 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., mutant) of the naturally occurring sequence.An example of such a case is DNA (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) where the protein is not naturally found (e.g., expression of a CRISPR / Cas RNA-guided polypeptide such as a CRISPR-Cas effector (e.g., a wild-type CRISPR-Cas effector, a variant CRISPR-Cas effector, a fusion CRISPR-Cas effector, etc.) in a eukaryotic cell). Thus, while the codon-optimized DNA may be recombinant and non-naturally occurring, the protein encoded by the DNA may have a wild-type amino acid sequence.

[0024] Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose amino acid sequence does not occur in nature. Instead, a "recombinant" polypeptide is encoded by a recombinant, non-naturally 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, mutant, etc.). Thus, a "recombinant" polypeptide is the result of human intervention, but can have a naturally occurring amino acid sequence.

[0025] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, artificial chromosome or cosmid, to which another DNA segment, or "insert," may be attached so as to bring about replication of the attached segment in a cell.

[0026] An "expression cassette" comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition, where the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence (or a coding sequence can also be said to be operably linked to a promoter) if the promoter affects its transcription or expression.

[0027] 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 typically generated for the purpose of expressing and / or propagating an 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.

[0028] A cell has been "genetically modified," "transformed," or "transfected" when exogenous DNA or RNA, such as a recombinant expression vector, has been introduced into the cell. The presence of exogenous DNA results in a permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the cell's genome. For example, in prokaryotes, yeast, and mammalian cells, 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 comprising a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived by mitosis from a single cell or common ancestor. A "cell line" is a clone of a primary cell that is capable of stable in vitro growth for many generations.

[0029] Suitable methods of genetic modification (also referred to as "transformation") include, for example, 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 microinjection, 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.

[0030] The choice of genetic modification method generally depends on the cell type to be transformed and the context in which the transformation occurs (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.

[0031] As used herein, a "target nucleic acid" is a polynucleotide (e.g., DNA such as genomic DNA) that includes a site ("target site" or "target sequence") targeted by an RNA-guided endonuclease polypeptide (e.g., a wild-type CRISPR-Cas effector, a variant CRISPR-Cas effector, a fusion CRISPR-Cas effector, etc.). The target sequence is a sequence to which the guide sequence of a CRISPR-Cas effector guide RNA of interest (e.g., a dual CRISPR-Cas effector guide RNA or a single-molecule CRISPR-Cas effector guide RNA) hybridizes. For example, the target site (or target sequence) 5'-GAGCAUAUC-3' within the target nucleic acid is targeted by (or bound by, or hybridizes to, or is complementary to) the sequence 5'-GAUAUGCUC-3'. Suitable hybridization conditions include physiological conditions normally present in a cell. With respect to 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 therefore not complementary to the guide RNA) is referred to as the "non-target strand" or "non-complementary strand."

[0032] "Cleavage" refers to the cleavage 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-strand and double-strand breaks are possible, and double-strand breaks can occur as a result of two different single-strand break events.

[0033] The terms "nuclease" and "endonuclease" are used interchangeably herein to refer to enzymes that have catalytic activity for cleaving nucleic acids (e.g., ribonuclease activity (ribonucleic acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[0034] A "cleavage domain" or "activity domain" or "nuclease domain" of a nuclease refers to a polypeptide sequence or domain within a nuclease that has catalytic activity for nucleic acid cleavage. A cleavage domain can be contained in a single polypeptide chain, or the cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain can consist of two or more isolated stretches of amino acids within a given polypeptide.

[0035] The term "stem cell" is used herein to refer to cells (e.g., plant stem cells, vertebrate stem cells) that have both the ability to self-renew and generate differentiated cell types (see Morrison et al. (1997) Cell 88:287-298). In the context of cell development, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell to which it is being compared. Thus, pluripotent stem cells (described below) can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which can differentiate into terminal cells (i.e., highly differentiated cells, e.g., neurons, cardiomyocytes, etc.), which can differentiate into further restricted cells (e.g., neuronal progenitor cells), which play characteristic roles in certain tissue types and may or may not retain the ability to proliferate further. Stem cells can be characterized by both the presence and absence of specific markers (e.g., proteins, RNA, etc.). Stem cells can also be identified by functional assays, both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny.

[0036] Stem cells of interest include pluripotent stem cells (PSCs). The term "pluripotent stem cells" or "PSCs" is used herein to mean stem cells that can produce all cell types of an organism. Thus, PSCs can give rise to cells of all germ layers of an organism (e.g., endoderm, mesoderm, and ectoderm of vertebrates). Pluripotent cells can form teratomas and contribute to ectoderm, mesoderm, or endoderm tissues in vivo. Plant pluripotent stem cells can give rise to all cell types of the plant (e.g., cells of roots, stems, leaves, etc.).

[0037] PSCs in animals can be derived in several different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov 6;282(5391):1145-7), whereas induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et al., Cell. 2007 Nov 30;131(5):861-72; Takahashi et al., Nat Protoc. 2007;2(12):3081-9; Yu et al., Science. 2007 Dec 21;318(5858):1917-20. Epub 2007 Nov 20). Because the term PSC refers to pluripotent stem cells regardless of their derivation, the term PSC encompasses the terms ESC and iPSC, as well as embryonic germ stem cells (EGSCs), another example of PSCs. PSCs may be in the form of established cell lines, they may be obtained directly from primary embryonic tissue, or they may be derived from somatic cells. PSCs may be target cells for the methods described herein.

[0038] "Embryonic stem cells" (ESCs) refer to PSCs isolated from the inner cell mass of an embryo, typically a blastocyst. ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g., hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.), HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International), Miz-hES1 (MizMedi Hospital-Seoul National University), HSF-1, HSF-6 (University of California at San Francisco), and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, such as humans, horses, cows, pigs, dogs, cats, rodents, such as mice, rats, hamsters, and primates (Thomson et al. (1998) Science 282:1145, Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844, Thomson et al. (1996) Biol. Reprod. 55:254, Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically grow as flat colonies with large nucleocytoplasmic ratios, defined borders, and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods for generating and characterizing ESCs can be found, for example, in U.S. Patent Nos. 7,029,913, 5,843,780, and 6,200,806, the disclosures of which are incorporated herein by reference. Methods for propagating hESCs in an undifferentiated form are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920.

[0039] "Embryonic germ stem cells" (EGSCs) or "embryonic germ cells" or "EG cells" refer to germ cells and / or germ cell precursor cells, e.g., PSCs derived from primordial germ cells, i.e., those that give rise to sperm and eggs. Embryonic germ cells (EG cells) are believed to have similar properties to embryonic stem cells, as described above. Examples of methods for generating and characterizing EG cells can be found, for example, in U.S. Patent No. 7,153,684, Matsui, Y., et al., (1992) Cell 70:841, Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98:113, Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726, and Koshimizu, U., et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference.

[0040] "Induced pluripotent stem cells" or "iPSCs" refer to PSCs derived from cells that are not PSCs (i.e., derived from cells that are differentiated into PSCs). iPSCs can be derived from several different cell types, including highly differentiated cells. iPSCs have an ES cell-like morphology and grow as flat colonies with a large nuclear-cytoplasmic ratio, defined borders, and prominent nucleoli. In addition, iPSCs express one or more key pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Publication Nos. US2009 / 0047263, US2009 / 0068742, US2009 / 0191159, US2009 / 0227032, US2009 / 0246875, and US2009 / 0304646, the disclosures of which are incorporated herein by reference. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) to reprogram the somatic cells to become pluripotent stem cells.

[0041] "Somatic cell" refers to any cell in an organism that does not normally give rise to all cell types in the organism in the absence of experimental manipulation. In other words, a somatic cell is a cell that is sufficiently differentiated so that it does not naturally generate cells of all three germ layers of the body, i.e., ectoderm, mesoderm, and endoderm. For example, somatic cells include both neurons and neural precursors, the latter of which may be capable of naturally giving rise to all or some cell types of the central nervous system, but are unable to give rise to cells of the mesodermal or endodermal lineages.

[0042] "Mitotic cell" means a cell undergoing mitosis. Mitosis is the process by which a eukaryotic cell separates the chromosomes in its nucleus into two identical sets in two separate nuclei. It is generally immediately followed by cytokinesis, which divides the nucleus, cytoplasm, organelles, and cell membrane into two cells containing approximately equal portions of these cellular components.

[0043] By "postmitotic cell" is meant a cell that has exited mitosis, i.e., is "quiescent," i.e., is no longer undergoing division. This quiescent state can be temporary, i.e., reversible, or permanent.

[0044] "Meiotic cell" means a cell undergoing meiosis, the process by which a cell divides its nuclear material for the purpose of producing gametes or spores. Unlike mitosis, in meiosis, chromosomes undergo a recombination step that shuffles genetic material between chromosomes. Additionally, the result of meiosis is four (genetically unique) haploid cells, compared to the two (genetically identical) diploid cells produced from mitosis.

[0045] In some cases, a component (e.g., a nucleic acid component (e.g., a CRISPR-Cas effector guide RNA), a protein component (e.g., a wild-type CRISPR-Cas effector polypeptide, a variant CRISPR-Cas effector polypeptide, a fusion CRISPR-Cas effector polypeptide, etc.) comprises a label moiety. As used herein, the terms "label," "detectable label," or "label moiety" refer to any moiety that provides signal detection and can vary widely depending on the particular nature of the assay. Label moieties of interest include both directly detectable labels (direct labels; e.g., fluorescent labels) and indirectly detectable labels (indirect labels; e.g., binding pair members). Fluorescent labels include any fluorescent label (e.g., a fluorescent dye (e.g., fluorescein, The label may be a fluorescent protein (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry, tomato, tangerine, and any fluorescent derivatives thereof), or the like. Detectable (direct or indirect) label moieties suitable for use in the methods include any moiety detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, a suitable indirect label includes biotin (a binding pair member), which can be bound by streptavidin (which itself can be directly or indirectly labeled). Labels also include radiolabels (direct labels) (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), enzymes (indirect labels) (e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.), fluorescent proteins (direct labels) (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivatives thereof), metal labels (direct labels), colorimetric labels, binding pair members, etc. "Binding pair partner" or "binding pair member" refers to one of a first and a second moiety, wherein the first and second moieties have specific binding affinity for each other. Suitable binding pairs include, but are not limited to, antigen / antibody (e.g., digoxigenin / anti-digoxigenin, dinitrophenyl (DNP) / anti-DNP, dansyl-X-anti-dansyl, fluorescein / anti-fluorescein, lucifer yellow / anti-lucifer yellow, and rhodamine anti-rhodamine), biotin / avidin (or biotin / streptavidin), and calmodulin-binding protein (CBP) / calmodulin. Any binding pair member may be suitable for use as an indirectly detectable labeling moiety.

[0046] Any given component, or combination of components, can be unlabeled or detectably labeled with a labeling moiety. In some cases, when more than one component is labeled, they can be labeled with labeling moieties that are distinguishable from one another.

[0047] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harvard 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 These can be found in standard textbooks such as (1998).

[0048] As used herein, the terms "treatment," "treating," and the like refer to obtaining a pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, e.g., a human, including (a) preventing the onset of the disease in a subject who is susceptible to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., preventing its development, and (c) relieving the disease, i.e., causing regression of the disease.

[0049] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to individual organisms, e.g., mammals, including, but not limited to, mice, monkeys, humans, non-human primates, ungulates, felines, dogs, cattle, sheep, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0050] 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.

[0051] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0052] Unless otherwise defined, 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 by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0053] It should 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, a reference to a "CRISPR-Cas effector polypeptide" includes a plurality of such polypeptides; a reference to a "guide RNA" includes a reference to one or more guide RNAs 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. Accordingly, this statement is intended to serve as a guideline precedent for use of exclusive terminology such as "solely," "only," and the like, or the use of "negative" limitations, with regard to the recitation of claim elements.

[0054] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can 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, can also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations 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 subcombination were individually and explicitly disclosed herein.

[0055] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should 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.

[0056] Detailed Description The present disclosure provides RNA-guided CRISPR-Cas effector proteins (variously referred to herein as "Cas14i" polypeptides, "Cas14j" polypeptides, "Cas14k" polypeptides, etc.), nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising a CRISPR-Cas effector polypeptide of the present disclosure and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid using a CRISPR-Cas effector polypeptide and a guide RNA of the present disclosure.

[0057] The present disclosure provides guide RNAs (herein referred to as "CRISPR-Cas effector guide RNAs") that bind to and provide sequence specificity for the CRISPR-Cas effector proteins of the present disclosure, nucleic acids encoding the CRISPR-Cas effector guide RNAs, and modified host cells containing the CRISPR-Cas effector guide RNAs and / or nucleic acids encoding same. The CRISPR-Cas effector guide RNAs are useful in a variety of applications, as provided.

[0058] composition CRISPR / CAS effector proteins and guide RNAs A CRISPR-Cas effector polypeptide of the present disclosure (e.g., a Cas14i polypeptide of the present disclosure, a Cas14j polypeptide of the present disclosure, etc.) interacts with (binds to) a corresponding guide RNA (e.g., a CRISPR-Cas effector guide RNA) to form a ribonucleoprotein (RNP) complex that is targeted to a specific site in a target nucleic acid through base pairing between the guide RNA and a target sequence within the target nucleic acid molecule. The guide RNA comprises a nucleotide sequence (guide sequence) that is complementary to the sequence of the target nucleic acid (target site). Thus, a CRISPR-Cas effector protein of the present disclosure forms a complex with the CRISPR-Cas effector guide RNA, and the guide RNA provides sequence specificity to the RNP complex via the guide sequence. The CRISPR-Cas effector protein of the complex provides site-specific activity. In other words, the CRISPR-Cas effector protein is guided to (e.g., stabilized at) a target site within a target nucleic acid sequence (e.g., a chromosomal or extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by its association with the guide RNA.

[0059] The disclosure provides compositions comprising a CRISPR-Cas effector polypeptide (and / or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide) (e.g., where the CRISPR-Cas effector polypeptide can be a naturally occurring protein, a nickase CRISPR-Cas effector protein, a catalytically inactive ("dead" CRISPR-Cas effector, also referred to herein as a "dCRISPR-Cas effector protein"), a fusion CRISPR-Cas effector protein, etc.). The disclosure provides compositions comprising a CRISPR-Cas effector guide RNA (and / or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA). The present disclosure provides compositions comprising (a) a CRISPR-Cas effector polypeptide (and / or a nucleic acid encoding a CRISPR-Cas effector polypeptide) (e.g., the CRISPR-Cas effector polypeptide can be a naturally occurring protein, a nickase CRISPR-Cas effector protein, a dCRISPR-Cas effector protein, a fusion CRISPR-Cas effector protein, etc.), and (b) a CRISPR-Cas effector guide RNA (and / or a nucleic acid encoding a CRISPR-Cas effector guide RNA).The present disclosure provides nucleic acid / protein complexes (RNP complexes) comprising (a) a CRISPR-Cas effector polypeptide of the present disclosure (e.g., the CRISPR-Cas effector polypeptide can be a naturally occurring protein, a nickase CRISPR-Cas effector protein, a Cdas12J protein, a fusion CRISPR-Cas effector protein, etc.), and (b) a CRISPR-Cas effector guide RNA.

[0060] CRISPR-Cas effector proteins A CRISPR-Cas effector polypeptide (this term is used interchangeably with the term "CRISPR-Cas effector protein") can bind to and / or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and / or a polypeptide associated with the target nucleic acid (e.g., methylation or acetylation of histone tails) (e.g., in some cases, the CRISPR-Cas effector protein includes an active fusion partner; in some cases, the CRISPR-Cas effector protein provides nuclease activity). In some cases, the CRISPR-Cas effector protein is a naturally occurring protein (e.g., one that occurs naturally in a bacteriophage). In other cases, the CRISPR-Cas effector protein is not a naturally occurring polypeptide (e.g., the CRISPR-Cas effector protein is a variant CRISPR-Cas effector protein, a fusion CRISPR-Cas effector protein, etc.).

[0061] The assay for determining whether a given protein interacts with a CRISPR-Cas effector guide RNA can be any convenient binding assay that tests for binding between a protein and a nucleic acid. Suitable binding assays (e.g., gel shift assays) will be known to those of skill in the art (e.g., assays that involve adding a CRISPR-Cas effector guide RNA and a protein to a target nucleic acid). The assay for determining whether a protein has activity (e.g., to determine whether the protein has nuclease activity and / or some heterologous activity that cleaves a target nucleic acid) can be any convenient assay (e.g., any convenient nucleic acid cleavage assay that tests for nucleic acid cleavage). Suitable assays (e.g., cleavage assays) will be known to those of skill in the art.

[0062] Natural CRISPR-Cas effector proteins function as endonucleases that catalyze double-stranded breaks at specific sequences in target double-stranded DNA (dsDNA). Sequence specificity is provided by an associated guide RNA that hybridizes to a target sequence within the target DNA. Natural CRISPR-Cas effector guide RNA is a crRNA, which contains (i) a guide sequence that hybridizes to a target sequence in the target DNA, and (ii) a protein-binding segment that includes a stem-loop (hairpin-dsRNA duplex) that binds to the CRISPR-Cas effector protein.

[0063] In some embodiments, the CRISPR-Cas effector proteins of the subject methods and / or compositions are (or are derived from) naturally occurring (wild-type) proteins. Examples of naturally occurring CRISPR-Cas effector proteins are shown in Figures 6A-6BB.

[0064] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6A and designated "PhageCas14J_k87_9374247_16." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6A. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6A. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6A. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6A. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6A except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6A (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0065] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6B and designated "PhageCas14J_LacPavin_0818_WC40_scaffold_407201_205." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6B. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6B. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6B. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6B. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6B except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6B (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0066] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6C and designated "PhageCas14J_BML_08042016_6_5m_scaffold_18_prodigal-single_54." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6C. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6C. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6C. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6C, except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6C (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0067] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6D and designated "Ga0194119_1000113823." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6D. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6D. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6D. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6D. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6D, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6D, and the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0068] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6E and designated "Ga0116197_10005458." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6E. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6E. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6E. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6E. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6E, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6E, where the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0069] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6F and designated "Ga0116179_10426881." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6F. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6F. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6F. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6F. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6F, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6F, and the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0070] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6G and designated "Ga0268285_10062095." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6G. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6G. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6G. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6G. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6G, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6G, where the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0071] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6H and designated "Ga0066868_100162752." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6H. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6H. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6H. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6H. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6H, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6H, and the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0072] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6I and designated "PhageCas14_SR-VP_2-4_scaffold_141_2548329_92." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 61. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 61. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 61. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 61, except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6I (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0073] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6J and designated "PhageCas14_SR-VP_4-6_scaffold_141_3640689_5." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6J. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6J. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6J. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6J. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6J except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6J (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0074] In some cases, the CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6K and designated "PhageCas14_RifSed." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6K. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6K. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6K. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6K. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6K, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6K, and the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0075] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6L and designated "PhageCas14_16ft_4_scaffold_2_465_16ft_4_Phage_29_13." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6L. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6L. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6L. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6L. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6L except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6L (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0076] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6M and designated "Ga0116179_10109322." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6M. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6M. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6M. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6M. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6M, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6M, where the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0077] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6N and designated "Ga0116179_10465782." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6N. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6N. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6N. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6N. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6N, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6N, where the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0078] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6O and designated "Ga0134101_10165752." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6O. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6O. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6O. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6O. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6O, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6O, where the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0079] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6P and designated "Ga0066665_100815632." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6P. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6P. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6P. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6P. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6P, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6P, and the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0080] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6Q and designated "Ga0224523_10070512." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6Q. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6Q. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6Q. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6Q. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6Q, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6Q, and the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0081] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6R and designated "Ga0247839_10583994." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6R. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6R. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6R. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6R. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6R, except that the sequence contains amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein. The CRISPR-Cas effector protein can bind to a guide RNA (with T replaced with U) or its reverse complement that includes a constant region comprising the nucleotide sequence shown in Figure 6R, and the guide RNA can include a target binding sequence, if desired, depending on the target nucleic acid.

[0082] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6S and designated "Cas14u9|PhageCas14|LacPavin_0818_WC55_scaffold_56344_prodigal-single_16." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6S. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6S. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6S. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6S, except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA (with T substituted for U) or its reverse complement, which comprises a constant region comprising the nucleotide sequence shown in Figure 6S, and the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0083] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6T and designated "Cas14u10|Ga0153798_100522201." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6T. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6T. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6T. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6T. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6T except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6T (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0084] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6U and designated "Cas14u_VU_u11|rifcsplowo2_12_scaffold_23_prodigal-single_23." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6U. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6U. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6U. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6U, except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6U (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0085] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6V and designated "Cas14u_VU_u12|SR-VP_4-6_scaffold_141_2630357_509." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6V. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6V. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6V. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6V. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6V except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6V (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0086] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6W and designated "Cas14u_VU_u13|gwd1_scaffold_1554_3." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6W. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6W. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6W. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6W. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6W except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6W (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0087] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6X and designated "Cas14u_VU_u14|pig_F100_scaffold_13388_4." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6X. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6X. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6X. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6X. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6X except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6X (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0088] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6Y and designated "Cas14u_VU_u15|pig_ID_3640_F65_scaffold_73762_2." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6Y. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6Y. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6Y. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6Y. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6Y except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6Y (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0089] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6Z and designated "Cas14u_VU_u16|pig_ID_1851_F40_2_scaffold_55126_1." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6Z. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6Z. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6Z. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6Z. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6Z except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6Z (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0090] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6AA and designated "Cas14u_VU_u17|pig_ID_3784_F96_scaffold_13509_10." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6AA. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6AA. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6AA. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6AA. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6AA, except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6AA (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0091] In some cases, a CRISPR-Cas effector protein (of the subject compositions and / or methods) comprises an amino acid sequence having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in FIG. 6BB and designated "Cas14u_VU_u18|SRR1747065_scaffold_28." For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6BB. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence depicted in Figure 6BB. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having 90% or greater sequence identity (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to the CRISPR-Cas effector amino acid sequence shown in Figure 6BB. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6BB. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence having the CRISPR-Cas effector protein sequence shown in Figure 6BB, except that the sequence includes amino acid substitutions (e.g., one, two, or three amino acid substitutions) that reduce the native catalytic activity of the protein.The CRISPR-Cas effector protein can bind to a guide RNA comprising a constant region comprising the nucleotide sequence shown in Figure 6BB (with T substituted for U) or its reverse complement, where the guide RNA can include a target binding sequence if desired, depending on the target nucleic acid.

[0092] In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 350 amino acids (aa) to about 550 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 350 aa to about 375 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 375 aa to about 400 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 390 aa to about 410 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 400 aa to about 410 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 400 aa to about 450 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 410 aa to about 425 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 425 aa to about 450 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 450 aa to about 500 aa. In some cases, the CRISPR-Cas effector proteins of the present disclosure have a length of about 450 aa to about 475 aa.

[0093] CRISPR-Cas effector variants A variant CRISPR-Cas effector protein has an amino acid sequence that differs by at least one amino acid (e.g., has a deletion, insertion, substitution, or fusion) when compared to the amino acid sequence of a corresponding wild-type CRISPR-Cas effector protein, e.g., when compared to the CRISPR-Cas effector amino acid sequence shown in any one of Figures 6A-6BB. In some cases, the CRISPR-Cas effector variant contains between one and ten amino acid substitutions compared to the CRISPR-Cas effector amino acid sequence shown in any one of Figures 6A-6BB. In some cases, the CRISPR-Cas effector variant contains between one and ten amino acid substitutions in the RuvC domain compared to the CRISPR-Cas effector amino acid sequence shown in any one of Figures 6A-6BB.

[0094] Variant-catalytic activity In some cases, the CRISPR-Cas effector protein is, for example, a variant CRISPR-Cas effector protein mutated relative to a native catalytically active sequence, and exhibits reduced cleavage activity (e.g., 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less) compared to the corresponding native sequence. In some cases, such a variant CRISPR-Cas effector protein is a catalytically "dead" protein (has substantially no cleavage activity) and may be referred to as a "dCRISPR-Cas effector." In some cases, the variant CRISPR-Cas effector protein is a nickase (which cleaves only one strand of a double-stranded target nucleic acid, e.g., double-stranded target DNA). As described in more detail herein, optionally, a CRISPR-Cas effector protein (optionally a CRISPR-Cas effector protein having wild-type cleavage activity, optionally a variant CRISPR-Cas effector having reduced cleavage activity, e.g., a dCRISPR-Cas effector or a nickase CRISPR-Cas effector) is fused (conjugated) to a heterologous polypeptide having an activity of interest (e.g., a catalytic activity of interest) to form a fusion protein (fused CRISPR-Cas effector protein).

[0095] Amino acid substitutions that result in a CRISPR-Cas effector polypeptide that binds to but does not cleave a target nucleic acid include, for example, substitutions of conserved amino acids among Cas14i, Cas14K, Cas14J, or Cas14u RuvC-I and / or RuvC-II and / or RuvC-III domains. Figure 8 provides an amino acid sequence alignment of Cas14i, Cas14K, Cas14J, and Cas14u amino acid sequences. Conserved amino acids are indicated. In some cases, a CRISPR-Cas effector polypeptide of the present disclosure includes one or more amino acid substitutions of (i) Asp corresponding to Asp at position 258 of the amino acid sequence shown in Figure 9, (ii) Glu corresponding to Glu at position 356 of the amino acid sequence shown in Figure 9, and (iii) Asp corresponding to Asp at position 435 of the amino acid sequence shown in Figure 9. Examples include (i) Asp at position 236 of the sequence shown in Figure 8 (e.g., Asp in the VGID sequence of Cas14i_ga0066868-100162752 in Figure 8, or a corresponding position in another CRISPR-Cas effector polypeptide), (ii) Glu at position 332 of the sequence shown in Figure 8 (e.g., Glu in the VAVENL sequence of Cas14i_ga0066868-100162752 in Figure 8, or a corresponding position in another CRISPR-Cas effector polypeptide), and (iii) Asp at position 415 of the sequence shown in Figure 8 (e.g., Asp in the NADVNAA sequence of Cas14i_ga0066868-100162752 in Figure 8, or a corresponding position in another CRISPR-Cas effector polypeptide).

[0096] Variant-fusion CRISPR-Cas effector polypeptides As described above, optionally, the CRISPR-Cas effector protein (optionally a CRISPR-Cas effector protein having wild-type cleavage activity, optionally a variant CRISPR-Cas effector having reduced cleavage activity, e.g., a dCRISPR-Cas effector or a nickase CRISPR-Cas effector) is fused (conjugated) to a heterologous polypeptide having an activity of interest (e.g., a catalytic activity of interest) to form a fusion protein. The heterologous polypeptide to which the CRISPR-Cas effector protein can be fused is referred to herein as a "fusion partner."

[0097] In some cases, the fusion partner is capable of modulating (e.g., inhibiting transcription, increasing transcription) transcription of the target DNA. For example, in some cases, the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcription repressor, a protein that functions via recruitment of a transcription inhibitor protein, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of histone modifiers such as those that modify histone acetylation and / or methylation, etc.). In some cases, the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that functions via recruitment of a transcription activator protein, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of histone modifiers such as those that modify histone acetylation and / or methylation, etc.).

[0098] In some cases, the fusion CRISPR-Cas effector protein comprises a heterologous polypeptide having an enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damaging activity, deaminating activity, dismutase activity, alkylating activity, depurinating activity, oxidizing activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity).

[0099] In some cases, the fusion CRISPR-Cas effector protein comprises a heterologous polypeptide having an enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity) that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid.

[0100] Examples of proteins (or fragments thereof) that can be used to increase transcription include transcription activators such as VP16, VP64, VP48, VP160, the p65 subdomain (e.g., from NFkB), and the activation domain of EDLL and / or the TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK; and Ten-Eleven These include, but are not limited to, DNA demethylases such as Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1.

[0101] Examples of proteins (or fragments thereof) that can be used to reduce transcription include transcription repressors such as Kruppel-associated box (KRAB or SKD); KOX1 repression domain; Mad mSIN3 interacting domain (SID); ERF repressor domain (ERD), SRDX repression domain (e.g., for repression in plants), etc.; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, and RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, and JARID1D / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, and HDAC11; HhaI DNA These include, but are not limited to, DNA methylases such as m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and peripheral mobilization elements such as Lamin A and Lamin B.

[0102] In some cases, the fusion partner has an enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by the fusion partner include nuclease activity, such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity, such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.); demethylase activity, such as that provided by a Ten-Eleven demethylase activity such as that provided by TET dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.), DNA repair activity, DNA damage activity, deamination activity such as that provided by deaminases (e.g., cytosine deaminase enzymes such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase and / or resolvase (e.g., G These include, but are not limited to, integrase activity such as that provided by a hyperactive mutant of Gin invertase, Gin invertase such as GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.; transposase activity; recombinase activity such as that provided by a recombinase (e.g., the catalytic domain of Gin recombinase); polymerase activity; ligase activity; helicase activity; photolyase activity; and glycosylase activity.

[0103] In some cases, the fusion partner has an enzymatic activity that modifies a protein (e.g., histone, RNA-binding protein, DNA-binding protein, etc.) associated with a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities (that modify a protein associated with a target nucleic acid) that can be provided by a fusion partner include histone methyltransferases (HMTs) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatin histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, etc., SET1A, SET1B, MLL1-5, ASH1, SYMD2, NS methyltransferase activity, such as that provided by D1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1), histone demethylases (e.g., lysine demethylase 1A (KDM1A, also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, demethylase activity such as that provided by histone acetylase transferases (e.g., catalytic cores / fragments of human acetyltransferases p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, etc.), histone deacetylases (e.g., These include, but are not limited to, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, and demyristoylating activity, such as those provided by HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.

[0104] Additional examples of suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domains (e.g., to generate chemically controllable fusion CRISPR-Cas effector proteins), and chloroplast transit peptides. Suitable chloroplast transit peptides include: These include, but are not limited to, TIFF0007649748000001.tif145164.

[0105] In some cases, the CRISPR-Cas effector fusion polypeptide of the present disclosure comprises (a) a CRISPR-Cas effector polypeptide of the present disclosure and (b) a chloroplast transit peptide. Thus, for example, a CRISPR-Cas effector polypeptide / guide RNA complex can be targeted to chloroplasts. In some cases, this targeting can be achieved by the presence of an N-terminal extension called a chloroplast transit peptide (CTP) or plastid transit peptide. If an expressed polypeptide is to be compartmentalized within a plant plastid (e.g., a chloroplast), a chromosomal transgene from a bacterial source must have a sequence encoding a CTP sequence fused to the sequence encoding the expressed polypeptide. Therefore, localization of an exogenous polypeptide to chloroplasts is often achieved by operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of the polynucleotide encoding the exogenous polypeptide. The CTP is removed in a processing step during translocation to the plastid. However, processing efficiency can be affected by the amino acid sequence of the CTP and nearby sequences at the amino terminus (NH2-terminus) of the peptide. Other options for targeting to chloroplasts that have been described are the maize cab-m7 signal sequence (U.S. Pat. No. 7,022,896, WO97 / 41228), the pea glutathione reductase signal sequence (WO97 / 41228), and the CTP described in US2009 / 029861.

[0106] In some cases, a CRISPR-Cas effector fusion polypeptide of the present disclosure can comprise (a) a CRISPR-Cas effector polypeptide of the present disclosure, and (b) an endosomal escape peptide. In some cases, the endosomal escape polypeptide has the amino acid sequence TIFF0007649748000002.tif4128, wherein each X is independently selected from lysine, histidine, and arginine. In some cases, the endosomal escape polypeptide has the amino acid sequence Contains TIFF0007649748000003.tif4128.

[0107] For examples of some of the above fusion partners (and others) used in the context of fusion with Cas9, zinc finger, and / or TALE proteins (for site-specific targeted nucleic acid modification, regulation of transcription, and / or targeted protein modification, e.g., histone modification), see, e.g., Nomura et al., J Am Chem Soc. 2007 Jul 18;129(28):8676-7; Rivenbark et al., Epigenetics. 2012 Apr;7(4):350-60; Nucleic Acids Res. 2016 Jul 8;44(12):5615-28; Gilbert et al., Cell. 2013 Jul 18;154(2):442-51; Kearns et al., Nat Methods. 2015 May;12(5):401-3; Mendenhall et al., Nat Biotechnol. 2013 Dec;31(12):1133-6, Hilton et al.,Nat Biotechnol.2015 May;33(5):510-7, Gordley et al.,Proc Natl Acad Sci US A.2009 Mar 31;106(13):5053-8, Akopian et al.,Proc Natl Acad Sci US A.2003 Jul 22;100(15):8688-91, Tan et.,al.,J Virol.2006 Feb;80(4):1939-48, Tan et al.,Proc Natl Acad Sci US A.2003 Oct 14;100(21):11997-2002, Papworth et al.,Proc Natl Acad Sci US A.2003 Feb 18;100(4):1621-6, Sanjana et al., Nat Protoc.2012 Jan 5;7(1):171-92, Beerli et al., Proc Natl Acad Sci US A.1998 Dec 8;95(25):14628-33, Snowden et al., Curr Biol.2002 Dec 23;12(24):2159-66, Xu et.al.,Xu et.al.,Cell Discov.See Komor et al., Nature. 2016 Apr 20;533(7603):420-4, Chaikind et al., Nucleic Acids Res. 2016 Aug 11, Choudhury at. al., Oncotarget. 2016 Jun 23, Du et al., Cold Spring Harb Protoc. 2016 Jan 4, Pham et al., Methods Mol Biol. 2016;1358:43-57, Balboa et al., Stem Cell Reports. 2015 Sep 8;5(3):448-59, Hara et al., Sci Rep. 2015 Jun 9;5:11221, Piatek et al., Plant Biotechnol J. 2015 May;13(4):578-89, Hu et al., Nucleic Acids Res. 2014 Apr;42(7):4375-90, Cheng et al., Cell Res. 2013 Oct;23(10):1163-71, and Maeder et al., Nat Methods. 2013 Oct;10(10):977-9.

[0108] Additional suitable heterologous polypeptides include, but are not limited to, polypeptides that directly and / or indirectly provide increased transcription and / or translation of a target nucleic acid (e.g., transcriptional activators or fragments thereof, proteins or fragments thereof that recruit transcriptional activators, small molecule / drug-responsive transcriptional and / or translational regulators, translational regulatory proteins, etc.). Non-limiting examples of heterologous polypeptides that achieve increased or decreased transcription include transcriptional activator and transcriptional repressor domains. In some such cases, a fused CRISPR-Cas effector polypeptide is targeted to a specific location (i.e., sequence) in a target nucleic acid by a guide nucleic acid (guide RNA), resulting in locus-specific regulation, such as blocking RNA polymerase binding to the promoter (selectively inhibiting the function of the transcriptional activator) and / or modifying the local chromatin state (e.g., when a fusion sequence that modifies the target nucleic acid or a polypeptide associated with the target nucleic acid is used). In some cases, the change is transient (e.g., transcriptional repression or activation). In some cases, the change is heritable (eg, when an epigenetic modification is made to the target nucleic acid or to a protein associated with the target nucleic acid, eg, a nucleosomal histone).

[0109] Non-limiting examples of heterologous polypeptides for use in 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 comprise an entire protein, or in some cases, a fragment of a protein (e.g., a functional domain).

[0110] The heterologous polypeptide of a subject fusion CRISPR-Cas effector polypeptide can be any domain (for purposes of this disclosure, this includes intramolecular and / or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.) that can interact with ssRNA, whether transiently or irreversibly, directly or indirectly, including endonucleases (e.g., RNase III from proteins such as SMG5 and SMG6, CRR22 DYW domain, Dicer, and PIN (PilT N-terminal) domain); proteins and protein domains involved in stimulating RNA cleavage (e.g., CPSF, CstF, CFIm, and CFIIm); exonucleases (e.g., XRN-1 or exonuclease T); deadenylases (e.g., HNT3); proteins and protein domains involved in nonsense-mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins and protein domains involved in RNA stabilization (e.g., PABP); proteins and protein domains involved in translational repression (e.g., Ago2 and Ago4); proteins and protein domains involved in translational stimulation (e.g., Staufen); proteins and protein domains involved in (e.g., capable of regulating) translation (e.g., translation factors such as initiation factors, elongation factors, and release factors, e.g., eIF4G); proteins and protein domains involved in RNA polyadenylation (e.g., PAP1, GLD-2, and Star-PAP); proteins and protein domains involved in RNA polyuridylation (e.g., CI D1 and terminal uridylate transferase); proteins and protein domains involved in RNA localization (e.g., from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains involved in the nuclear retention of RNA (e.g., Rrp6); proteins and protein domains involved in the nuclear export of RNA (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains involved in the repression of RNA splicing (e.g., PTB, Sam68, and hnRNP A1);These include, but are not limited to, effector domains selected from the group including proteins and protein domains involved in stimulating RNA splicing (e.g., serine / arginine-rich (SR) domains); proteins and protein domains involved in reducing transcription efficiency (e.g., FUS (TLS)); and proteins and protein domains involved in stimulating transcription (e.g., CDK7 and HIV Tat). Alternatively, the effector domain may be selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains with nonsense-mediated RNA activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of regulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridylation of RNA; proteins and protein domains with RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains with RNA nuclear export activity; proteins and protein domains capable of suppressing RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in detail in WO2012 / 068627, which is incorporated herein by reference in its entirety;

[0111] Some RNA splicing factors that can be used (in whole or as fragments) as heterologous polypeptides in fusion CRISPR-Cas effector polypeptides have a modular organization, with distinct sequence-specific RNA-binding modules and splicing effector domains. For example, members of the serine / arginine-rich (SR) protein family contain an N-terminal RNA recognition motif (RRM) that binds to exon splicing enhancers (ESEs) in pre-mRNA and a C-terminal RS domain that promotes exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to exon splicing silencers (ESSs) through its RRM domain and inhibits exon inclusion through its C-terminal glycine-rich domain. Some splicing factors can regulate the use of alternative splice sites (SSs) by binding to regulatory sequences between the two alternative sites. For example, ASF / SF2 can recognize an ESE and promote the use of an intron-proximal site, whereas hnRNP A1 can bind to an ESS and redirect splicing to the use of an intron-distal site. One use of such factors is to generate ESFs that regulate alternative splicing of endogenous genes, particularly disease-related genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites, encoding proteins with opposing functions. The long splicing isoform, Bcl-xL, is a potent apoptosis inhibitor expressed in long-lived, postmitotic cells and is upregulated in many cancer cells, protecting them from apoptotic signals. The short isoform, Bcl-xS, is a pro-apoptotic isoform expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple cω elements located either in the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites). For further examples, see WO2010 / 075303, the entire contents of which are incorporated herein by reference.

[0112] Further suitable fusion partners include, but are not limited to, proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., lamin A, lamin B, etc.), protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).

[0113] Examples of various additional heterologous polypeptides (or fragments thereof) suitable for the subject fusion CRISPR-Cas effector polypeptides include, but are not limited to, those described in the following applications (these publications relate to other CRISPR endonucleases, such as Cas9, but the fusion partners described can alternatively be used with CRISPR-Cas effectors): PCT patent applications: WO2010 / 075303, WO2012 / 068627, and WO2013 / 155555; and, e.g., U.S. Patent and Patent Application Nos. 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, 8,697,No. 359, No. 2014 / 0068797, No. 2014 / 0170753, No. 2014 / 0179006, No. 2014 / 0179770, No. 2014 / 0186843, No. 2014 / 0186919, No. 2014 / 0186958, No. 2014 / 0189896, No. 2014 / 0227787, No. 2014 / 0234972, No. 2014 / 0242664, No. 2014 / 0242699, No. 2014 / No. 0242700, No. 2014 / 0242702, No. 2014 / 0248702, No. 2014 / 0256046, No. 2014 / 0273037, No. 2014 / 0273226, No. 2014 / 027323 No. 0, No. 2014 / 0273231, No. 2014 / 0273232, No. 2014 / 0273233, No. 2014 / 0273234, No. 2014 / 0273235, No. 2014 / 0287938, No. 20 No. 14 / 0295556, No. 2014 / 0295557, No. 2014 / 0298547, No. 2014 / 0304853, No. 2014 / 0309487, No. 2014 / 0310828, No. 2014 / 031 No. 0830, No. 2014 / 0315985, No. 2014 / 0335063, No. 2014 / 0335620, No. 2014 / 0342456, No. 2014 / 0342457, No. 2014 / 0342458, Nos. 2014 / 0349400, 2014 / 0349405, 2014 / 0356867, 2014 / 0356956, 2014 / 0356958, 2014 / 0356959, 2014 / 0357523, 2014 / 0357530, 2014 / 0364333, and 2014 / 0377868, all of which are incorporated herein by reference in their entirety.

[0114] In some cases, the heterologous polypeptide (fusion partner) provides subcellular localization. That is, the heterologous polypeptide comprises a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence that retains the fusion protein outside the nucleus, e.g., a nuclear export sequence (NES), a sequence that retains the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an ER retention signal, etc.). In some embodiments, the CRISPR-Cas effector fusion polypeptide does not comprise an NLS (which may be advantageous, for example, when the target nucleic acid is RNA present in the cytosol) so that the protein is not targeted to the nucleus. In some embodiments, the heterologous polypeptide can be provided with a tag (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag, etc.) to facilitate tracking and / or purification (i.e., the heterologous polypeptide is detectably labeled).

[0115] In some cases, the CRISPR-Cas effector protein (e.g., a wild-type CRISPR-Cas effector protein, a variant CRISPR-Cas effector protein, a fusion CRISPR-Cas effector protein, a dCRISPR-Cas effector protein, etc.) comprises (is fused to) a nuclear localization signal (NLS) (e.g., in some cases, two or more, three or more, four or more, or five or more NLSs). Thus, in some cases, the CRISPR-Cas effector polypeptide comprises one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some cases, the one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) the N-terminus and / or C-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is located at the N-terminus and an NLS is located at the C-terminus.

[0116] In some cases, the CRISPR-Cas effector protein (e.g., a wild-type CRISPR-Cas effector protein, a variant CRISPR-Cas effector protein, a fusion CRISPR-Cas effector protein, a dCRISPR-Cas effector protein, etc.) comprises (is fused to) one to ten NLSs (e.g., one to nine, one to eight, one to seven, one to six, one to five, two to ten, two to nine, two to eight, two to seven, two to six, or two to five NLSs). In some cases, the CRISPR-Cas effector protein (e.g., a wild-type CRISPR-Cas effector protein, a variant CRISPR-Cas effector protein, a fusion CRISPR-Cas effector protein, a dCRISPR-Cas effector protein, etc.) comprises (is fused to) two to five NLSs (e.g., two to four or two to three NLSs).

[0117] Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 14); an NLS from nucleoplasmin (e.g., the sequence nucleoplasmin bipartite NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 16) or RQRRNELKRSP (SEQ ID NO: 17); hRNPA1 M9 NLS with TIFF0007649748000005.tif4128; sequence of the IBB domain from importin-alpha TIFF0007649748000006.tif4130; the sequences of VSRKRPRP (SEQ ID NO: 20) and PPKKARED (SEQ ID NO: 21) of fibroid T protein; the sequence of PQPKKKPL (SEQ ID NO: 22) of human p53; the sequence of SALIKKKKKMAP (SEQ ID NO: 23) of mouse c-abl IV; the sequences of DRLRR (SEQ ID NO: 24) and PKQKKRK (SEQ ID NO: 25) of influenza virus NS1; the sequence of RKLKKKIKKL (SEQ ID NO: 26) of hepatitis virus delta antigen; the sequence of REKKKFLKRR (SEQ ID NO: 27) of mouse Mx1 protein; the sequence of human poly(ADP-ribose) polymerase TIFF0007649748000007.tif4128; and the sequence of the steroid hormone receptor (human) glucocorticoid Included are NLS sequences derived from TIFF0007649748000008.tif4128. Generally, NLS (or multiple NLSs) are strong enough to drive the accumulation of detectable amounts of CRISPR-Cas effector protein in the nucleus of eukaryotic cells.Detection of accumulation in nucleus can be carried out by any suitable technique.For example, detectable marker can be fused with CRISPR-Cas effector protein, thereby making its location in the cell visible.Cell nucleus can also be isolated from cell, and then its contents can be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot or enzyme activity assay.Accumulation in nucleus can also be determined indirectly.

[0118] In some cases, the CRISPR-Cas effector fusion polypeptide contains a "protein transduction domain" or PTD (also known as a CPP—cell-penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD bound to another molecule and / or nanoparticle, which can range from small polar molecules to large macromolecules, facilitates membrane crossing of molecules, for example, moving from the extracellular space to the intracellular space or from the cytosol into an organelle. In some embodiments, the PTD is covalently attached to the amino acid terminus of the polypeptide (e.g., linked to a wild-type CRISPR-Cas effector to generate a fusion protein, or linked to a variant CRISPR-Cas effector protein, e.g., a dCRISPR-Cas effector, a nickase CRISPR-Cas effector, or a fusion CRISPR-Cas effector protein to generate a fusion protein). In some embodiments, the PTD is covalently linked to the carboxyl terminus of the polypeptide (e.g., linked to a wild-type CRISPR-Cas effector to generate a fusion protein, or linked to a variant CRISPR-Cas effector protein, such as a dCRISPR-Cas effector, a nickase CRISPR-Cas effector, or a fusion CRISPR-Cas effector protein to generate a fusion protein). In some cases, the PTD is inserted internally into the CRISPR-Cas effector fusion polypeptide at a suitable insertion site (i.e., not at the N- or C-terminus of the CRISPR-Cas effector fusion polypeptide). In some cases, the subject CRISPR-Cas effector fusion polypeptide comprises (conjugated to, fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, the PTD comprises a nuclear localization signal (NLS) (e.g., optionally two or more, three or more, four or more, or five or more NLSs).Thus, in some cases, the CRISPR-Cas effector fusion polypeptide comprises one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some embodiments, the PTD is covalently linked to a nucleic acid (e.g., a CRISPR-Cas effector guide nucleic acid, a polynucleotide encoding a CRISPR-Cas effector guide nucleic acid, a polynucleotide encoding a CRISPR-Cas effector fusion polypeptide, a donor polynucleotide, etc.). Examples of PTDs include a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT, containing YGRKKRRQRRR (SEQ ID NO: 112)); a polyarginine sequence containing a sufficient number of arginines (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines) for direct entry into cells; a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a 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); polylysine (Wender et al. al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 30); transportan. TIFF0007649748000009.tif11128; and RQIKIWFQNRRMKWKK (SEQ ID NO: 33). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 34), RKKRRQRRR (SEQ ID NO: 35), and arginine homopolymers of 3 to 50 arginine residues. Exemplary PTD domain amino acid sequences include the following: TIFF0007649748000010.tif18128. In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6):371-381). ACPPs contain a polycationic CPP (e.g., Arg9 or "R9") connected to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero, thereby inhibiting cellular attachment and uptake. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesive properties, thus "activating" the ACPP to cross membranes.

[0119] Linkers (e.g., for fusion partners) In some embodiments, the CRISPR-Cas effector protein of interest can be fused to the fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide can have any of a variety of amino acid sequences. Proteins can be joined by a generally flexible spacer peptide, although other chemical linkages are not excluded. Suitable linkers include polypeptides between 4 and 40 amino acids in length, or between 4 and 25 amino acids in length. These linkers can be produced by linking proteins using synthetic, linker-encoding oligonucleotides, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with some degree of flexibility can be used. The linking peptide can have virtually any amino acid sequence, keeping in mind that preferred linkers generally have sequences that result in flexible peptides. The use of small amino acids, such as glycine and alanine, is useful in creating flexible peptides. Creating such sequences is routine for those of skill in the art. A variety of different linkers are commercially available and may be suitable for use.

[0120] Examples of linker polypeptides include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , G.S.G.S.G.S. n (SEQ ID NO: 40), GGSGGS n (SEQ ID NO: 41), and GGGS n (SEQ ID NO: 42), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers. Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 43), GGSGG (SEQ ID NO: 44), GSGSG (SEQ ID NO: 45), GSGGG (SEQ ID NO: 46), GGGSG (SEQ ID NO: 47), GSSSG (SEQ ID NO: 48), and the like. Those skilled in the art will recognize that the design of a peptide conjugated with any desired element can include a linker that is fully or partially flexible, whereby the linker can include a flexible linker as well as one or more moieties that impart a less flexible structure.

[0121] Detectable Label In some cases, the CRISPR-Cas effector polypeptides of the present disclosure comprise a detectable label. Suitable detectable labels and / or moieties capable of providing a detectable signal can include, but are not limited to, enzymes, radioisotopes, members of specific binding pairs, fluorophores, fluorescent proteins, quantum dots, etc.

[0122] Suitable fluorescent proteins include green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2 (12), mRFP1, pocilloporin, Renilla GFP, and Monster. Examples of fluorescent proteins include, but are not limited to, GFP, paGFP, Kaede and kindling proteins, phycobiliproteins, and phycobiliprotein conjugates, including B-phycoerythrin, R-phycoerythrin, and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, and mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). Suitable for use are any of the various fluorescent and chromatic proteins from anthozoan species, such as those described in Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0123] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.

[0124] CRISPR-Cas effector guide RNA A nucleic acid molecule that binds to a CRISPR-Cas effector protein to form a ribonucleoprotein complex (RNP) and targets the complex to a specific location within a target nucleic acid (e.g., target DNA) is referred to herein as a "CRISPR-Cas effector guide RNA" or simply "guide RNA." In some cases, a hybrid DNA / RNA may be made such that the CRISPR-Cas effector guide RNA contains DNA bases in addition to RNA bases, but it should be understood that the term "CRISPR-Cas effector guide RNA" is still used herein to encompass such molecules.

[0125] A CRISPR-Cas effector guide RNA can be said to contain two segments: a targeting segment and a protein-binding segment. The protein-binding segment is also referred to herein as the "constant region" of the guide RNA. The targeting segment of a CRISPR-Cas effector guide RNA contains a nucleotide sequence (guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (target site) within a target nucleic acid (e.g., a target dsDNA, 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 subject CRISPR-Cas effector guide RNA may contain two complementary stretches of nucleotides that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex). Site-specific binding and / or cleavage of a target nucleic acid (e.g., genomic DNA, dsDNA, RNA, etc.) can occur at a location (e.g., a target sequence at a target locus) determined by base-pairing complementarity between the CRISPR-Cas effector guide RNA (the guide sequence of the CRISPR-Cas effector guide RNA) and the target nucleic acid.

[0126] The CRISPR-Cas effector guide RNA and CRISPR-Cas effector protein (e.g., a wild-type CRISPR-Cas effector protein, a variant CRISPR-Cas effector protein, a fusion CRISPR-Cas effector polypeptide, etc.) form a complex (e.g., associated via non-covalent interactions). The CRISPR-Cas effector guide RNA provides target specificity to the complex by including a targeting segment that includes a guide sequence (a nucleotide sequence complementary to the sequence of a target nucleic acid). The CRISPR-Cas effector protein of the complex provides site-specific activity (e.g., cleavage activity provided by the CRISPR-Cas effector protein and / or activity provided by a fusion partner, in the case of a fusion CRISPR-Cas effector protein). In other words, the CRISPR-Cas effector protein is guided to a target nucleic acid sequence (e.g., a target sequence) by its association with the CRISPR-Cas effector guide RNA.

[0127] The "guide sequence," also referred to as the "targeting sequence" of a CRISPR-Cas effector guide RNA, can be modified such that the CRISPR-Cas effector guide RNA can target a CRISPR-Cas effector protein (e.g., a naturally occurring CRISPR-Cas effector protein, a fusion CRISPR-Cas effector polypeptide, etc.) to any desired sequence in any desired target nucleic acid, with the exception that a PAM sequence (e.g., as described herein) can be taken into account. Thus, for example, a CRISPR-Cas effector guide RNA can have a guide sequence that is complementary to (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, a chromosomal sequence, a eukaryotic RNA, etc.).

[0128] Guide sequence of CRISPR-Cas effector guide RNA The target CRISPR-Cas effector guide RNA comprises a guide sequence (i.e., targeting sequence), which is a nucleotide sequence complementary to a sequence (target site) in the target nucleic acid. In other words, the guide sequence of the CRISPR-Cas effector guide RNA can interact with the target nucleic acid (e.g., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA)) in a sequence-specific manner by hybridization (i.e., base pairing). The guide sequence of the CRISPR-Cas effector guide RNA can be modified (e.g., by genetic engineering) / designed (e.g., taking into account PAM, for example, when targeting a dsDNA target) to hybridize with any desired target sequence within the target nucleic acid (e.g., eukaryotic target nucleic acid such as genomic DNA).

[0129] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or greater (e.g., 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100%.

[0130] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over the seven contiguous 3'-most nucleotides of the target site of the target nucleic acid.

[0131] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or greater (e.g., 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17 or more (e.g., 18 or greater, 19 or greater, 20 or greater, 21 or greater, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17 or more (e.g., 18 or greater, 19 or greater, 20 or greater, 21 or greater, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17 or more (e.g., 18 or greater, 19 or greater, 20 or greater, 21 or greater, 22 or greater) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 17 or more (e.g., 18 or greater, 19 or greater, 20 or greater, 21 or greater, 22 or greater) contiguous nucleotides.

[0132] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or greater (e.g., 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19 or more (e.g., 20% or greater, 21% or greater, 22% or greater) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19 or more (e.g., 20% or greater, 21% or greater, 22% or greater) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides.

[0133] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or greater (e.g., 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 17-25 contiguous nucleotides.

[0134] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or greater (e.g., 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 19-25 contiguous nucleotides.

[0135] In some cases, the guide sequence has a length in the range of 17-30 nucleotides (nt) (e.g., 17-25, 17-22, 17-20, 19-30, 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some cases, the guide sequence has a length in the range of 17-25 nucleotides (nt) (e.g., 17-22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt). In some cases, the guide sequence has a length of 17 nt or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 19 nt or more (e.g., 20 nt or more, 21 nt or more, or 22 nt or more; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 17 nt. In some cases, the guide sequence has a length of 18 nt. In some cases, the guide sequence has a length of 19 nt. In some cases, the guide sequence has a length of 20 nt. In some cases, the guide sequence has a length of 21 nt. In some cases, the guide sequence has a length of 22 nt. In some cases, the guide sequence has a length of 23 nt.

[0136] In some cases, the guide sequence (also referred to as a "spacer sequence") has a length of 15 to 50 nucleotides (e.g., 15 nucleotides (nt) to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 35 nt, 35 nt to 40 nt, 40 nt to 45 nt, or 45 nt to 50 nt).

[0137] Protein-binding segments of CRISPR-Cas effector guide RNAs The protein binding segment ("constant region") of the target CRISPR-Cas effector guide RNA interacts with CRISPR-Cas effector protein. The CRISPR-Cas effector guide RNA guides the bound CRISPR-Cas effector protein to a specific nucleotide sequence in the target nucleic acid via the above-mentioned guide sequence. The protein binding segment of the CRISPR-Cas effector guide RNA can comprise two stretches of nucleotides that are complementary to each other and hybridize to form a double-stranded RNA duplex (dsRNA duplex). Thus, in some cases, the protein binding segment comprises a dsRNA duplex.

[0138] In some cases, the dsRNA duplex region comprises a range of 5 to 25 base pairs (bp) (e.g., 5 to 22, 5 to 20, 5 to 18, 5 to 15, 5 to 12, 5 to 10, 5 to 8, 8 to 25, 8 to 22, 8 to 18, 8 to 15, 8 to 12, 12 to 25, 12 to 22, 12 to 18, 12 to 15, 13 to 25, 13 to 22, 13 to 18, 13 to 15, 14 to 25, 14 to 22, 14 to 18, 14 to 15, 15 to 25, 15 to 22, 15 to 18, 17 to 25, 17 to 22, or 17 to 18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the dsRNA duplex region comprises a range of 6 to 15 base pairs (bp) (e.g., 6 to 12, 6 to 10, or 6 to 8 bp, e.g., 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the duplex region comprises 5 or more bp (e.g., 6 or more, 7 or more, or 8 or more bp). In some cases, the duplex region comprises 6 or more bp (e.g., 7 or more, or 8 or more bp). In some cases, not all nucleotides in the duplex region are paired, and thus the duplex-forming region may comprise a bulge. As used herein, the term "bulge" is used to refer to a stretch of nucleotides (which may be a single nucleotide) that do not contribute to the double-stranded duplex and are surrounded 5' and 3' by contributing nucleotides, and as such, the bulge is considered part of the duplex region. In some cases, the dsRNA comprises one or more bulges (e.g., two or more, three or more, four or more bulges). In some cases, the dsRNA duplex contains two or more bulges (e.g., three or more, four or more bulges). In some cases, the dsRNA duplex contains one to five bulges (e.g., one to four, one to three, two to five, two to four, or two to three bulges).

[0139] Thus, in some cases, stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70% to 100% complementarity to each other (e.g., 75% to 100%, 80% to 10%, 85% to 100%, 90% to 100%, 95% to 100% complementarity). In some cases, stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70% to 100% complementarity to each other (e.g., 75% to 100%, 80% to 10%, 85% to 100%, 90% to 100%, 95% to 100% complementarity). In some cases, stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 85% to 100% complementarity to each other (e.g., 90% to 100%, 95% to 100% complementarity). In some cases, the stretches of nucleotides that hybridize to each other to form the dsRNA duplex have 70%-95% complementarity to each other (e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95% complementarity).

[0140] In other words, in some embodiments, dsRNA duplexes comprising two stretches of nucleotides have 70% to 100% complementarity with each other (e.g., 75% to 100%, 80% to 10%, 85% to 100%, 90% to 100%, 95% to 100% complementarity). In some cases, dsRNA duplexes comprising two stretches of nucleotides have 85% to 100% complementarity with each other (e.g., 90% to 100%, 95% to 100% complementarity). In some cases, dsRNA duplexes comprising two stretches of nucleotides have 70% to 95% complementarity with each other (e.g., 75% to 95%, 80% to 95%, 85% to 95%, 90% to 95% complementarity).

[0141] The duplex region of a subject CRISPR-Cas effector guide RNA can include one or more (one, two, three, four, five, etc.) mutations relative to the naturally occurring duplex region. For example, in some cases, base pairs can be maintained, but the nucleotides contributed to the base pair from each segment can be different. In some cases, the duplex region of a subject CRISPR-Cas effector guide RNA includes more base pairs, fewer base pairs, smaller bulges, larger bulges, fewer bulges, more bulges, or any convenient combination thereof, compared to the naturally occurring duplex region (of the naturally occurring CRISPR-Cas effector guide RNA).

[0142] Examples of various Cas9 guide RNAs can be found in the art, and in some cases, modifications similar to those introduced into Cas9 effector guide RNAs can also be introduced into the CRISPR-Cas effector guide RNAs of the present disclosure (e.g., mutations to the dsRNA duplex region, extensions at the 5' or 3' ends to add stabilization to provide for interaction with another protein, etc.). For example, Jinek et al.,Science.2012 Aug 17;337(6096):816-21, Chylinski et al.,RNA Biol.2013 May;10(5):726-37, Ma et al.,Biomed Res Int.2013;2013:270805, Hou et al.,Proc Natl Acad Sci US A.2013 Sep 24;110(39):15644-9, Jinek et al.,Elife.2013;2:e00471, Pattanayak et al.,Nat Biotechnol.2013 Sep;31(9):839-43, Qi et al,Cell.2013 Feb 28;152(5):1173-83, Wang et al.,Cell.2013 May 9;153(4):910-8, Auer et al.,Genome Res.2013 Oct 31, Chen et al.,Nucleic Acids Res.2013 Nov 1;41(20):e19, Cheng et al.,Cell Res.2013 Oct;23(10):1163-71, Cho et al.,Genetics.2013 Nov;195(3):1177-80, DiCarlo et al., Nucleic Acids Res.2013 Apr;41(7):4336-43, Dickinson et al., Nat Methods.2013 Oct;10(10):1028-34, Ebina et al., Sci Rep.2013;3:2510, Fujii et al. al,Nucleic Acids Res.2013 Nov 1;41(20):e187, Hu et al.,Cell Res.2013 Nov;23(11):1322-5, Jiang et al.,Nucleic Acids Res.2013 Nov 1;41(20):e188、Larson et al.,Nat Protoc.2013 Nov;8(11):2180-96、Mali et.at.,Nat Methods.2013 Oct;10(10):957-63、Nakayama et al.,Genesis.2013 Dec;51(12):835-43、Ran et al.,Nat Protoc.2013 Nov;8(11):2281-308、Ran et al.,Cell.2013 Sep 12;154(6):1380-9、Upadhyay et al.,G3(Bethesda).2013 Dec 9;3(12):2233-8、Walsh et al.,Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15514-5、Xie et al.,Mol Plant.2013 Oct 9、Yang et al.,Cell.2013 Sep 12;154(6):1370-9、Briner et al.,Mol Cell.2014 Oct 23;56(2):333-9, and U.S. Patents and Patent Applications Nos. 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, 8,697,359, 2014 / 0068797, 2014 / 0170753, 2014 / 0179006, 2014 / 0179770, 2014 / 0186843, and 2014 / 018691 No. 9, No. 2014 / 0186958, No. 2014 / 0189896, No. 2014 / 0227787, No. 2014 / 0234972, No. 2014 / 0242664, No. 2014 / 0242699, No. 2014 / 0242700, No. 201 No. 4 / 0242702, No. 2014 / 0248702, No. 2014 / 0256046, No. 2014 / 0273037, No. 2014 / 0273226, No. 2014 / 0273230, No. 2014 / 0273231, No. 2014 / 02732 No. 32, No. 2014 / 0273233, No. 2014 / 0273234, No. 2014 / 0273235, No. 2014 / 0287938, No. 2014 / 0295556, No. 2014 / 0295557, No. 2014 / 0298547, No. 20 No. 14 / 0304853, No. 2014 / 0309487, No. 2014 / 0310828, No. 2014 / 0310830, No. 2014 / 0315985, No. 2014 / 0335063, No. 2014 / 0335620, No. 2014 / 0342 456, 2014 / 0342457, 2014 / 0342458, 2014 / 0349400, 2014 / 0349405, 2014 / 0356867, 2014 / 0356956, 2014 / 0356958, 2014 / 0356959, 2014 / 0357523, 2014 / 0357530, 2014 / 0364333, and 2014 / 0377868, all of which are incorporated by reference in their entirety.

[0143] An example of a constant region suitable for inclusion in a CRISPR-Cas effector guide RNA is provided in Figure 7 (e.g., T is substituted with U). A CRISPR-Cas effector guide RNA can include a constant region having one to five nucleotide substitutions compared to any one of the nucleotide sequences shown in Figure 7. A CRISPR-Cas effector guide RNA of the present disclosure can include a constant region having at least 85%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% nucleotide sequence identity to any one of the crRNA nucleotide sequences shown in Figure 7 (e.g., T is substituted with U).

[0144] The nucleotide sequence (in which T is substituted with U) can be combined with a selected spacer sequence (the spacer sequence comprising a target nucleic acid binding sequence ("guide sequence")) of 15-50 nucleotides (e.g., 15 nucleotides (nt) to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 35 nt, 35 nt to 40 nt, 40 nt to 45 nt, or 45 nt to 50 nt in length). In some cases, the spacer sequence is 35-38 nucleotides in length. For example, any one of the nucleotide sequences (in which T is substituted with U) shown in FIG. 7 can be included in a guide RNA comprising an (N)n constant region, where N is any nucleotide and n is an integer between 15 and 50 (e.g., 15-20, 20-25, 25-30, 30-35, 35-38, 35-40, 40-45, or 45-50).

[0145] CRISPR-CAS effector system The present disclosure provides a CRISPR-Cas effector system, which includes (a) a CRISPR-Cas effector polypeptide and a CRISPR-Cas effector guide RNA of the present disclosure, (b) a CRISPR-Cas effector polypeptide, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid of the present disclosure, (c) a CRISPR-Cas effector fusion polypeptide and a CRISPR-Cas effector guide RNA of the present disclosure, and (d) a CRISPR-Cas effector fusion polypeptide, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid of the present disclosure. (e) an mRNA encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a CRISPR-Cas effector guide RNA; (f) an mRNA encoding a CRISPR-Cas effector polypeptide of the present disclosure, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (g) an mRNA encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a CRISPR-Cas effector guide RNA. (h) mRNA encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (i) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (j) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; (k) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (l) a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA;and a recombinant expression vector comprising a nucleotide sequence encoding a donor template nucleic acid; (m) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (n) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (o) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (p) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; The present invention can include a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (q) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a second CRISPR-Cas effector guide RNA; or (r) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a second CRISPR-Cas effector guide RNA, or a variation of one of (a) through (r).

[0146] nucleic acid The present disclosure provides one or more nucleic acids comprising one or more of a donor polynucleotide sequence, a nucleotide sequence encoding a CRISPR-Cas effector polypeptide (e.g., a wild-type CRISPR-Cas effector protein, a nickase CRISPR-Cas effector protein, a dCRISPR-Cas effector protein, a fusion CRISPR-Cas effector protein, etc.), a CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide. The present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide. The present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide. The present disclosure provides a recombinant expression vector comprising (a) a nucleotide sequence encoding a CRISPR-Cas effector polypeptide, and (b) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA(s). The present disclosure provides recombinant expression vectors comprising (a) a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide, and (b) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA(s). In some cases, the nucleotide sequence encoding the CRISPR-Cas effector protein and / or the nucleotide sequence encoding the CRISPR-Cas effector guide RNA are operably linked to a promoter that is operable in a selected cell type (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a primate cell, a rodent cell, a human cell, etc.).

[0147] In some cases, the nucleotide sequence encoding the CRISPR-Cas effector polypeptide of the present disclosure is codon-optimized. This type of optimization can involve altering the CRISPR-Cas effector-encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while still encoding the same protein. Thus, the codons can be altered, but the encoded protein remains unchanged. For example, if the intended target cell is a human cell, a human-codon-optimized CRISPR-Cas effector-encoding nucleotide sequence can be used. As another non-limiting example, if the intended host cell is a mouse cell, a mouse-codon-optimized CRISPR-Cas effector-encoding nucleotide sequence can be generated. As another non-limiting example, if the intended host cell is a plant cell, a plant-codon-optimized CRISPR-Cas effector-encoding nucleotide sequence can be generated. As another non-limiting example, if the intended host cell is an insect cell, an insect-codon-optimized CRISPR-Cas effector-encoding nucleotide sequence can be generated.

[0148] The present disclosure provides one or more recombinant expression vectors (optionally in different recombinant expression vectors, and optionally in the same recombinant expression vector) that include: (i) a nucleotide sequence of a donor template nucleic acid, where the donor template includes a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome); (ii) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA (e.g., operably linked to a promoter operable in a target cell, such as a eukaryotic cell) that hybridizes to a target sequence at a target locus in the targeted genome; and (iii) a nucleotide sequence encoding a CRISPR-Cas effector protein (e.g., operably linked to a promoter operable in a target cell, such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors (optionally in different recombinant expression vectors, and optionally in the same recombinant expression vector) that include: (i) a nucleotide sequence of a donor template nucleic acid, where the donor template includes a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome); and (ii) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA (e.g., operably linked to a promoter operable in a target cell, such as a eukaryotic cell) that hybridizes to a target sequence at a target locus in the targeted genome. The present disclosure provides one or more recombinant expression vectors (optionally in different recombinant expression vectors, and optionally in the same recombinant expression vector) that include (i) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA (e.g., operably linked to a promoter operable in a target cell, such as a eukaryotic cell) that hybridizes to a target sequence at a target locus in a targeted genome, and (ii) a nucleotide sequence encoding a CRISPR-Cas effector protein (e.g., operably linked to a promoter operable in a target cell, such as a eukaryotic cell).

[0149] Suitable expression vectors include viral expression vectors (e.g., 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 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655), adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997,Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997,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 WO93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828, Mendelson et al., Virol. (1988) 166:154-165, and Flotte et al. al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997, Takahashi et al., J Virol 73:7812 7816, 1999), retroviral vectors (e.g., viral vectors based on murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). In some cases, the recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some cases, the recombinant expression vector of the present disclosure is a recombinant lentiviral vector. In some cases, the recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[0150] Depending on the host / vector system utilized, any of a number of suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., may be used in the expression vector.

[0151] In some embodiments, the nucleotide sequence encoding the CRISPR-Cas effector guide RNA is operably linked to a regulatory element, e.g., a transcriptional control element such as a promoter. In some embodiments, the nucleotide sequence encoding the CRISPR-Cas effector protein or CRISPR-Cas effector fusion polypeptide is operably linked to a regulatory element, e.g., a transcriptional repression element such as a promoter.

[0152] 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., 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, such as hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cells, bone marrow (BM) CD34(+) cells, etc.).

[0153] Non-limiting examples of eukaryotic promoters (promoters functional in eukaryotic cells) include EF1α, those derived from immediate early cytomegalovirus (CMV), herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) derived from retroviruses, and mouse metallothionein-I. Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. Expression vectors may also contain a ribosome binding site for translation initiation and a transcription terminator. Expression vectors may also contain appropriate sequences for amplifying expression. Expression vectors may also contain nucleotide sequences encoding protein tags (e.g., 6xHis tags, hemagglutinin tags, fluorescent proteins, etc.) that can be fused with CRISPR-Cas effector proteins, thus resulting in fusion CRISPR-Cas effector polypeptides.

[0154] In some embodiments, the nucleotide sequence encoding the CRISPR-Cas effector guide RNA and / or the CRISPR-Cas effector fusion polypeptide is operably linked to an inducible promoter. In some embodiments, the nucleotide sequence encoding the CRISPR-Cas effector guide RNA and / or the CRISPR-Cas effector fusion protein is operably linked to a constitutive promoter.

[0155] The promoter may be a constitutively active promoter (i.e., a promoter that is constitutively active / "ON" state), an inducible promoter (i.e., a promoter whose active / "ON" or inactive / "OFF" state is controlled by an external stimulus, e.g., a particular temperature, compound, or the presence of a protein), a spatially constrained promoter (i.e., a transcriptional control element, enhancer, etc.) (e.g., a tissue-specific promoter, cell-type-specific promoter, etc.), or a temporally constrained promoter (i.e., the promoter is "ON" or "OFF" state during a particular stage of embryonic development or during a particular stage of a biological process, e.g., the hair follicle cycle in mice).

[0156] Suitable promoters may be derived from viruses, and are therefore referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. 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, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31 (17)), the human H1 promoter (H1), and the like.

[0157] In some cases, the nucleotide sequence encoding the CRISPR-Cas effector guide RNA is operably linked to (under the control of) a promoter operable in eukaryotic cells (e.g., a U6 promoter, an enhanced U6 promoter, an H1 promoter, etc.). As will be understood by those skilled 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 Pol III promoter, the RNA may need to be mutated if several Ts are in a row (encoding Us in the RNA). This is because a string of Ts (e.g., five Ts) in DNA can act as a terminator for polymerase III (Pol III). Therefore, to ensure transcription of the guide RNA in eukaryotic cells, it may sometimes be necessary to modify the sequence encoding the guide RNA to eliminate the execution of Ts. In some cases, the nucleotide sequence encoding the CRISPR-Cas effector protein (e.g., a wild-type CRISPR-Cas effector protein, a nickase CRISPR-Cas effector protein, a dCRISPR-Cas effector protein, a fusion CRISPR-Cas effector protein, etc.) is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EF1α promoter, an estrogen receptor-regulated promoter, etc.).

[0158] Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose-inducible promoter, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc. Thus, inducible promoters can be regulated by molecules including, but not limited to, doxycycline, estrogen and / or estrogen analogs, IPTG, etc.

[0159] Inducible promoters suitable for use include any inducible promoter described herein or known to those of skill in the art. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems including tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, gaecdysone receptor, and steroid / retinoic acid receptor-regulated promoters). These promoters include, but are not limited to, chemically / biochemically regulated and physically regulated promoters such as promoters from the id / thyroid receptor superfamily, metal-regulated promoters (e.g., promoters derived from metallothioneins (proteins that bind and sequester metal ions) from yeast, mouse, and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0160] In some cases, the promoter is a spatially constrained promoter (i.e., a cell-type specific promoter, a tissue-specific promoter, etc.), such that in a multicellular organism, the promoter is active (i.e., "ON") in a specific subset of cells. A spatially constrained promoter may also be referred to as an enhancer, a transcriptional control element, a regulatory sequence, etc. Any convenient spatially constrained promoter can be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell, prokaryotic cell).

[0161] In some cases, the promoter is a reversible promoter.Suitable reversible promoters, including reversibly inducible promoters, are known in the art.Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes.The modification of a reversible promoter derived from a first organism (e.g., the first organism is a prokaryote and the second organism is a eukaryote, the first organism is a eukaryote and the second organism is a prokaryote, etc.) for use in a second organism is well known in the art. Examples of such reversible promoters and systems based on such reversible promoters but also including additional regulatory proteins include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (AlcR)), tetracycline-regulated promoters (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogen-associated regulated promoters (e.g., salicylic acid-regulated promoter, ethylene-regulated promoter, benzothiadiazole-regulated promoter, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0162] Methods for introducing nucleic acids (e.g., nucleic acids comprising donor polynucleotide sequences, one or more nucleic acids encoding CRISPR-Cas effector proteins and / or CRISPR-Cas effector guide RNAs, etc.) into host cells are known in the art, and any convenient method can be used to introduce nucleic acids (e.g., expression constructs) into cells. Suitable methods include, for example, viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0163] Introduction of the recombinant expression vector into cells can occur in any culture medium and under any culture conditions that promote cell survival. Introduction of the recombinant expression vector into target cells can be performed in vivo or ex vivo. Introduction of the recombinant expression vector into target cells can be performed in vitro.

[0164] In some embodiments, CRISPR-Cas effector protein can be provided as RNA.RNA can be provided by direct chemical synthesis, or can be transcribed in vitro from DNA (for example, encoding CRISPR-Cas effector protein).Once synthesized, RNA can be introduced into cells by any of the well-known techniques for introducing nucleic acid into cells (for example, microinjection, electroporation, transfection, etc.).

[0165] Nucleic acids can be provided to cells using well-developed transfection techniques (see, e.g., Angel and Yanik (2010) PLoS ONE 5(7):e11756), as well as commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA transfection kits from Stemgent, and TransIT®-mRNA transfection kits from Mirus Bio LLC. See also Beumer et al. (2008) PNAS 105(50):19821-19826.

[0166] The vector can be directly provided to target host cells.In other words, the cell is contacted with a vector containing the target nucleic acid (for example, the recombinant expression vector that has a donor template sequence and encodes a CRISPR-Cas effector guide RNA, the recombinant expression vector that encodes a CRISPR-Cas effector protein, etc.), and the vector is thereby taken up by the cell.The method for contacting the cell with the nucleic acid vector that is a plasmid includes electroporation, calcium chloride transfection, microinjection, and lipofection, which are well known in the art.In the case of viral vector delivery, the cell can be contacted with a viral particle that contains the target viral expression vector.

[0167] Retroviruses, such as lentiviruses, are suitable for use in the methods of the present disclosure. Commonly used retroviral vectors are "defective," i.e., unable to produce viral proteins necessary for productive infection. Rather, vector replication requires growth in a packaging cell line. To produce viral particles containing a nucleic acid of interest, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by the packaging cell line. Different packaging cell lines provide different envelope proteins (ecotropic, amphotropic, or xenotropic) that are incorporated into the capsid, and this envelope protein determines the specificity of the viral particle to the cell (ecotropic for mouse and rat, amphotropic for most mammalian cell types, including human, dog, and mouse, and xenotropic for most mammalian cell types except mouse cells). Using an appropriate packaging cell line can ensure that the cell is targeted by the packaged viral particle. Methods for introducing a subject expression vector into a packaging cell line and for harvesting viral particles produced by the packaging line are well known in the art. Nucleic acids can also be introduced by direct microinjection (e.g., injection of RNA).

[0168] The vector used to provide a nucleic acid encoding a CRISPR-Cas effector guide RNA and / or a CRISPR-Cas effector polypeptide to a target host cell can include a suitable promoter for driving expression of the nucleic acid of interest, i.e., transcriptional activation. In other words, in some cases, the nucleic acid of interest is operably linked to a promoter. This may include a ubiquitously acting promoter, such as the CMV-β-actin promoter, or an inducible promoter, such as a promoter that is active in a specific cell population or responds to the presence of a drug such as tetracycline. By transcriptional activation, it is intended that transcription is increased 10-fold, 100-fold, or more usually 1000-fold over the basal level in the target cell. In addition, the vector used to provide a nucleic acid encoding a CRISPR-Cas effector guide RNA and / or a CRISPR-Cas effector protein to a cell can include a nucleic acid sequence encoding a selectable marker in the target cell to identify cells that have incorporated the CRISPR-Cas effector guide RNA and / or the CRISPR-Cas effector protein.

[0169] A nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide or a CRISPR-Cas effector fusion polypeptide may optionally be RNA. Thus, a CRISPR-Cas effector fusion protein can be introduced into a cell as RNA. Methods for introducing RNA into a cell are known in the art and may include, for example, direct injection, transfection, or any other method used for introducing DNA. A CRISPR-Cas effector protein may alternatively be provided to a cell as a polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases the solubility of the product. This domain may be linked to the polypeptide through a defined protease cleavage site, e.g., a TEV sequence, that is cleaved by TEV protease. The linker may also contain one or more flexible sequences, e.g., 1 to 10 glycine residues. In some embodiments, cleavage of the fusion protein is performed in a buffer that maintains the solubility of the product, e.g., in the presence of 0.5 to 2 M urea, in the presence of a solubility-enhancing polypeptide and / or polynucleotide, etc. Domains of interest include endosomal degradative domains, such as influenza HA domains, and other polypeptides that aid in production, such as IF2 domains, GST domains, GRPE domains, etc. Polypeptides can be formulated for improved stability. For example, peptides can be PEGylated, where the polyethyleneoxy groups provide improved longevity in the bloodstream.

[0170] Additionally or alternatively, the CRISPR-Cas effector polypeptides of the present disclosure can be fused to a polypeptide permeation domain to facilitate cellular uptake. Several permeation domains are known in the art and can be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. For example, a permeation peptide can have the amino acid sequence The penetrating peptide may be derived from the third alpha helix of the Drosophila melanogaster transcription factor Antennapedia, called penetratin, containing TIFF0007649748000011.tif4128. As another example, the penetrating peptide may contain the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of the native tat protein. Other penetrating domains include poly-arginine motifs, such as the region of amino acids 34-56 of the HIV-1 rev protein, nona-arginine, octa-arginine, etc. (See, e.g., Futaki et al. (2003) Curr Protein Pept Sci. 2003 Apr;4(2):87-9 and 446, and Wender et al. (2000) Proc. Natl. Acad. Sci. USA 2000 Nov. 21;97(24):13003-8, published U.S. patent applications 2003 / 0220334, 2003 / 0083256, 2003 / 0032593, and 2003 / 0022831, which are specifically incorporated by reference herein for their teachings of translocating peptides and peptoids.) The nona-arginine (R9) sequence is one of the more efficient PTDs characterized (Wender et al. 2000, Uemura et al. 2002). The site at which the fusion is made may be selected in order to optimize the biological activity, secretion, or binding characteristics of the polypeptide. Optimal sites can be determined by routine experimentation.

[0171] The CRISPR-Cas effector polypeptides of the disclosure can be produced in vitro, or by eukaryotic cells, or by prokaryotic cells, which may be further processed by unfolding, e.g., heat denaturation, dithiothreitol reduction, etc., and further refolded using methods known in the art.

[0172] Modifications of interest that do not alter the primary sequence include chemical derivatization of the polypeptide, such as acylation, acetylation, carboxylation, amidation, etc. Also included are glycosylation modifications, such as those made by modifying the glycosylation pattern of a polypeptide during its synthesis and processing or during further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. Also encompassed are sequences having phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0173] Also suitable for inclusion in embodiments of the present disclosure are nucleic acids (e.g., encoding CRISPR-Cas effector guide RNAs, encoding CRISPR-Cas effector fusion proteins, etc.) and proteins (e.g., CRISPR-Cas effector fusion proteins derived from wild-type or variant proteins) that have been modified using conventional molecular biology techniques and synthetic chemistry to improve their resistance to proteolysis, alter target sequence specificity, optimize solubility properties, modify protein activity (e.g., transcriptional regulatory activity, enzymatic activity, etc.), or make them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acid residues. D-amino acids can be substituted for some or all amino acid residues.

[0174] The CRISPR-Cas effector polypeptides of the present disclosure can be prepared by in vitro synthesis using conventional methods known in the art. Various commercially available synthesizers are available, such as automated synthesizers from Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, natural amino acids can be substituted with unnatural amino acids. The specific sequence and preparation method are determined by convenience, economy, required purity, etc.

[0175] If desired, various groups may be introduced into the peptide during synthesis or expression, allowing for linkage to other molecules or surfaces. Thus, cysteine ​​can be used to create thioethers, histidine for linkage to metal ion complexes, carboxyl groups for amide or ester formation, amino groups for amide formation, etc.

[0176] The CRISPR-Cas effector polypeptides of the present disclosure may also be isolated and purified according to conventional methods of recombinant synthesis. A lysate of the expression host may be prepared, and the lysate may be purified using high-performance liquid chromatography (HPLC), exclusion chromatography, gel electroporation, affinity chromatography, or other purification techniques. For the most part, the compositions used will represent 20% or more by weight of the desired product, more usually 75% or more, preferably 95% or more, and for therapeutic purposes, usually 99.5% by weight, relative to contaminants associated with the method of preparation and purification of the product. Percentages are typically based on total protein. Thus, in some cases, the CRISPR-Cas effector polypeptides or CRISPR-Cas effector fusion polypeptides of the present disclosure are at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-CRISPR-Cas effector proteins, other macromolecules, etc.).

[0177] To induce cleavage or any desired modification in a target nucleic acid (e.g., genomic DNA) or to induce any desired modification in a polypeptide associated with the target nucleic acid, the CRISPR-Cas effector guide RNA and / or CRISPR-Cas effector polypeptide and / or donor template sequence of the present disclosure, whether introduced as a nucleic acid or as a polypeptide, are provided to the cell for a period of about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period of about 30 minutes to about 24 hours, which may be repeated at a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency of about every day to about every 4 days. The agent(s) may be provided to the cells of the subject one or more times, e.g., 1, 2, 3, or 4 or more times, and the cells are incubated with the agent(s) for a period of time, e.g., 16-24 hours, after each contact event, after which time the medium is replaced with fresh medium and the cells are further cultured.

[0178] When two or more different targeting complexes (e.g., two different CRISPR-Cas effector guide RNAs that are complementary to different sequences within the same or different target nucleic acid) are provided to a cell, the complexes can be provided simultaneously (e.g., as two polypeptides and / or nucleic acids) or delivered simultaneously. Alternatively, they can be provided sequentially, e.g., a targeting complex is provided first, followed by a second targeting complex, or vice versa.

[0179] To improve delivery of DNA vectors to target cells, DNA can be protected from damage and its entry into cells, for example, by using lipoplexes and polyplexes. Therefore, in some cases, the nucleic acid of the present disclosure (e.g., the recombinant expression vector of the present disclosure) can be coated with lipids in organized structures such as micelles or liposomes. When the organized structure is complexed with DNA, it is called a lipoplex. There are three types of lipids: anionic (negatively charged), neutral, or cationic (positively charged). Lipoplexes utilizing cationic lipids have proven useful for gene transfer. Due to their positive charge, cationic lipids naturally complex with negatively charged DNA. Also, as a result of their charge, they interact with cell membranes. Endocytosis of the lipoplex then occurs, releasing the DNA into the cytoplasm. The cationic lipids protect the DNA from degradation by the cell.

[0180] Polymer-DNA complexes are called polyplexes. Most polyplexes are composed of cationic polymers, and their production is regulated by ionic interactions. One major difference between the way polyplexes and lipoplexes work is that polyplexes cannot release their DNA payload into the cytoplasm; therefore, cotransfection with an endosomolytic agent, such as an inactivated adenovirus, which dissolves endosomes created during endocytosis, is essential. However, this is not always the case; polymers such as polyethyleneimine, as well as chitosan and trimethylchitosan, have their own methods of disrupting endosomes.

[0181] Dendrimers, highly branched macromolecules with spherical shapes, can also be used to genetically modify stem cells. The surface of dendrimer particles can be functionalized to modify their properties. In particular, cationic dendrimers (i.e., those with a positive surface charge) can be constructed. When in the presence of genetic material such as a DNA plasmid, charge complementarity results in a transient association of the nucleic acid with the cationic dendrimer. Upon reaching its destination, the dendrimer-nucleic acid complex can be taken up by the cell by endocytosis.

[0182] In some cases, a nucleic acid (e.g., an expression vector) of the present disclosure comprises an insertion site for a guide sequence of interest. For example, a nucleic acid can comprise an insertion site for a guide sequence of interest, where the insertion site is immediately adjacent to a nucleotide sequence encoding a portion of the CRISPR-Cas effector guide RNA that remains unchanged when the guide sequence is changed to hybridize to a desired target sequence (e.g., a sequence that contributes to the CRISPR-Cas effector binding aspect of the guide RNA, e.g., a sequence that contributes to the dsRNA duplex(es) of the CRISPR-Cas effector guide RNA; this portion of the guide RNA may also be referred to as the "scaffold" or "constant region" of the guide RNA). Thus, in some cases, a nucleic acid of interest (e.g., an expression vector) comprises a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, except that the portion encoding the guide sequence portion of the guide RNA is the insertion sequence (insertion site). An insertion site is any nucleotide sequence used for insertion of a desired sequence. "Insertion sites" for use in various techniques are known to those of skill in the art, and any convenient insertion site can be used. The insertion site can be for any method for manipulating nucleic acid sequences. For example, in some cases, the insertion site is a multiple cloning site (MCS) (e.g., a site containing one or more restriction enzyme recognition sequences), a site for ligation-independent cloning, a site for recombination-based cloning (e.g., att site-based recombination), a nucleotide sequence recognized by CRISPR / Cas (e.g., Cas9)-based technologies, etc.

[0183] The insertion site can be any desired length and can depend on the type of insertion site (e.g., whether (and how many) the site includes one or more restriction enzyme recognition sequences, whether the site includes a target site for a CRISPR / Cas protein, etc.). In some cases, the insertion site for the nucleic acid of interest is 3 or more nucleotides (nt) in length (e.g., 5 or more, 8 or more, 10 or more, 15 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 25 or more, or 30 or more nt in length). In some cases, the length of the insertion site of the nucleic acid of interest is in the range of 2 to 50 nucleotides (nt) (e.g., 2 to 40 nt, 2 to 30 nt, 2 to 25 nt, 2 to 20 nt, 5 to 50 nt, 5 to 40 nt, 5 to 30 nt, 5 to 25 nt, 5 to 20 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 25 nt, 10 to 20 nt, 17 to 50 nt, 17 to 40 nt, 17 to 30 nt, 17 to 25 nt). In some cases, the length of the insertion site of the nucleic acid of interest is in the range of 5 to 40 nt.

[0184] Nucleic acid modification In some embodiments, a nucleic acid of interest (e.g., a CRISPR-Cas effector guide RNA) has one or more modifications, such as base modifications, backbone modifications, etc., to provide the nucleic acid with new or enhanced characteristics (e.g., improved stability). A nucleoside is a base-sugar combination. The base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide is a nucleoside that further includes a phosphate group covalently attached to the sugar portion of the nucleoside. In the case of nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming an oligonucleotide, the phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound can then be further joined to form a circular compound, although linear compounds are preferred. In addition, linear compounds may have internal nucleotide base complementarity and therefore may fold in such a way as to produce fully or partially double-stranded compounds. Within oligonucleotides, the phosphate groups are generally considered to form the internucleoside backbone of the oligonucleotide. The usual linkage or backbone of RNA and DNA is the 3' to 5' phosphodiester linkage.

[0185] Suitable nucleic acid modifications include, but are not limited to, 2'O-methyl modified nucleotides, 2'fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and 5' caps (e.g., 7-methylguanylate caps (m7G)). Further details and additional modifications are described below.

[0186] 2'-O-methyl modified nucleotides (also called 2'-O-methyl RNA) are a naturally occurring modification of RNA found in tRNA and other small RNA molecules that arise as a post-transcriptional modification. Oligonucleotides containing 2'-O-methyl RNA can be directly synthesized. This modification increases the Tm of the RNA:RNA duplex but results in only minor changes in RNA:DNA stability. It is stable to attack by single-stranded ribonucleases and is typically 5-10 times less susceptible to DNases than DNA. It is commonly used in antisense oligos as a means of increasing stability and binding affinity for target messages.

[0187] 2' fluoro-modified nucleotides (e.g., 2' fluoro bases) have a fluorine-modified ribose that increases binding affinity (Tm) and also confers some relative nuclease resistance when compared to natural RNA. These modifications are commonly used in ribozymes and siRNAs to improve stability in serum or other biological fluids.

[0188] LNA bases have a modification to the ribose backbone that locks the base at the C3'-terminal position, favoring an RNA A-type helix duplex geometry. This modification significantly increases Tm and is highly nuclease-resistant. Multiple LNA insertions can be placed in oligos at any position except the 3' end. Applications ranging from antisense oligos to hybridization probes for SNP detection and allele-specific PCR have been described. Due to the significant increase in Tm conferred by LNAs, they can also increase primer-dimer formation and self-hairpin formation. In some cases, the number of LNAs incorporated into a single oligo is 10 or fewer bases.

[0189] Phosphorothioate (PS) linkages (i.e., phosphorothioate linkages) substitute sulfur atoms for non-bridging oxygen atoms in the phosphate backbone of nucleic acids (e.g., oligos). This modification confers resistance to nuclease degradation to internucleotide linkages. Phosphorothioate linkages can be introduced between the last 3-5 nucleotides at the 5' or 3' end of an oligo to inhibit exonuclease degradation. Inclusion of phosphorothioate linkages within an oligo (e.g., throughout the oligo) also helps reduce attack by endonucleases.

[0190] In some embodiments, the nucleic acid of interest has one or more nucleotides that are 2'-O-methyl modified nucleotides. In some embodiments, the nucleic acid of interest (e.g., dsRNA, siNA, etc.) has one or more 2' fluoro-modified nucleotides. In some embodiments, the nucleic acid of interest (e.g., dsRNA, siNA, etc.) has one or more LNA bases. In some embodiments, the nucleic acid of interest (e.g., dsRNA, siNA, etc.) has one or more nucleotides linked by phosphorothioate linkages (i.e., the nucleic acid of interest has one or more phosphorothioate linkages). In some embodiments, the nucleic acid of interest (e.g., dsRNA, siNA, etc.) has a 5' cap (e.g., a 7-methylguanylate cap (m7G)). In some embodiments, the nucleic acid of interest (e.g., dsRNA, siNA, etc.) has a combination of modified nucleotides. For example, the nucleic acid of interest (e.g., dsRNA, siNA, etc.) can have a 5' cap (e.g., a 7-methylguanylate cap (m7G)) in addition to having one or more nucleotides with other modifications (e.g., 2'-O-methyl nucleotides and / or 2' fluoro-modified nucleotides and / or LNA bases and / or phosphorothioate linkages).

[0191] Modified backbones and modified internucleoside linkages Examples of suitable nucleic acids (e.g., CRISPR-Cas effector guide RNAs) containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0192] Suitable modified oligonucleotide backbones containing phosphate atoms include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidate and aminoalkyl phosphoramidate), phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates, which typically have 3'-5' linkages, their 2'-5' linked analogs, and those with inverted polarity, in which one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages. Preferred oligonucleotides with inverted polarity contain a single 3'-3' linkage at the 3'-most internucleotide linkage, i.e., a single inverted nucleoside residue that may be basic (either lacking a nucleobase or having a hydroxyl group instead).Various salts (e.g., potassium or sodium), mixed salts, and free acid forms are also included.

[0193] In some embodiments, the nucleic acids of interest contain one or more phosphorothioate and / or heteroatom internucleoside linkages, particularly -CH-NH-O-CH-, -CH-N(CH)-O-CH- (known as a methylene(methylimino) or MMI backbone), -CH-ON(CH)-CH-, -CH-N(CH)-N(CH)-CH-, and -ON(CH)-CH-CH- (natural phosphodiester internucleotide linkages are represented as -OP(=O)(OH)-O-CH-). MMI-type internucleoside linkages are disclosed in the above-referenced U.S. Pat. No. 5,489,677, the disclosure of which is incorporated herein by reference in its entirety. Suitable amide internucleoside linkages are disclosed in U.S. Pat. No. 5,602,240, the disclosure of which is incorporated herein by reference in its entirety.

[0194] Also suitable is the nucleic acid having morpholino backbone structure, as described in U.S. Patent No. 5,034,506.For example, in some embodiments, the nucleic acid of interest comprises a 6-membered morpholino ring instead of a ribose ring.In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkages replace phosphodiester linkages.

[0195] Suitable modified polynucleotide backbones that do not contain phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonic acid and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH constituent moieties.

[0196] Imitation The nucleic acid of interest may be a nucleic acid mimic. When applied to polynucleotides, the term "mimetic" is intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups; replacement of only the furanose ring is also referred to in the art as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNAs, the sugar backbone of a polynucleotide is replaced with an amide backbone, particularly an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone.

[0197] One polynucleotide mimic that has been reported to have excellent hybridization properties is peptide nucleic acid (PNA). The backbone in PNA compounds is two or more linked aminoethylglycine units, giving PNA an amide-containing backbone. Heterocyclic base moieties are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents describing the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262 (the disclosures of which are incorporated herein by reference in their entireties).

[0198] Another class of polynucleotide mimics being studied is based on linked morpholino units (morpholino nucleic acids) with a heterocyclic base attached to the morpholino ring. Several linking groups have been reported to link the morpholino monomer units in morpholino nucleic acids. One class of linking group was selected to obtain nonionic oligomeric compounds. Nonionic morpholino-based oligomeric compounds are less likely to have undesirable interactions with cellular proteins. Morpholino-based polynucleotides are nonionic mimics of oligonucleotides that are less likely to form undesirable interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Patent No. 5,034,506, the disclosure of which is incorporated herein by reference in its entirety. A variety of compounds within the morpholino class of polynucleotides have been prepared with a variety of different linking groups joining the monomeric subunits.

[0199] Another class of polynucleotide mimics is called cyclohexenyl nucleic acid (CeNA). The furanose ring normally present in DNA / RNA molecules is replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers have been prepared and used for the synthesis of oligomeric compounds according to classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides with specific CeNA modifications have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602, the disclosure of which is incorporated herein by reference in its entirety). In general, the incorporation of CeNA monomers into DNA strands increases the stability of DNA / RNA hybrids. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. Studies incorporating the CeNA structure into native nucleic acid structures have progressed with facile conformational adaptation demonstrated by NMR and circular dichroism.

[0200] A further modification includes locked nucleic acids (LNAs), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of which is incorporated herein by reference in its entirety). LNAs and LNA analogs exhibit very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility properties. Potent and non-toxic antisense oligonucleotides containing LNAs have been described (see, e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638, the disclosure of which is incorporated herein by reference in its entirety).

[0201] The synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, along with their oligomerization and nucleic acid recognition properties, have been described (e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630, the disclosure of which is incorporated herein by reference in its entirety). LNAs and their preparation are also described in WO98 / 39352 and WO99 / 14226, and U.S. Application Nos. 2012 / 0165514, 2010 / 0216983, 2009 / 0041809, 2006 / 0117410, 2004 / 0014959, 2002 / 0094555, and 2002 / 0086998, the disclosures of which are incorporated herein by reference in their entireties.

[0202] modified sugar moiety The subject nucleic acids can also contain one or more substituted sugar moieties, including sugar substituents selected from OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. O((CH2) n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON((CH2) n CH3)2 is particularly preferred, where n and m are from 1 to about 10. Other preferred polynucleotides include C1 to C 10The sugar substituents include lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacological properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety), i.e., alkoxyalkoxy groups. Further suitable modifications include the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE), illustrated in the Examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0203] Other suitable sugar substituents include methoxy (-O-CH), aminopropoxy (-OCHCHCHNH), allyl (-CH-CH=CH), -O-allyl (-O-CH-CH=CH), and fluoro (F). The 2'-sugar substituent may be at the arabino (up) or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3'-terminal nucleoside or in 2'-5' linked oligonucleotides, and the 5' position of the 5'-terminal nucleotide. Oligomeric compounds may also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar.

[0204] Base modifications and substitutions Nucleic acids of interest may also contain modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-hydroxymethylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine. cytosine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0205] Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993, the disclosures of which are incorporated herein by reference in their entirety. Some of these nucleobases are useful for increasing the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278, the disclosure of which is incorporated herein by reference in its entirety), and is a suitable base substitution, for example, when combined with a 2'-O-methoxyethyl sugar modification.

[0206] Conjugates Another possible modification of a nucleic acid of interest involves chemically linking one or more moieties or conjugates to the polynucleotide that improve the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties or conjugates can include conjugate groups covalently attached to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that improve the pharmacodynamic properties of oligomers, and groups that improve the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that improve pharmacodynamic properties include groups that improve uptake, resistance to degradation, and / or enhance sequence-specific hybridization with the target nucleic acid. Groups that improve pharmacokinetic properties include groups that improve the uptake, distribution, metabolism, or excretion of the nucleic acid of interest.

[0207] Conjugate moieties include cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), choline acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.Lipid moieties include, but are not limited to, lipid moieties such as those listed above (Beckmann, 1996, 277, 923-937).

[0208] The conjugate may comprise a "protein transduction domain" or PTD (also known as a CPP - cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule and / or nanoparticle, which may range from a small polar molecule to a large macromolecule, facilitates membrane crossing of the molecule, for example, moving from the extracellular space to the intracellular space or from the cytosol into an organelle (e.g., the nucleus). In some embodiments, the PTD is covalently attached to the 3' end of the exogenous polynucleotide. In some embodiments, the PTD is covalently attached to the 5' end of the exogenous polynucleotide. Exemplary PTDs include a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT, including YGRKKRRQRRR (SEQ ID NO: 34)), a polyarginine sequence containing a number of arginines sufficient for direct entry into cells (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), a 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), polylysine (Wender et al. (2004) Pharm. Research 21:1248-1256), and the like. al.(2000)Proc.Natl.Acad.Sci.USA 97:13003-13008), TIFF0007649748000012.tif24128. Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 34), RKKRRQRRR (SEQ ID NO: 35), and arginine homopolymers of 3 to 50 arginine residues. Exemplary PTD domain amino acid sequences include the following: TIFF0007649748000013.tif17128. In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6):371-381). ACPPs contain a polycationic CPP (e.g., Arg9 or "R9") connected to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero, thereby inhibiting cellular attachment and uptake. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesive properties, thus "activating" the ACPP to cross membranes.

[0209] Introduction of constructs into target cells A CRISPR-Cas effector guide RNA (or a nucleic acid comprising a nucleotide sequence encoding same) and / or a CRISPR-Cas effector polypeptide of the disclosure (or a nucleic acid comprising a nucleotide sequence encoding same) and / or a CRISPR-Cas effector fusion polypeptide of the disclosure (or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the disclosure) and / or a donor polynucleotide (donor template) can be introduced into a host cell by any of a variety of well-known methods.

[0210] Any of a variety of compounds and methods can be used to deliver the CRISPR-Cas effector system of the present disclosure to a target cell (e.g., a CRISPR-Cas effector system can include (a) a CRISPR-Cas effector polypeptide and a CRISPR-Cas effector guide RNA of the present disclosure, (b) a CRISPR-Cas effector polypeptide, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid of the present disclosure, (c) a CRISPR-Cas effector fusion polypeptide and a CRISPR-Cas effector guide RNA of the present disclosure, and a donor template nucleic acid of the present disclosure). (d) a CRISPR-Cas effector fusion polypeptide, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid of the present disclosure; (e) an mRNA encoding a CRISPR-Cas effector polypeptide and a CRISPR-Cas effector guide RNA of the present disclosure; (f) an mRNA encoding a CRISPR-Cas effector polypeptide, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid of the present disclosure; and (g) an mRNA encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure. (h) mRNA encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (i) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (j) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; (k) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (l) a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA;and a recombinant expression vector comprising a nucleotide sequence encoding a donor template nucleic acid; (m) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (n) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (o) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (p) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; The CRISPR-Cas effector fusion system can include a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide, a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (q) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a second CRISPR-Cas effector guide RNA; or (r) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a second CRISPR-Cas effector guide RNA, or some variation of one of (a)-(r). As a non-limiting example, the CRISPR-Cas effector system of the present disclosure can be combined with a lipid. As another non-limiting example, the CRISPR-Cas effector system of the present disclosure can be combined with or formulated into a particle. ,

[0211] Methods for introducing nucleic acids into host cells are known in the art, and any convenient method can be used to introduce a nucleic acid of interest (e.g., an expression construct / vector) into a target cell (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a human cell, etc.). Suitable methods include, for example, 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 microinjection, nanoparticle-mediated nucleic acid delivery (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.

[0212] In some cases, the CRISPR-Cas effector polypeptides of the present disclosure are provided as nucleic acids (e.g., mRNA, DNA, plasmids, expression vectors, viral vectors, etc.) encoding the CRISPR-Cas effector polypeptides. In some cases, the CRISPR-Cas effector polypeptides of the present disclosure are provided directly as proteins (e.g., without or with associated guide RNAs, i.e., as ribonucleoprotein complexes). The CRISPR-Cas effector polypeptides of the present disclosure can be introduced into (provided to) cells by any convenient method, and such methods are known to those of skill in the art. As an illustrative example, the CRISPR-Cas effector polypeptides of the present disclosure can be directly injected into cells (e.g., with or without a CRISPR-Cas effector guide RNA or a nucleic acid encoding a CRISPR-Cas effector guide RNA, and with or without a donor polynucleotide). As another example, a preformed complex of a CRISPR-Cas effector polypeptide of the present disclosure and a CRISPR-Cas effector guide RNA (RNP) can be introduced into a cell (e.g., a eukaryotic cell) (e.g., via injection, via nucleofection, conjugated to one or more components, e.g., conjugated to a CRISPR-Cas effector protein, conjugated to a guide RNA, conjugated to a CRISPR-Cas effector polypeptide and guide RNA of the present disclosure, via a protein transduction domain (PTD), etc.).

[0213] In some cases, a CRISPR-Cas effector fusion polypeptide of the present disclosure (e.g., a dCRISPR-Cas effector fused to a fusion partner, a nickase CRISPR-Cas effector fused to a fusion partner, etc.) is provided as a nucleic acid (e.g., mRNA, DNA, plasmid, expression vector, viral vector, etc.) encoding the CRISPR-Cas effector fusion polypeptide. In some cases, a CRISPR-Cas effector fusion polypeptide of the present disclosure is provided directly as a protein (e.g., without or with an associated guide RNA, i.e., as a ribonucleoprotein complex). A CRISPR-Cas effector fusion polypeptide of the present disclosure can be introduced into (provided to) a cell by any convenient method, and such methods are known to those of skill in the art. As an illustrative example, a CRISPR-Cas effector fusion polypeptide of the present disclosure can be directly injected into a cell (e.g., with or without a nucleic acid encoding a CRISPR-Cas effector guide RNA and with or without a donor polynucleotide). As another example, a preformed complex of a CRISPR-Cas effector fusion polypeptide of the present disclosure and a CRISPR-Cas effector guide RNA (RNP) can be introduced into a cell (e.g., via injection, via nucleofection, conjugated to one or more components, e.g., conjugated to a CRISPR-Cas effector fusion protein, conjugated to a guide RNA, conjugated to a CRISPR-Cas effector fusion polypeptide of the present disclosure and a guide RNA, via a protein transduction domain (PTD), etc.).

[0214] In some cases, a nucleic acid (e.g., a CRISPR-Cas effector guide RNA, a nucleic acid comprising a nucleotide sequence encoding a 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 CRISPR-Cas effector polypeptide, a CRISPR-Cas effector fusion polypeptide) in or associated with a particle. In some cases, a CRISPR-Cas effector system of the present disclosure is delivered to a cell in or associated with a particle. The terms "particle" and "nanoparticle" can be used interchangeably where appropriate. A recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide and / or a CRISPR-Cas effector guide RNA of the present disclosure, an mRNA comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a guide RNA can be delivered simultaneously using a particle or lipid envelope. For example, the CRISPR-Cas effector polypeptide and CRISPR-Cas effector 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 a lipid or lipidoid and a hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, e.g., where the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), and / or the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG), and / or the particle further comprises cholesterol (e.g., particles from Formulation 1 = DOTAP 100, DMPC 0, PEG 0, cholesterol 0; Formulation No. 2 = DOTAP 90, DMPC 0, PEG 10, cholesterol 0; Formulation No. 3 = DOTAP 90, DMPC 0, PEG 5, cholesterol 5).For example, particles can be formed using a multi-step process, in which the CRISPR-Cas effector polypeptide and CRISPR-Cas effector guide RNA are mixed together, e.g., in a 1:1 molar ratio, e.g., at room temperature, for e.g., 30 minutes, e.g., in sterile, nuclease-free 1x phosphate buffered saline (PBS); separately, DOTAP, DMPC, PEG, and cholesterol are dissolved in alcohol, e.g., 100% ethanol, as applicable to the formulation, and the two solutions are mixed together to form particles containing the complexes).

[0215] The CRISPR-Cas effector polypeptides of the present disclosure (or mRNAs comprising a nucleotide sequence encoding the CRISPR-Cas effector polypeptides of the present disclosure, or recombinant expression vectors comprising a nucleotide sequence encoding the CRISPR-Cas effector polypeptides of the present disclosure) and / or CRISPR-Cas effector guide RNAs (or nucleic acids, such as one or more expression vectors encoding the CRISPR-Cas effector guide RNAs) can be co-delivered using particles or lipid envelopes. For example, biodegradable core-shell nanoparticles having a poly(β-amino ester) (PBAE) core encapsulated by a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self-assembling bioadhesive polymers are used, and such particles / nanoparticles can be applied, for example, to oral delivery of peptides to the brain, intravenous delivery of peptides, and nasal delivery of peptides. Other embodiments, such as oral absorption and intraocular delivery of hydrophobic drugs, are also contemplated. Molecular envelope technology can be used, requiring an engineered polymer envelope to be protected and delivered to the site of disease. A dose of about 5 mg / kg can be used in single or multiple doses depending on various factors, eg, the target tissue.

[0216] Lipidoid compounds (e.g., as described in U.S. Patent Application No. 2011 / 0293703) are also useful in administering polynucleotides and can be used to deliver a CRISPR-Cas effector polypeptide of the present disclosure, a CRISPR-Cas effector fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a CRISPR-Cas effector system of the present disclosure (e.g., a CRISPR-Cas effector system that comprises (a) a CRISPR-Cas effector polypeptide of the present disclosure, and CRISPR-Cas effector guide RNA, (b) a CRISPR-Cas effector polypeptide, CRISPR-Cas effector guide RNA, and donor template nucleic acid of the present disclosure, (c) a CRISPR-Cas effector fusion polypeptide and CRISPR-Cas effector guide RNA of the present disclosure, (d) a CRISPR-Cas effector fusion polypeptide, CRISPR-Cas effector guide RNA, and donor template nucleic acid of the present disclosure, (e) a CRISPR-Cas effector polypeptide of the present disclosure, (f) mRNA, CRISPR-Cas effector guide RNA, and donor template nucleic acid encoding a CRISPR-Cas effector polypeptide of the present disclosure; (g) mRNA, CRISPR-Cas effector guide RNA, and donor template nucleic acid encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure; (h) mRNA, CRISPR-Cas effector guide RNA, and donor template nucleic acid encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure; (i) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (j) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid;(k) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (l) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; (m) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (n) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (o) a recombinant expression vector encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure (p) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (q) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a second CRISPR-Cas effector guide RNA; or (r) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a second CRISPR-Cas effector guide RNA;or some variation of one of (a)-(r). In one embodiment, the amino alcohol lipidoid compound is combined with a drug to be delivered to a cell or subject to form a microparticle, nanoparticle, liposome, or micelle. The amino alcohol lipidoid compound can be combined with other amino alcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. These particles can then, optionally, be combined with pharmaceutical excipients to form pharmaceutical compositions.

[0217] Poly(beta-amino alcohols) (PBAAs) can be used to deliver a CRISPR-Cas effector polypeptide of the present disclosure, a CRISPR-Cas effector fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a CRISPR-Cas effector system of the present disclosure to a target cell. U.S. Patent Publication No. 2013 / 0302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that have been prepared using combinatorial polymerization.

[0218] Sugar-based particles, e.g., GalNAc, can be used as described with reference to WO2014 / 118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136(49), 16958-16961), to deliver a CRISPR-Cas effector polypeptide of the present disclosure, a CRISPR-Cas effector fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a CRISPR-Cas effector system of the present disclosure to a target cell.

[0219] In some cases, lipid nanoparticles (LNPs) are used to deliver the CRISPR-Cas effector polypeptide of the present disclosure, the CRISPR-Cas effector fusion polypeptide of the present disclosure, the RNP of the present disclosure, the nucleic acid of the present disclosure, or the CRISPR-Cas effector system of the present disclosure to target cells.Negatively charged polymers such as RNA can be loaded into LNPs at low pH values ​​(for example, pH 4), and ionized lipids exhibit positive charges.However, at physiological pH values, LNPs exhibit low surface charge, which corresponds to longer circulation time. The focus is on four ionizable cationic lipids: 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). Preparation of LNPs is described, for example, in Rosin et al. (2011) Molecular Therapy 19:1286-2200. The cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2''-(methoxypolyethylene glycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(omega-methoxy-poly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristoyloxylpropyl-3-amine (PEG-C-DOMG) may also be used.Nucleic acids (e.g., CRISPR-Cas effector guide RNAs, nucleic acids of the present disclosure, etc.) can be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG in a 40:10:40:10 molar ratio), optionally incorporating 0.2% SP-DiOC18.

[0220] Spherical nucleic acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver a CRISPR-Cas effector polypeptide of this disclosure, a CRISPR-Cas effector fusion polypeptide of this disclosure, an RNP of this disclosure, a nucleic acid of this disclosure, or a CRISPR-Cas effector system of this disclosure to a target cell. For example, 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 2012 7:635-638, Zhang et al. 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, 209ra152 (2013); and Mirkin, et al., Small, 10:186-192.

[0221] Self-assembled nanoparticles bearing RNA can be constructed from polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of the polyethylene glycol (PEG).

[0222] Generally, a "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering a CRISPR-Cas effector polypeptide of this disclosure, a CRISPR-Cas effector fusion polypeptide of this disclosure, an RNP of this disclosure, a nucleic acid of this disclosure, or a CRISPR-Cas effector system of this disclosure to a target cell have a diameter of 500 nm or less, e.g., 25 nm to 35 nm, 35 nm to 50 nm, 50 nm to 75 nm, 75 nm to 100 nm, 100 nm to 150 nm, 150 nm to 200 nm, 200 nm to 300 nm, 300 nm to 400 nm, or 400 nm to 500 nm. In some cases, nanoparticles suitable for use in delivering a CRISPR-Cas effector polypeptide of this disclosure, a CRISPR-Cas effector fusion polypeptide of this disclosure, an RNP of this disclosure, a nucleic acid of this disclosure, or a CRISPR-Cas effector system of this disclosure to a target cell have a diameter of 25 nm to 200 nm. In some cases, nanoparticles suitable for use in delivering a CRISPR-Cas effector polypeptide of this disclosure, a CRISPR-Cas effector fusion polypeptide of this disclosure, an RNP of this disclosure, a nucleic acid of this disclosure, or a CRISPR-Cas effector system of this disclosure to a target cell have a diameter of 100 nm or less. In some cases, nanoparticles suitable for use in delivering a CRISPR-Cas effector polypeptide of this disclosure, a CRISPR-Cas effector fusion polypeptide of this disclosure, an RNP of this disclosure, a nucleic acid of this disclosure, or a CRISPR-Cas effector system of this disclosure to a target cell have a diameter of 35 nm to 60 nm.

[0223] Nanoparticles suitable for use in delivering the disclosed CRISPR-Cas effector polypeptides, disclosed CRISPR-Cas effector fusion polypeptides, disclosed RNPs, disclosed nucleic acids, or disclosed CRISPR-Cas effector systems to target cells can be provided in different forms, such as solid nanoparticles (e.g., metals such as silver, gold, iron, and titanium), non-metallic, lipid-based solids, polymers, nanoparticle suspensions, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles), can be prepared. Nanoparticles made of semiconductor materials can also be labeled quantum dots if they are small enough (typically 10 nm or less) for quantization of electronic energy levels to occur. Such nanoparticles are used in biomedical applications such as drug carriers or imaging agents and can be adapted for similar purposes in the present disclosure.

[0224] Semi-solid and soft nanoparticles are also suitable for use in delivering a CRISPR-Cas effector polypeptide of the present disclosure, a CRISPR-Cas effector fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a CRISPR-Cas effector system of the present disclosure to a target cell. A prototypical nanoparticle of semi-solid nature is a liposome.

[0225] In some cases, exosomes are used to deliver a CRISPR-Cas effector polypeptide of this disclosure, a CRISPR-Cas effector fusion polypeptide of this disclosure, an RNP of this disclosure, a nucleic acid of this disclosure, or a CRISPR-Cas effector system of this disclosure to a target cell. Exosomes are endogenous nanovesicles that can transport RNA and proteins, and deliver RNA to the brain and other target organs.

[0226] In some cases, liposomes are used to deliver the CRISPR-Cas effector polypeptides, CRISPR-Cas effector fusion polypeptides, RNPs, nucleic acids, or CRISPR-Cas effector systems of the present disclosure to target cells. Liposomes are spherical vesicular structures composed of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids, but phospholipids are most commonly used to generate liposomes. Liposome formation occurs spontaneously when a lipid membrane is mixed with an aqueous solution, but it can also be promoted by applying force in the form of shaking using a homogenizer, sonicator, or extrusion device. Several other additives may be added to liposomes to modify their structure and properties. For example, either cholesterol or sphingomyelin may be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of the liposome's internal cargo. Liposomal formulations may be composed primarily of lipids such as natural phospholipids and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialogangliosides.

[0227] Stable nucleic acid-lipid particles (SNALPs) can be used to deliver the disclosed CRISPR-Cas effector polypeptides, disclosed CRISPR-Cas effector fusion polypeptides, disclosed RNPs, disclosed nucleic acids, or disclosed CRISPR-Cas effector systems to target cells. The SNALP formulation can include the lipids 3-N-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in a molar ratio of 2:40:10:48. SNALP liposomes can be prepared by combining D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), cholesterol, and siRNA using a lipid / siRNA ratio of 25:1 and a 48 / 40 / 10 / 2 molar ratio of cholesterol / D-Lin-DMA / DSPC / PEG-C-DMA. The resulting SNALP liposomes can be approximately 80-100 nm in size. SNALP can contain synthetic cholesterol (Sigma-Aldrich, St. Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane. SNALPs can include synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids Inc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

[0228] Other cationic lipids, such as the amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), can be used to deliver the CRISPR-Cas effector polypeptides of the present disclosure, the CRISPR-Cas effector fusion polypeptides of the present disclosure, the RNPs of the present disclosure, the nucleic acids of the present disclosure, or the CRISPR-Cas effector systems of the present disclosure to target cells. Preformed vesicles having the following lipid composition can be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in a 40 / 10 / 40 / 10 molar ratio, respectively, and a FVII siRNA / total lipid ratio of approximately 0.05 (w / w). To ensure a narrow particle size distribution in the 70-90 nm range and a low polydispersity index of 0.11 ± 0.04 (n = 56), particles can be extruded up to three times through an 80 nm membrane before adding guide RNA. Particles containing highly potent amino lipids 16 may be used, and the molar ratio of the four lipid components 16, DSPC, cholesterol, and PEG-lipid (50 / 10 / 38.5 / 1.5) can be further optimized to enhance in vivo activity.

[0229] Lipids can be combined with the disclosed CRISPR-Cas effector system or its component(s) or the nucleic acid encoding same to form lipid nanoparticles (LNPs). Suitable lipids include, but are not limited to, DLin-KC2-DMA4, C12-200, and the co-lipids disteroylphosphatidylcholine, cholesterol, and PEG-DMG can be combined with the disclosed CRISPR-Cas effector system or its components using a spontaneous vesicle formation procedure. The molar ratio of the components can be approximately 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / disteroylphosphatidylcholine / cholesterol / PEG-DMG).

[0230] The CRISPR-Cas effector systems or components thereof of the present disclosure may be delivered encapsulated in PLGA microspheres, as further described in U.S. Published Application Nos. 2013 / 0252281, 2013 / 0245107, and 2013 / 0244279.

[0231] Supercharged proteins can be used to deliver the disclosed CRISPR-Cas effector polypeptides, the disclosed CRISPR-Cas effector fusion polypeptides, the disclosed RNPs, the disclosed nucleic acids, or the disclosed CRISPR-Cas effector systems to target cells. Supercharged proteins are a class of engineered or naturally occurring proteins that have an unusually high net positive or negative theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to resist thermally or chemically induced aggregation. Superpositively charged proteins can also 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.

[0232] Cell-penetrating peptides (CPPs) can be used to deliver the CRISPR-Cas effector polypeptides of this disclosure, the CRISPR-Cas effector fusion polypeptides of this disclosure, the RNPs of this disclosure, the nucleic acids of this disclosure, or the CRISPR-Cas effector systems of this disclosure to target cells. 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 a sequence that contains an alternating pattern of polar / charged amino acids and nonpolar, hydrophobic amino acids.

[0233] The implantable device can be used to deliver a CRISPR-Cas effector polypeptide of the present disclosure, a CRISPR-Cas effector fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure (e.g., a CRISPR-Cas effector guide RNA, a nucleic acid encoding a CRISPR-Cas effector guide RNA, a nucleic acid encoding a CRISPR-Cas effector polypeptide, a donor template, etc.), or a CRISPR-Cas effector 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 the circulation, a target cell in a tissue, a target cell within an organ, etc.). Implantable devices suitable for use in delivering a CRISPR-Cas effector polypeptide of this disclosure, a CRISPR-Cas effector fusion polypeptide of this disclosure, an RNP of this disclosure, a nucleic acid of this disclosure, or a CRISPR-Cas effector system of this disclosure to a target cell (e.g., a target cell in vivo, where the target cell is a target cell in the circulation, a target cell in a tissue, a target cell within an organ, etc.) can include a container (e.g., a reservoir, matrix, etc.) that contains the CRISPR-Cas effector polypeptide, CRISPR-Cas effector fusion polypeptide, RNP, or CRISPR-Cas effector system (or a component thereof, e.g., a nucleic acid of this disclosure).

[0234] Suitable implantable devices may include, for example, a polymer substrate, such as a matrix, used as the device body, and optionally additional scaffolding materials, such as metals or additional polymers, as well as materials to improve visibility and imaging. Implantable delivery devices may be advantageous in providing localized and prolonged release, with the delivered polypeptide and / or nucleic acid being released directly into the target site, e.g., the extracellular matrix (ECM), the vasculature surrounding a tumor, diseased tissue, etc. Suitable implantable delivery devices include devices suitable for delivery to cavities such as the peritoneal cavity and / or for use in any other type of administration where the drug delivery system is not tethered or attached, and include a biostable and / or biodegradable and / or bioabsorbable polymer substrate (e.g., which may optionally be a matrix). In some cases, suitable implantable drug delivery devices include degradable polymers, with bulk erosion being the primary release mechanism. In some cases, suitable implantable drug delivery devices contain non-degradable or slowly degrading polymers, and the primary release mechanism is diffusion rather than bulk erosion, whereby the outer portion functions as a membrane and the inner portion functions as a drug reservoir that is virtually unaffected by the environment for extended periods (e.g., from about one week to about several months). Combinations of different polymers with different release mechanisms may optionally be used. The concentration gradient can be maintained effectively constant for a significant period of the total release period, so that the diffusion rate is effectively constant (referred to as "zero-mode" diffusion). The term "constant" refers to a diffusion rate that is maintained above a low threshold of therapeutic effect but may still optionally be characterized by an initial burst and / or may fluctuate (e.g., increase and decrease to a certain extent). The diffusion rate can be maintained in this manner for extended periods and considered constant at a certain level to optimize the therapeutic effective period, e.g., the effective silencing period.

[0235] In some cases, implantable delivery systems are designed to shield the nucleotide-based therapeutic agent from degradation, either chemical in nature or due to attack from enzymes and other factors within the subject's body.

[0236] The site for device implantation, or target site, can be selected for maximum therapeutic effect. For example, the delivery device can be implanted within or proximal to the tumor environment or blood supply associated with the tumor. Target locations can include, for example, 1) the brain at sites of degeneration such as Parkinson's disease or Alzheimer's disease in the basal ganglia, white matter, and gray matter; 2) the spine, as in amyotrophic lateral sclerosis (ALS); 3) the cervix; 4) active and chronically inflamed joints; 5) the dermis, as in psoriasis; 6) sympathetic and sensory nerve sites for analgesic effects; 7) bone; 8) sites of acute or chronic infection; 9) within the vagina; 10) the cochlea - auditory system, membranous labyrinth of the inner ear, vestibular system; 11) the uterine lining; 12) the uterine lining; 13) the uterine lining; 14) the uterine lining; 15) the uterine lining; 16) the uterine lining; 17) the uterine lining; 18) the uterine lining; 19) the uterine lining; 20) the uterine lining; 21) the uterine lining; 22) the uterine lining; 23) the uterine lining; 24) the uterine lining; 25) the uterine lining; 26) the uterine lining; 27) the uterine lining; 28) the uterine lining; 29) the uterine lining; 30) the uterine lining; 31) the uterine lining; 32) the uterine lining; 33) the uterine lining; 34) the 1) in the trachea, 12) in the heart, annulus, epicardium, 13) the urinary tract or bladder, 14) the biliary system, 15) parenchymal tissues including, but not limited to, the kidney, liver, and spleen, 16) lymph nodes, 17) salivary glands, 18) gums, 19) in (within) joints, 20) in the eye, 21) brain tissue, 22) ventricles, 23) cavities including the peritoneal cavity (for example, but not limited to, in the case of ovarian cancer), 24) in the esophagus, and 25) in the rectum, and 26) in the vasculature.

[0237] The method of insertion, such as implantation, may optionally already be used for other types of tissue implantation and / or insertion and / or tissue sampling, optionally without modification or optionally with only minor modifications to such methods, optionally including, but not limited to, brachytherapy, biopsy, endoscopy with and / or without ultrasound, e.g., stereotactic radioscopy of brain tissue, laparoscopy, including implantation of a laparoscope into joints, abdominal organs, bladder walls, and body cavities.

[0238] Modified host cells The present disclosure provides modified cells comprising a CRISPR-Cas effector polypeptide of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure. The present disclosure provides modified cells comprising a CRISPR-Cas effector polypeptide of the present disclosure, where the modified cell is a cell that does not normally comprise a CRISPR-Cas effector polypeptide of the present disclosure. The present disclosure provides modified cells (e.g., genetically modified cells) comprising a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure. The present disclosure provides genetically modified cells genetically modified with an mRNA comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure. The present disclosure provides genetically modified cells genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a 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 CRISPR-Cas effector polypeptide of the present disclosure, and (b) a nucleotide sequence encoding a CRISPR-Cas effector 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 CRISPR-Cas effector polypeptide of the present disclosure, (b) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA of the present disclosure, and (c) a nucleotide sequence encoding a donor template.

[0239] Cells that function as recipients of the CRISPR-Cas effector polypeptides of the present disclosure and / or nucleic acids comprising nucleotide sequences encoding the CRISPR-Cas effector polypeptides of the present disclosure and / or CRISPR-Cas effector guide RNAs of the present disclosure can be any of a variety of cells, including, for example, in vitro cells, in vivo cells, ex vivo cells, primary cells, cancer cells, animal cells, plant cells, algal cells, fungal cells, etc. Cells that function as recipients of the CRISPR-Cas effector polypeptides of the present disclosure and / or nucleic acids comprising nucleotide sequences encoding the CRISPR-Cas effector polypeptides of the present disclosure and / or CRISPR-Cas effector guide RNAs of the present disclosure are referred to as "host cells" or "target cells." Host cells or target cells can be recipients of the CRISPR-Cas effector systems of the present disclosure. Host cells or target cells can be recipients of the CRISPR-Cas effector RNPs of the present disclosure. A host cell or target cell can be the recipient of a single component of the CRISPR-Cas effector system of the present disclosure.

[0240] Non-limiting examples of cells (target cells) include prokaryotic cells, fungal cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells derived from plants (e.g., cells derived from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, club mosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, etc.), and the like. Examples of cells include cells from various organisms, including but not limited to: seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells from mammals (e.g., ungulates (e.g., pigs, cows, goats, sheep), rodents (e.g., rats, mice), non-human primates, humans, felines (e.g., cats), canines (e.g., dogs), etc.). In some cases, the cells are not derived from a naturally occurring organism (e.g., the cells may be synthetically produced, also referred to as artificial cells).

[0241] The cell can be an in vitro cell (e.g., an established cultured cell line). The cell can be an ex vivo cell (a cultured cell from an individual). The cell can be an in vivo cell (e.g., a cell within an individual). The cell can be an isolated cell. The cell can be a cell inside an organism. The cell can be an organism. The cell can be a cell in cell culture (e.g., an in vitro cell culture). The cell can be one of a population of cells. The cell can be a prokaryotic cell or derived from a prokaryotic cell. The cell can be a bacterial cell or derived from a bacterial cell. The cell can be an archaeal cell or derived from an archaeal cell. The cell can be a eukaryotic cell or derived from a eukaryotic cell. The cell can be a plant cell or derived from a plant cell. The cell can be an animal cell or derived from an animal cell. The cell can be a vertebrate cell or derived from a vertebrate cell. The cell can be a vertebrate cell or derived from a vertebrate. The cell may be a mammalian cell or may be derived from a mammalian cell. The cell may be a rodent cell or may be derived from a rodent cell. The cell may be a human cell or may be derived from a human cell. The cell may be a microbial cell or may be derived from a microbial cell. The cell may be a fungal cell or may be derived from a fungal cell. The cell may be an insect cell. The cell may be an arthropod cell. The cell may be a protozoan cell. The cell may be a helminth cell.

[0242] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.), somatic cells such as fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, etc.

[0243] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autologous expanded 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, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postnatal stem cells.

[0244] In some cases, the cell is an immune cell, a neuron, an epithelial cell, an 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).

[0245] In some cases, the cells are stem cells. Stem cells include adult stem cells, which are also referred to as somatic stem cells.

[0246] Adult stem cells reside in differentiated tissues but retain the property of self-renewal and the ability to give rise to multiple cell types, usually cell types typical of the tissue they are found in. Many 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, mammalian stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, olfactory stem cells, neural crest stem cells, etc.

[0247] Stem cells of interest include mammalian stem cells, where the term "mammal" refers to any animal classified as a mammal, including humans, non-human primates, domestic and farm animals, and zoo, laboratory, sport, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (e.g., mouse, rat) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0248] The stem cells may express one or more stem cell markers, such as SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[0249] In some embodiments, the stem cells are hematopoietic stem cells (HSCs). HSCs are mesodermally derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs express CD34 + and CD3 - HSCs are characterized as being capable of repopulating erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewal cell division and can be induced to differentiate into the same lineages as those found in vivo. Thus, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0250] In other embodiments, the stem cells are neural stem cells (NSCs). Neural stem cells (NSCs) are capable of differentiating into neurons and glia (including oligodendrocytes and astrocytes). Neural stem cells are pluripotent stem cells capable of multiple divisions and, under certain conditions, can produce daughter cells that are neural stem cells, or neural progenitor cells that may be neuroblasts or glioblasts, e.g., cells committed to becoming one or more types of neurons and glial cells, respectively. Methods for obtaining NSCs are known in the art.

[0251] In another embodiment, the stem cells are mesenchymal stem cells (MSCs). MSCs, originally derived from embryonic mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, bone marrow matrix, and tendon. Methods for isolating MSCs are known in the art, and any known method can be used to obtain MSCs. See, for example, U.S. Patent No. 5,736,396, which describes the isolation of human MSCs.

[0252] The cell is optionally a plant cell. The plant cell may be a monocotyledonous plant cell. The cell may be a dicotyledonous plant cell.

[0253] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, such as barley, bean (dry food), canola, corn, cotton (pima), cotton (upland), flaxseed, hay (alfalfa), hay (non-alfalfa), oat, peanut, rice, sorghum, soybean, sugar beet, sugarcane, sunflower (oil), sunflower (non-oil), sweet potato, tobacco (burley), tobacco (flue-cured), tomato, wheat (durum), wheat (spring), wheat (winter), etc. In another example, the cells may be derived from, for example, alfalfa sprouts, aloe leaves, arrowroot, arrowheads, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet stalks, beets, bitter melon, bok choy, broccoli, broccoli rabe (rapini), Brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopal), pumpkin, cardoon, carrot, cauliflower, celery, chayote, Chinese artichoke (kronu), Chinese cabbage, Chinese cabbage, Chinese chives, Chinese leek, Chinese spinach, Chinese chrysanthemum (tung)ho), collard greens, corn stalks, sweet corn, cucumber, radish, edible dandelion greens, taro, pea sprouts (pea leaves), touki (winter melon), eggplant, endive, esculenta, osmanthus, shepherd's purse, frisee, mustard greens (Chinese mustard), kailan, galangal (Siamese, Thai fresh ginger), garlic, ginger, burdock, greens, Hanover salad greens, ouau sotto le, Jerusalem artichoke, jicama, kale greens, kohlrabi, Querite lettuce, Bibb lettuce, Boston lettuce, Boston red lettuce, Green leaf lettuce, Iceberg lettuce, Sunny lettuce, Oakleaf green lettuce, Oakleaf red lettuce, Processed lettuce, Red leaf lettuce, Romaine lettuce, Ruby romaine lettuce, Russian red mustard lettuce, Linkok, Loboc, Cowpea, Lotus root, Marsh lettuce, Agave leaf lettuce, Yam, Mescu Orchid mix, mizuna, moap (luffa), moo, mokua (fuzzy squash), mushroom, mustard, yam, okra, water spinach, spring onion, opo (long squash), decorative corn, decorative gourd, parsley, parsnip, beans, capsicum (bell type), capsicum, pumpkin, radicchio, radish sprouts, radish, rape greens, rape greens, rhubarb, romaine (baby red), rutabaga, hamcho (sea bean), loofah (tokado) Vegetable crop cells include, but are not limited to, spinach, squash, straw bale, sugarcane, sweet potato, Swiss chard, tamarind, taro, taro leaves, taro sprouts, tatsoi, tepeguaje (leucaena), tindora, tomatillo, tomato, tomato (cherry), tomato (grape type), tomato (plum type), turmeric, turnip greens, turnip, water chestnut, yampi, yams, rapeseed, yuca (cassava), etc.

[0254] The cell is optionally an arthropod cell. For example, the cell is an arthropod cell, e.g., a cell from an arthropod order, such as Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, eoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasm atodea), cockroaches (Blattaria), termites (Isoptera), mantodea, paraneuroptera, psolocoptera (Psocoptera), thrips (Thysanoptera), lice (Phthiraptera), Hemiptera, endopterygota or holometabola, hymenoptera The cell may be a cell of a suborder, family, subfamily, group, subgroup, or species of the order Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera, Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[0255] The cell optionally is an insect animal cell, for example, the cell optionally is a mosquito, grasshopper, bedbug, fly, flea, bee, wasp, ant, louse, moth, or beetle cell.

[0256] kit The present disclosure provides kits that include a CRISPR-Cas effector system of the present disclosure or components of a CRISPR-Cas effector system of the present disclosure.

[0257] The kits of the present disclosure include (a) a CRISPR-Cas effector polypeptide and a CRISPR-Cas effector guide RNA of the present disclosure, (b) a CRISPR-Cas effector polypeptide, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid of the present disclosure, (c) a CRISPR-Cas effector fusion polypeptide and a CRISPR-Cas effector guide RNA of the present disclosure, (d) a CRISPR-Cas effector fusion polypeptide, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid of the present disclosure. (e) mRNA encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a CRISPR-Cas effector guide RNA; (f) mRNA encoding a CRISPR-Cas effector polypeptide of the present disclosure, a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (g) mRNA encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a CRISPR-Cas effector guide RNA; (h) mRNA encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a CRISPR-Cas effector guide RNA; (i) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (j) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; (k) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure and a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (l) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a donor template nucleic acid;(m) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (n) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid; (o) a first recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (p) a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, The present invention can include a donor template nucleic acid, (q) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a second recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, and a donor template nucleic acid, (r) a recombinant expression vector comprising a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first CRISPR-Cas effector guide RNA, and a nucleotide sequence encoding a second CRISPR-Cas effector guide RNA, or a variation of one of (a) through (r).

[0258] Kits of the present disclosure can include (a) the above-described components of a CRISPR-Cas effector system of the present disclosure, or components that may comprise a CRISPR-Cas effector system of the present disclosure, and (b) one or more additional reagents, such as (i) buffers, (ii) protease inhibitors, (iii) nuclease inhibitors, (iv) reagents necessary for developing or visualizing a detectable label, (v) positive and / or negative control target DNA, (vi) positive and / or negative control CRISPR-Cas effector guide RNAs, etc. Kits of the present disclosure can include (a) the above-described components of a CRISPR-Cas effector system of the present disclosure, or components that may comprise a CRISPR-Cas effector system of the present disclosure, and (b) a therapeutic agent.

[0259] The kits of the present disclosure can include a recombinant expression vector comprising (a) an insertion site for inserting a nucleic acid comprising a nucleotide sequence encoding a portion of a CRISPR-Cas effector guide RNA that hybridizes to a target nucleotide sequence in a target nucleic acid, and (b) a nucleotide sequence encoding a CRISPR-Cas effector-binding portion of the CRISPR-Cas effector guide RNA.The kits of the present disclosure can include a recombinant expression vector comprising (a) an insertion site for inserting a nucleic acid comprising a nucleotide sequence encoding a portion of a CRISPR-Cas effector guide RNA that hybridizes to a target nucleotide sequence in a target nucleic acid, (b) a nucleotide sequence encoding the CRISPR-Cas effector-binding portion of the CRISPR-Cas effector guide RNA, and (c) a nucleotide sequence encoding a CRISPR-Cas effector polypeptide of the present disclosure.

[0260] usefulness The CRISPR-Cas effector polypeptides of the present disclosure, or the CRISPR-Cas effector fusion polypeptides of the present disclosure, are utilized in a variety of ways (e.g., in combination with a CRISPR-Cas effector guide RNA, and optionally further in combination with a donor template). For example, the CRISPR-Cas effector polypeptides of the present disclosure can be used to (i) modify (e.g., cleave, e.g., nick, methylate, etc.) a target nucleic acid (DNA or RNA, single-stranded or double-stranded), (ii) regulate transcription of the target nucleic acid, (iii) label the target nucleic acid, (iv) bind to the target nucleic acid (e.g., for purposes of isolation, labeling, imaging, tracking, etc.), or (v) modify a polypeptide (e.g., histone) associated with the target nucleic acid. Thus, the present disclosure provides methods for modifying a target nucleic acid. In some cases, the disclosed methods for modifying a target nucleic acid include contacting the target nucleic acid with (a) a CRISPR-Cas effector polypeptide of the disclosure, and (b) one or more (e.g., two) CRISPR-Cas effector guide RNAs. In some cases, the disclosed methods for modifying a target nucleic acid include contacting the target nucleic acid with (a) a CRISPR-Cas effector polypeptide of the disclosure, (b) a CRISPR-Cas effector guide RNA, and (c) a donor nucleic acid (e.g., a donor template). In some cases, the contacting step is performed in an in vitro cell. In some cases, the contacting step is performed in an in vivo cell. In some cases, the contacting step is performed in an ex vivo cell.

[0261] Because methods using CRISPR-Cas effector polypeptides involve binding of the CRISPR-Cas effector polypeptide to a specific region in a target nucleic acid (by being targeted there by an associated CRISPR-Cas effector guide RNA), these methods are generally referred to herein as binding methods (e.g., target nucleic acid binding methods). However, it should be understood that in some cases, a binding method may result in nothing other than binding of the target nucleic acid, while in other cases, the method may have a different end result (e.g., the method may result in modification of the target nucleic acid, such as cleavage / methylation, modulation of transcription from the target nucleic acid, modulation of translation of the target nucleic acid, genome editing, modulation of a protein associated with the target nucleic acid, isolation of the target nucleic acid, etc.).

[0262] Additionally, Jinek et al.,Science.2012 Aug 17;337(6096):816-21 and Chylinski et al.,RNA Biol.2013 May;10(5):726-37;Ma et al.,Biomed Res Int.2013;2013:270805;Hou et al.,Proc Natl Acad Sci US A.2013 Sep 24;110(39):15644-9;Jinek et al al.,Elife.2013;2:e00471;Pattanayak et al.,Nat Biotechnol.2013 Sep;31(9):839-43;Qi et al.,Cell.2013 Feb 28;152(5):1173-83;Wang et al.,Cell.2013 May 9;153(4):910-8;Auer et al.,Genome Res.2013 Oct 31;Chen et al.,Nucleic Acids Res.2013 Nov 1;41(20):e19;Cheng et al.,Cell Res.2013 Oct;23(10):1163-71 Cho et al al.,Genetics.2013 Nov;195(3):1177-80;DiCarlo et al.,Nucleic Acids Res.2013 Apr;41(7):4336-43;Dickinson et al.,Nat Methods.2013 Oct;10(10):1028-34;Ebina et al.,Sci Rep.2013;3:2510;Fujii et al,Nucleic Acids Res.2013 Nov 1;41(20):e187;Hu et al.,Cell Res.2013 Nov;23(11):1322-5;Jiang et al.,Nucleic Acids Res 1;41(20):e188;Larson et al.,Nat Protoc.2013 Nov;8(11):2180-96;2013 Dec;51(12):835-43、Ran et al.,Nat Protoc.2013 Nov;8(11):2281-308、Ran et al.,Cell.2013 Sep 12;154(6):1380-9、Upadhyay et al.,G3(Bethesda).2013 Dec 9;3(12):2233-8、Walsh et al.,Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15514-5、Xie et al.,Mol Plant.2013 Oct 9、Yang et al.,Cell.2013 Sep 12;154(6):1370-9, and U.S. Patents and Patent Applications Nos. 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, 8,697,359, 2014 / 0068797, 2014 / 0170753, 2014 / 0179006, 2014 / 0179770, 2014 / 0186843, 2014 / 0186 No. 919, No. 2014 / 0186958, No. 2014 / 0189896, No. 2014 / 0227787, No. 2014 / 0234972, No. 2014 / 0242664, No. 2014 / 0242699, No. 2014 / 0242700, No. 2 No. 014 / 0242702, No. 2014 / 0248702, No. 2014 / 0256046, No. 2014 / 0273037, No. 2014 / 0273226, No. 2014 / 0273230, No. 2014 / 0273231, No. 2014 / 027 No. 3232, No. 2014 / 0273233, No. 2014 / 0273234, No. 2014 / 0273235, No. 2014 / 0287938, No. 2014 / 0295556, No. 2014 / 0295557, No. 2014 / 0298547, No. 2014 / 0304853, 2014 / 0309487, 2014 / 0310828, 2014 / 0310830, 2014 / 0315985, 2014 / 0335063, 2014 / 0335620, 2014 / 03 See Nos. 42456, 2014 / 0342457, 2014 / 0342458, 2014 / 0349400, 2014 / 0349405, 2014 / 0356867, 2014 / 0356956, 2014 / 0356958, 2014 / 0356959, 2014 / 0357523, 2014 / 0357530, 2014 / 0364333, and 2014 / 0377868, each of which is incorporated by reference herein in its entirety.

[0263] For example, the present disclosure provides, but is not limited to, methods for cleaving a target nucleic acid, editing a target nucleic acid, modulating transcription from a target nucleic acid, isolating a target nucleic acid, binding a target nucleic acid, imaging a target nucleic acid, modifying a target nucleic acid, and the like.

[0264] As used herein, the terms / phrases "contacting a target nucleic acid," and "contacting a target nucleic acid," e.g., with a CRISPR-Cas effector polypeptide or with a CRISPR-Cas effector fusion polypeptide, encompass all methods for contacting a target nucleic acid. For example, a CRISPR-Cas effector polypeptide may be provided to a cell as a protein, RNA (encoding the CRISPR-Cas effector polypeptide), or DNA (encoding the CRISPR-Cas effector polypeptide), while a CRISPR-Cas effector guide RNA may be provided as a guide RNA or as a nucleic acid encoding the guide RNA. Thus, for example, when a method is performed in a cell (e.g., inside a cell in vitro, inside a cell in vivo, inside a cell ex vivo), a method that includes contacting a target nucleic acid encompasses the introduction into the cell of any or all of the components in their active / final state (e.g., in the form of a protein(s) of a CRISPR-Cas effector polypeptide, in the form of a protein of a CRISPR-Cas effector fusion polypeptide, and optionally in the form of an RNA of a guide RNA), and also encompasses the introduction into the cell of one or more nucleic acids encoding one or more of the components (e.g., nucleic acid(s) comprising nucleotide sequence(s) encoding a CRISPR-Cas effector polypeptide or a CRISPR-Cas effector fusion polypeptide, nucleic acid(s) comprising nucleotide sequence(s) encoding a guide RNA(s), nucleic acid(s) comprising a nucleotide sequence encoding a donor template, etc.). Methods can also be performed outside a cell in vitro, and thus methods that involve contacting a target nucleic acid encompass contacting anywhere (unless otherwise specified), e.g., outside a cell in vitro, inside a cell in vitro, inside a cell in vivo, inside a cell ex vivo, etc.

[0265] In some cases, the disclosed methods for modifying a target nucleic acid include introducing into a target cell a nucleic acid comprising a CRISPR-Cas effector locus, e.g., a nucleotide sequence encoding a CRISPR-Cas effector polypeptide, and about 1 kilobase (kb) to 5 kb in length of nucleotide sequences surrounding the CRISPR-Cas effector-encoding nucleotide sequence from a cell that comprises a CRISPR-Cas effector locus (e.g., in some cases, the cell in its native state (as it naturally occurs) comprises a CRISPR-Cas effector locus), and the target cell does not normally (in its natural state) comprise a CRISPR-Cas effector locus. However, one or more spacer sequences encoding guide sequences of the encoded crRNA(s) can be modified such that one or more target sequences of interest are targeted. Thus, for example, in some cases, a disclosed method for modifying a target nucleic acid includes introducing into a target cell a CRISPR-Cas effector locus, e.g., a nucleic acid obtained from a source cell (e.g., in some cases, the cell in its native state (the state in which it occurs in nature) contains a CRISPR-Cas effector locus), where the nucleic acid has a length of 100 nucleotides (nt) to 5 kb (e.g., 100 nt to 500 nt, 500 nt to 1 kb, 1 kb to 1.5 kb, 1.5 kb to 2 kb, 2 kb to 2.5 kb, 2.5 kb to 3 kb, 3 kb to 3.5 kb, 3.5 kb to 4 kb, or 4 kb to 5 kb) and includes a nucleotide sequence encoding a CRISPR-Cas effector polypeptide. As noted above, in such cases, one or more spacer sequences encoding guide sequences of the encoded crRNA(s) can be modified to target one or more target sequences of interest. In some cases, the method includes introducing into a target cell i) a CRISPR-Cas effector locus and ii) a donor DNA template. In some cases, the target nucleic acid is in a cell-free composition in vitro. In some cases, the target nucleic acid is present in a target cell. In some cases, the target nucleic acid is present in a target cell, and the target cell is a prokaryotic cell. In some cases, the target nucleic acid is present in a target cell, and the target cell is a eukaryotic cell.In some cases, the target nucleic acid is present in a target cell, and the target cell is a mammalian cell. In some cases, the target nucleic acid is present in a target cell, and the target cell is a plant cell.

[0266] In some cases, the disclosed methods for modifying a target nucleic acid include contacting the target nucleic acid with a CRISPR-Cas effector polypeptide of the present disclosure or with a CRISPR-Cas effector fusion polypeptide of the present disclosure. In some cases, the disclosed ...

Claims

1. (a) a CRISPR-Cas effector polypeptide or a nucleic acid molecule encoding the CRISPR-Cas effector polypeptide, wherein the CRISPR-Cas effector polypeptide comprises an amino acid sequence having 90% or more amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 57; (b) a CRISPR-Cas effector guide RNA, or one or more DNA molecules encoding said CRISPR-Cas effector guide RNA; A composition comprising: The CRISPR-Cas effector guide RNA is i) a region that binds to a CRISPR-Cas effector polypeptide, and ii) a guide sequence that hybridizes to the target sequence Including, The CRISPR-Cas effector polypeptide interacts with a CRISPR-Cas effector guide RNA to form a ribonucleoprotein complex that is targeted to the target sequence via base pairing between the guide RNA and the target sequence. The composition.

2. The composition of claim 1, wherein the CRISPR-Cas effector polypeptide comprises an amino acid sequence having 95% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

57.

3. 2. The composition of claim 1, wherein the CRISPR-Cas effector guide RNA comprises a nucleotide sequence having 90%, 95%, 98%, 99%, or 100% nucleotide sequence identity to the crRNA sequence set forth in SEQ ID NO:

58.

4. The composition of any one of claims 1 to 3, wherein the CRISPR-Cas effector polypeptide is fused to a nuclear localization signal (NLS).

5. 5. The composition of any one of claims 1 to 4, wherein the composition comprises a lipid; (a) and (b) are in a liposome; (a) and (b) are in a particle; or the composition comprises a buffer, a nuclease inhibitor, and a protease inhibitor.

6. The composition of any one of claims 1 to 5, wherein the CRISPR-Cas effector polypeptide is a nickase capable of cleaving only one strand of a double-stranded target nucleic acid molecule or a catalytically inactive CRISPR-Cas effector polypeptide (dCRISPR-Cas effector).

7. The composition of any one of claims 1 to 6, further comprising a DNA donor template.

8. A CRISPR-Cas effector fusion polypeptide comprising a CRISPR-Cas effector polypeptide fused to a heterologous polypeptide, wherein the CRISPR-Cas effector polypeptide comprises an amino acid sequence having 90% or greater amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:57, and the CRISPR-Cas effector fusion polypeptide interacts with a CRISPR-Cas effector guide RNA to form a ribonucleoprotein complex that is targeted to the target sequence via base pairing between the guide RNA and the target sequence. The CRISPR-Cas effector fusion polypeptide.

9. The CRISPR-Cas effector fusion polypeptide of claim 8, wherein the CRISPR-Cas effector polypeptide comprises an amino acid sequence having 95% or more identity to the amino acid sequence set forth in SEQ ID NO:

57.

10. The CRISPR-Cas effector fusion polypeptide of claim 8 or 9, wherein the CRISPR-Cas effector polypeptide is a nickase capable of cleaving only one strand of a double-stranded target nucleic acid molecule or is a catalytically inactive CRISPR-Cas effector polypeptide (dCRISPR-Cas effector).

11. The CRISPR-Cas effector fusion polypeptide of any one of claims 8 to 10, wherein the heterologous polypeptide is fused to the N-terminus and / or C-terminus of the CRISPR-Cas effector polypeptide.

12. The CRISPR-Cas effector fusion polypeptide of any one of claims 8 to 11, comprising a nuclear localization signal (NLS).

13. (i) the heterologous polypeptide is a targeting polypeptide that provides binding to a cell surface moiety on a target cell or cell type; (ii) the heterologous polypeptide exhibits an enzymatic activity that modifies a target DNA; (iii) the heterologous polypeptide exhibits an enzymatic activity that modifies a target polypeptide associated with a target nucleic acid; (iv) the heterologous polypeptide is an endosomal escape polypeptide; (v) the heterologous polypeptide is a chloroplast transit peptide; (vi) the heterologous polypeptide comprises a protein transduction domain; (vii) the heterologous polypeptide increases or decreases transcription; or (viii) the heterologous polypeptide is a protein-binding domain; A CRISPR-Cas effector fusion polypeptide according to any one of claims 8 to 12.

14. 14. The CRISPR-Cas effector fusion polypeptide of claim 13, wherein the heterologous polypeptide exhibits one or more enzymatic activities selected from nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damaging activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

15. 14. The CRISPR-Cas effector fusion polypeptide of claim 13, wherein the heterologous polypeptide exhibits one or more enzymatic activities selected from histone modifying activity, or methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylating activity, demyristoylating activity, glycosylation activity (e.g. from an O-GlcNAc transferase), and deglycosylating activity.

16. The CRISPR-Cas effector fusion polypeptide of claim 13, wherein the heterologous polypeptide is a transcriptional repressor domain or a transcriptional activation domain.

17. A nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector fusion polypeptide of any one of claims 8 to 16.

18. 18. The nucleic acid of claim 17, which is a recombinant adeno-associated viral vector, a recombinant retroviral vector, or a recombinant lentiviral vector.

19. (a) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; (b) a nucleotide sequence encoding a CRISPR-Cas effector polypeptide, wherein the CRISPR-Cas effector polypeptide comprises an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57; One or more nucleic acids comprising: The CRISPR-Cas effector guide RNA is i) a region that binds to a CRISPR-Cas effector polypeptide, and ii) a guide sequence that hybridizes to the target sequence Including, The CRISPR-Cas effector polypeptide interacts with a CRISPR-Cas effector guide RNA to form a ribonucleoprotein complex that is targeted to the target sequence via base pairing between the guide RNA and the target sequence. The one or more nucleic acids.

20. 20. The one or more nucleic acids of claim 19, wherein the one or more nucleic acids are one or more recombinant expression vectors.

21. 21. The one or more nucleic acids of claim 20, wherein the one or more recombinant expression vectors are selected from one or more recombinant adeno-associated viral vectors, one or more recombinant retroviral vectors, or one or more recombinant lentiviral vectors.

22. 22. A cell comprising a composition according to any one of claims 1 to 7, a CRISPR-Cas effector fusion polypeptide according to any one of claims 8 to 16, a nucleic acid according to claim 17 or 18, or one or more nucleic acids according to any one of claims 19 to 21, wherein the cell is a eukaryotic cell.

23. A cell comprising a CRISPR-Cas effector fusion polypeptide according to any one of claims 8 to 16 or a nucleic acid according to claim 17.

24. A method for modifying a target nucleic acid, comprising contacting said target nucleic acid with a composition according to any one of claims 1 to 7, the contacting results in modification of the target nucleic acid by the CRISPR-Cas effector polypeptide; and The contacting is performed inside a cell in vitro, inside a cell ex vivo, or outside a cell in vitro. The method.

25. 1. A method for modulating transcription from a target DNA, modifying a target nucleic acid, or modifying a protein associated with a target nucleic acid, comprising the steps of: The method further comprises: (a) a CRISPR-Cas effector fusion polypeptide according to any one of claims 8 to 16, and (b) a CRISPR-Cas effector guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid. contacting the The contacting is performed inside a cell in vitro, inside a cell ex vivo, or outside a cell in vitro. The method.

26. 26. The method of claim 25, wherein the CRISPR-Cas effector guide RNA comprises a nucleotide sequence having 85%, 90%, 95%, or 100% identity to the crRNA sequence set forth in SEQ ID NO:

58.

27. A composition according to any one of claims 1 to 7, a CRISPR-Cas effector fusion polypeptide according to any one of claims 8 to 16, a nucleic acid according to claim 17 or 18, or one or more nucleic acids according to any one of claims 19 to 21, for use in the treatment and / or prevention of a disease.

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