WO2024011176A2 - Modified crispr-cas effector polypeptides and methods of use thereof - Google Patents

Modified crispr-cas effector polypeptides and methods of use thereof Download PDF

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WO2024011176A2
WO2024011176A2 PCT/US2023/069709 US2023069709W WO2024011176A2 WO 2024011176 A2 WO2024011176 A2 WO 2024011176A2 US 2023069709 W US2023069709 W US 2023069709W WO 2024011176 A2 WO2024011176 A2 WO 2024011176A2
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polypeptide
cell
crispr
nucleic acid
cas effector
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PCT/US2023/069709
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French (fr)
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WO2024011176A3 (en
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Jennifer A. Doudna
Tanja KORTEMME
Benjamin Jagger
Cody KRIVACIC
Kai Chen
Spiros Liras
Victor RUSU
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The Regents Of The University Of California
Evercrisp Biosciences
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Publication of WO2024011176A2 publication Critical patent/WO2024011176A2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • a Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 459WO_SEQ_LIST.xml” created on July 3, 2023 and having a size of 124,952 bytes.
  • the contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
  • CRISPR-Cas systems comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid.
  • CRISPR-Cas systems can bind to and modify a targeted nucleic acid.
  • the programmable nature of these CRISPR-Cas effector systems has facilitated their use as a versatile technology for use in, e.g., gene editing.
  • Many therapeutic approaches involve delivery of a nucleic acid vector or messenger RNA encoding a CRISPR- Cas protein, rather than delivery of the CRISPR-Cas protein per se. Delivery of nucleic acid vectors encoding a CRISPR-Cas protein, however, has certain disadvantages, namely possible insertional mutagenesis and the possibility of off-target gene editing.
  • the present disclosure provides fusion polypeptides comprising a CRISPR-Cas effector polypeptide and a cell penetrating polypeptide.
  • the present disclosure provides compositions comprising a fusion polypeptide of the present disclosure and a guide nucleic acid.
  • the present disclosure provides methods of modifying a target nucleic acid in a eukaryotic cell.
  • the present disclosure provides for a fusion polypeptide comprising a CRISPR-Cas effector polypeptide and a cell penetrating polypeptide (CPP), wherein the CPP is an A22p polypeptide.
  • CPP cell penetrating polypeptide
  • the present disclosure provides for a fusion polypeptide comprising two or more nuclear localization sequences (NLSs), a CRISPR-Cas effector polypeptide, and a CPP, wherein the CPP is a Bac7 polypeptide.
  • the present disclosure provides for a fusion polypeptide comprising two or more NLSs, a CRISPR-Cas effector polypeptide, and a CPP, wherein the CPP is a CATat2 polypeptide.
  • the present disclosure provides for a fusion polypeptide comprising two or more NLSs, a CRISPR-Cas effector polypeptide, and a CPP, wherein the CPP is a VP22 polypeptide.
  • the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
  • the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO: 2), or a variant thereof.
  • the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO:3), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NO:5),or a variant thereof.
  • the VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO: 6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof.
  • the fusion polypeptide comprises two or more copies of the CPP. In some embodiments, the fusion polypeptide comprises a linker between the CPP and the CRISPR-Cas effector polypeptide. In some embodiments, the linker between the CPP and the CRISPR-Cas effector polypeptide is a proteolytically cleavable.
  • the two or more NLSs comprise the amino acid sequence K(K/R)X(K/R), where X is any amino acid.
  • the two or more NLSs comprise the amino acid sequence PKKKRKV (SEQ ID NO:8).
  • the two or more NLS are at the N-terminus of the CRISPR-Cas effector polypeptide.
  • the CPP is at the C- terminus of the CRISPR-Cas effector polypeptide.
  • the CPP is inserted internally within the CRISPR-Cas effector polypeptide.
  • the two or more NLSs are inserted at both the N- and C-termini.
  • the two or more NLSs are inserted at both the N- and C-termini and are inserted internally within the CRISPR-Cas effector polypeptide.
  • the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a Type V CRISPR- Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is catalytically active. In some embodiments, the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
  • the fusion polypeptide further comprises at least one additional heterologous polypeptide.
  • the at least one additional heterologous polypeptide is a deaminase, a base editor, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.
  • the present disclosure provides for a composition
  • a composition comprising: a) a fusion polypeptide, or a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
  • the guide nucleic acid is a single-molecule guide nucleic acid.
  • the composition further comprises a donor nucleic acid.
  • the present disclosure provides a nucleic acid that comprises a nucleotide sequence encoding the fusion polypeptide.
  • the nucleotide sequence is operably linked to a transcriptional control clement, optionally wherein the transcriptional control element is a promoter.
  • the nucleic acid is a recombinant expression vector.
  • the recombinant expression vector comprises the nucleic acid.
  • a cell comprises the fusion polypeptide. In some embodiments, a cell comprises the recombinant expression vector. In some embodiments, the cell (either of the cells above) is a eukaryotic cell. In some embodiments, the cell (either of the cells above) is in vitro. In some embodiments, the cell is in vivo.
  • the present disclosure provides for a method of modifying a target nucleic acid comprising contacting the target nucleic acid (e.g., in a eukaryotic cell) with a) a fusion polypeptide; and b) a guide nucleic acid.
  • the contacting includes introducing into a cell (e.g., a eukaryotic cell): a) the fusion polypeptide or a nucleic acid encoding the fusion polypeptide; and b) the guide nucleic acid or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
  • the nucleic acid encoding the fusion polypeptide and/or the nucleic acid encoding the guide nucleic acid is a recombinant expression vector. In some embodiments, the method comprises introducing a recombinant expression vector into the eukaryotic cell.
  • the method comprises introducing into the cell a donor nucleic acid.
  • the cell is in vitro.
  • the cell is in vivo.
  • the cell is a mammalian cell, an insect cell, an avian cell, a reptile cell, an amphibian cell, an arachnid cell, a protozoan cell, or a plant cell.
  • the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.
  • the modifying comprises genome editing.
  • the present disclosure provides for a fusion polypeptide comprising: a) a CRISPR-Cas effector polypeptide; and b) a CPP, wherein the CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidsl058 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069;
  • the CPP is an A22p polypeptide, a Bac7 polypeptide, a CATat2 polypeptide, or a VP22 polypeptide.
  • the present disclosure provides for a fusion polypeptide comprising, in order from N-terminus to C-terminus: a) a first NLS; b) a second NLS; c) a CRISPR-Cas effector polypeptide; d) at least two CPPs, optionally wherein one or more independently selected peptide linkers are interposed between any two of polypeptides (a) through (d), optionally wherein the CPPs are separated by a peptide linker.
  • the at least two CPPs are A22p polypeptides.
  • the A22p polypeptides comprise the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
  • the at least two CPPs are Bac7 polypeptides.
  • the Bac7 polypeptides comprise the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof.
  • the at least two CPPs are CATat2 polypeptides.
  • the CATat2 polypeptides comprise the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG), or a variant thereof.
  • the at least two CPPs are VP22 polypeptides.
  • the VP22 polypeptides comprise the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NOG) or DAATATRGRSA ASRPTERPRAPARSASRPRRVD (SEQ ID NOG), or a variant thereof.
  • FIG. 1A-1D schematically depict an assay for testing delivery and editing efficiency.
  • FIG. 1A (SEQ ID NOG).
  • FIG. IB (SEQ ID NO:102).
  • FIG. ID GPNAT (SEQ ID NO:103) - GIHGVPAAT (SEQ ID NO: 104) - SLEVLFQ (SEQ ID NO: 105).
  • FIG. 2 depicts the effect of CATat2 cell-penetrating peptide (CPP) on delivery of Cas9- guidc RNA ribonuclcoprotcin (RNP) into neural progenitor cells (NPCs), and gene editing in the NPCs, in vitro.
  • CPP CATat2 cell-penetrating peptide
  • RNP Cas9- guidc RNA ribonuclcoprotcin
  • NPCs neural progenitor cells
  • FIG. 3 depicts the effect of VP22 CPP on delivery of Cas9-guide RNA RNP into NPCs, and gene editing in the NPCs, in vitro.
  • FIG. 4 depicts the effect of A22p CPP on delivery of Cas9-guide RNA RNP into NPCs, and gene editing in the NPCs, in vitro.
  • FIG. 5 depicts the effect of Bac7 CPP on delivery of Cas9-guide RNA RNP into NPCs, and gene editing in the NPCs, in vitro.
  • FIG. 6A-6D depict sites of insertion of a CPP into a Cas9 protein (FIG. 6A); and testing of a Bac7-inserted Cas9 (FIG. 6B-6D).
  • FIG. 6B Top:(SEQ ID NOs:106); Bottom: GPNAT (SEQ ID NO: 103) - GIHGVPAAT (SEQ ID NO: 104) - SLEVLFQ (SEQ ID NO: 105).
  • FIG. 7 schematically depicts CRISPR-Cas fusion polypeptides with variable numbers of C-terminal CPPs. (SEQ ID NO:1)
  • FIG. 8A-8E depict various CRISPR-Cas fusion polypeptides with variable numbers of C-terminal A22p CPPs.
  • FIG. 9 depicts a comparison of nucleofection delivery of a nucleic acid encoding a CRISPR-Cas fusion polypeptide and direct delivery of a CRISPR-Cas fusion polypeptide.
  • FIG. 10 depicts delivery of RNPs comprising various CRISPR-Cas fusion polypeptides with A22p as the CPP.
  • FIG. 11 depicts delivery of RNPs comprising various CRISPR-Cas fusion polypeptides with A22p as the CPP.
  • FIG. 12A-12B depict RNP delivery of Lb Cast 2a using CPPs.
  • FIG. 13 depicts RNP delivery of Lb Casl2a using CPPs.
  • FIG. 14A-14J provide amino acid sequences of various CRISPR-Cas effector polypeptides.
  • Heterologous refers to an amino acid sequence that is not found in the native polypeptide.
  • a fusion CRISPR-Cas effector polypeptide comprises: a) a CRISPR-Cas effector polypeptide; and b) one or more heterologous polypeptides, where the heterologous polypeptide comprises an amino acid sequence from a protein other than a CRISPR-Cas effector polypeptide.
  • Heterologous refers to a nucleotide sequence that is not found in the native nucleic acid.
  • a heterologous guide nucleotide sequence (present in a targeting segment) that can hybridize with a target nucleotide sequence (target region) of a target nucleic acid is a nucleotide sequence that is not found in nature in a guide nucleic acid together with a binding segment that can bind to a CRISPR-Cas effector polypeptide of the present disclosure.
  • a heterologous target nucleotide sequence (present in a heterologous targeting segment) is from a different source than a binding nucleotide sequence (present in a binding segment) that can bind to a CRISPR-Cas effector polypeptide of the present disclosure.
  • a guide nucleic acid may comprise a guide nucleotide sequence (present in a targeting segment) that can hybridize with a target nucleotide sequence present in a eukaryotic target nucleic acid.
  • a guide nucleic acid of the present disclosure can be generated by human intervention and can comprise a nucleotide sequence not found in a naturally- occurring guide nucleic acid.
  • nucleic acid refers to a nucleic acid, cell, protein, or organism that is found in nature.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides or combinations thereof. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • polynucleotide and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and nongene tically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence.
  • Polypeptides as described herein also include polypeptides having various amino acid additions, deletions, or substitutions relative to the native amino acid sequence of a polypeptide of the present disclosure.
  • polypeptides that are homologs of a polypeptide of the present disclosure contain non-conservative changes of certain amino acids relative to the native sequence of a polypeptide of the present disclosure.
  • polypeptides that are homologs of a polypeptide of the present disclosure contain conservative changes of certain amino acids relative to the native sequence of a polypeptide of the present disclosure, and thus may be referred to as conservatively modified variants.
  • a conservatively modified variant may include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well-known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.
  • the following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • a modification of an amino acid to produce a chemically similar amino acid may be referred to as an analogous amino acid.
  • a polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.
  • FASTA is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).
  • DNA regulatory sequences refer to transcriptional and translational control sequences, such as promoters, enhancers, poly adenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.
  • transformation is used interchangeably herein with “genetic modification” and refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (e.g., DNA exogenous to the cell) into the cell.
  • Genetic change (“modification”) can be accomplished either by incorporation of the new nucleic acid into the genome of the host cell, or by transient or stable maintenance of the new nucleic acid as an episomal element.
  • a permanent genetic change is generally achieved by introduction of new DNA into the genome of the cell.
  • chromosomes In prokaryotic cells, permanent changes can be introduced into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell.
  • Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.
  • the choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. 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.
  • “Operably linked” refers to a juxtaposition wherein the components so described arc in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • heterologous promoter and “heterologous control regions” refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature.
  • a “transcriptional control region heterologous to a coding region” is a transcriptional control region that is not normally associated with the coding region in nature.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, c.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms "individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, nonhuman primates, ungulates, felines, canines, bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, “about 100” means an amount of from 90- 110. Where about is used in the context of a range, the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, “from about 100 to about 1000” means that the range extends from 90 to 1100.
  • a and/or B is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used herein a phrase such as “A. B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • isolated and purified refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment).
  • isolated when used in reference to an isolated protein, refers to a protein that has been removed from the culture medium of the host cell that expressed the protein. As such an isolated protein is free of extraneous or unwanted compounds (e.g., nucleic acids, native bacterial or other proteins, etc.).
  • the present disclosure provides fusion polypeptides comprising a CRISPR-Cas effector polypeptide and one or more heterologous polypeptides, where at least one of the one or more heterologous polypeptides is one that facilitates cellular uptake and endosomal escape of a complex comprising the fusion polypeptide.
  • the present disclosure provides compositions comprising a fusion polypeptide of the present disclosure and a guide nucleic acid.
  • a heterologous polypeptide that facilitates cellular uptake and endosomal escape of a complex comprising a fusion polypeptide of the present disclosure is referred to herein as a “cell penetrating polypeptide” (CPP).
  • a suitable CPP facilitates cellular uptake and endosomal escape of a fusion polypeptide of the present disclosure.
  • a suitable CPP is one that, when present in a fusion polypeptide comprising a CRISPR-Cas effector polypeptide, provides for an increase in cellular uptake of the fusion polypeptide (and thus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% (or two-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or more than 50-fold, compared to the cellular update of the same CRISPR-Cas effector polypeptide not fused to the CPP, or not fused to the CPP in the same configuration.
  • a CPP can be at or near the N-terminus of a CRISPR-Cas effector polypeptide; for example, a CPP can be at the N-terminus of the CRISPR-Cas effector polypeptide, or can be within 10 amino acids (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of the N-terminus of the CRISPR-Cas effector polypeptide.
  • a CPP can be at or near the C-terminus of a CRISPR-Cas effector polypeptide; for example, a CPP can be at the C terminus of the CRISPR-Cas effector polypeptide, or can be within 10 amino acids (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of the C-terminus of the CRISPR-Cas effector polypeptide.
  • a CPP can be inserted internally within a CRISPR-Cas effector polypeptide.
  • a fusion polypeptide can comprise a single CPP.
  • a fusion polypeptide can comprise 2 CPPs.
  • a fusion polypeptide can comprise 3 CPPs.
  • a fusion polypeptide can comprise 4 CPPs.
  • a fusion polypeptide can comprise more than 4 CPPs.
  • a fusion polypeptide of the present disclosure comprises: i) a single CPP at or near the N-terminus of the CRISPR-Cas effector polypeptide; and ii) a single CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide.
  • a fusion polypeptide comprises: i) 2 CPPs at or near the N-terminus of the CRISPR-Cas effector polypeptide; and ii) a single CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide.
  • a fusion polypeptide comprises: i) a single at or near the N-terminus of the CRISPR-Cas effector polypeptide; and ii) 2 CPPs at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises 2 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises 3 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide.
  • a fusion polypeptide comprises 4 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises 5 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide.
  • a CPP can have a length of from about 10 amino acids to about 60 amino acids; e.g., a CPP can have a length of from 10 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids, from 20 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, from 45 amino acids to 50 amino acids, from 50 amino acids to 55 amino acids, or from 55 amino acids to 60 amino acids.
  • a CPP has a length of from 10 amino acids to 15 amino acids (e.g., 10, 11, 12, 13, 14, or 15 amino acids) In some cases, a CPP has a length of from 15 amino acids to 20 amino acids (e.g., 15, 16, 17, 18, 19, or 20 amino acids). In some cases, a CPP has a length of from 20 amino acids to 25 amino acids (e.g., 20, 21, 22, 23, 24, or 25 amino acids). In some cases, a CPP has a length of from 25 amino acids to 30 amino acids (e.g., 25, 26, 27, 28, 29, or 30 amino acids). In some cases, a CPP has a length of from 30 amino acids to 35 amino acids (e.g., 30, 31, 32, 33, 34, or 35 amino acids).
  • Suitable CPPs include, but are not limited to, an A22p polypeptide, a Bac7 polypeptide, a CATat2 polypeptide, a VP22 polypeptide, an MPG polypeptide, a TP10 polypeptide, a pVEC polypeptide, a pep-1 polypeptide, a Mel-Pl polypeptide, an ATRAM polypeptide, an Aurein 1.2 polypeptide, a buforin II polypeptide, a ZF5.3 polypeptide, an aPP6Rl polypeptide, and the like.
  • the CPP is an A22p polypeptide, where the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
  • a variant of an A22p polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1).
  • an A22p polypeptide has a length of 22 amino acids.
  • the CPP is a Bac7 polypeptide, where the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NOG), or a variant thereof.
  • a variant of a Bac7 polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2).
  • a Bac7 polypeptide has a length of 24 amino acids.
  • the CPP is a CATat2 polypeptide, where the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), or a variant thereof.
  • a variant of a CATat2 polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG).
  • a CATat2 polypeptide has a length of 13 amino acids.
  • a CATat2 polypeptide has the following amino acid sequence: KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NOG).
  • a CATat2 polypeptide has a length of from 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38, 39, or 40 amino acids). In some cases, a CATat2 polypeptide has the following amino acid sequence: KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG). In some cases, a CATat2 polypeptide has a length of from 40 amino acids to 45 amino acids (e.g., 40, 41, 42, 43, 44, or 45 amino acids).
  • the CPP is a VP22 polypeptide.
  • a VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NOG), or a variant thereof.
  • a variant of a VP22 polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID N0:6).
  • a VP22 polypeptide has a length of 32 amino acids.
  • a VP22 polypeptide comprises the amino acid sequence: DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7); and has a length of from 30 amino acids to 35 amino acids (e.g., 30, 31, 32, 33, 34, or 35 amino acids).
  • a fusion polypeptide of the present disclosure can include, in addition to the CRISPR- Cas effector polypeptide and the CPP, a nuclear localization signal (NLS).
  • NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:8); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:9)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 10) or RQRRNELKRSP (SEQ ID NO: 11); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 12); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKD
  • NLS are of sufficient strength to drive accumulation of the fusion protein in a detectable amount in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.
  • a fusion polypeptide of the present disclosure includes (is fused to) a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs).
  • NLS nuclear localization signal
  • a fusion polypeptide of the present disclosure includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs).
  • one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N- terminus and/or the C-terminus.
  • one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C- terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C- terminus.
  • a fusion polypeptide of the present disclosure comprises a single NLS at the N-terminus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide. In some cases, a fusion polypeptide of the present disclosure comprises a single NLS at the C-terminus of the CRISPR- Cas effector polypeptide present in the fusion polypeptide. In some cases, a fusion polypeptide of the present disclosure comprises two NLSs at the N-terminus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide.
  • a fusion polypeptide of the present disclosure comprises 2 NLSs at the C-terminus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide.
  • the following are non-limiting examples of fusion polypeptides comprising both a CPP and an NLS.
  • a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) a single CPP at the C- terminus.
  • a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 2 CPPs at the C-terminus.
  • a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 3 CPPs at the C-terminus.
  • a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N- terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 4 CPPs at the C-terminus.
  • a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 5 CPPs at the C-terminus.
  • a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) a single NLS at the C-terminus.
  • a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 2 NLSs at the C-terminus.
  • a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 3 NLSs at the C-terminus.
  • a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 4 NLSs at the C-terminus.
  • a CPP is inserted internally within a CRISPR-Cas effector polypeptide.
  • a CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidslO58 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246
  • a CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidsl058 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246 and 1247; and xvi) amino acids 1247 and 1248 based on the Spy Cas9 amino acid sequence depicted in FIG.
  • a CPP is inserted between amino acids 204 and 205 based on the Spy Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide (e.g., in another type 11 CRISPR-Cas effector polypeptide).
  • a CPP is inserted between amino acids 205 and 206 based on the Spy Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide (e.g., in another type II CRISPR- Cas effector polypeptide).
  • a fusion polypeptide of the present disclosure comprises at least one CPP and a CRISPR-Cas effector polypeptide, and may further include one or more NLSs.
  • Suitable CRISPR-Cas effector polypeptides include Type II CRISPR-Cas effector polypeptides, Type III CRISPR Cas effector polypeptides, Type V CRISPR Cas effector polypeptides, and Type VI CRISPR-Cas effector polypeptides.
  • Suitable CRISPR-Cas effector polypeptides include fusion polypeptides that comprise: i) a CRISPR-Cas effector polypeptide; and ii) a heterologous polypeptide other than a CPP and an NLS.
  • a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in any one of FIG. 14A-14J.
  • CRISPR-Cas effector polypeptides are known to those skilled in the art, and any CRISPR-Cas effector polypeptide can be used in a fusion polypeptide of the present disclosure.
  • the amino acid sequences depicted in FIG. 14A-14J are provided as examples.
  • the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide.
  • the type II CRISPR-Cas effector polypeptide is a Cas9 polypeptide, e.g., Staphylococcus aureus Cas9, Streptococcus pyogenes Cas9 (SpCas9), etc.
  • the CRISPR- Cas effector polypeptide is a variant of a wild-type SpCas9 and comprises one or more of the following substitutions: A61R, Lil HR, A1322R, D1135L, S1136W, G1218K, E1219Q, N1317R, R1333P, R1335A, and T1337R.
  • the CRISPR-Cas effector polypeptide is an SpG polypeptide or a SpRY polypeptide; see, e.g., Walton et al. (2020) Science 368:290, and WO 2019/051097.
  • a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes DI 135V, R1135Q, and T1137R substitutions, relative to wild-type SpCas9.
  • a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes DI 135V, R1335Q, T1337R, and G1218R substitutions, relative to wild-type SpCas9.
  • a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes D1135L, S1136W, G1218K, E1219Q, R1335A, and T1337R substitutions, relative to wild-type SpCas9.
  • a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes L1111R, A1322R, D1135L, S1136W, G1218K, E1219Q, R1335A, and T1337R substitutions, relative to wild-type SpCas9.
  • a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes A61R, L1111R, A1322R, D1135L, S1136W, G1218K, E1219Q, N1317R, R1333P, R1335A, and T1337R substitutions, relative to wild-type SpCas9.
  • the amino acid sequence of a wild-type SpCas9 polypeptide is provided in FIG. 14A.
  • one or more surface Cys residues of a CRISPR-Cas effector polypeptide are substituted.
  • a Cys at position 80 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with an amino acid other than a Cys.
  • a Cys at position 80 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with a Ser.
  • a Cys at position 574 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with an amino acid other than a Cys.
  • a Cys at position 574 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with a Ser.
  • a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14A, where amino acid 80 is other than a Cys, e.g., where amino acid 80 is a Ser.
  • a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG.
  • the CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide, e.g., a Casl2a, a Casl2b, a Casl2c, a Casl2d, or a Casl2c polypeptide.
  • the CRISPR-Cas effector polypeptide is a type VI CRISPR-Cas effector polypeptide, e.g., a Casl3a polypeptide, a Casl3b polypeptide, a Casl3c polypeptide, or a Casl3d polypeptide.
  • the CRISPR-Cas effector polypeptide is a Casl4 polypeptide.
  • the CRISPR-Cas effector polypeptide is a Casl4a polypeptide, a Casl4b polypeptide, or a Casl4c polypeptide.
  • the CRISPR-Cas effector polypeptide is a Cas7-ll polypeptide; see, e.g., Ozcan et al. (2021) Nature 597:720.
  • the CRISPR-Cas effector polypeptide is a CRISPRi polypeptide; see, e.g., Qi et al. (2013) Cell 152:1173; and Jensen et al. (2021) Genome Research doi:10.1101/gr.275607.121.
  • the CRISPR-Cas effector polypeptide is a CRISPRa polypeptide; see, e.g., Jensen et al.
  • the CRISPR-Cas effector polypeptide is a CRISPRoff polypeptide. See, e.g., Nunez et al. (2021) Cell 184:2503. In some cases, the CRISPR-Cas effector polypeptide is a nickase. In some cases, the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
  • a CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide, such as a Casl3a polypeptide, a Casl3b polypeptide, a Casl3c polypeptide, a Casl3d polypeptide, a Casl3e polypeptide, a Casl3f polypeptide, a Casl3X polypeptide, or a Casl3Y polypeptide.
  • a CRISPR-Cas effector polypeptide is a Type III CRISPR-Cas effector polypeptide.
  • a suitable Type III CRISPR-Cas effector polypeptide is a Cas7-l l polypeptide (see, e.g., Ozcan et al. (2021) Nature 597:710).
  • a CRISPR-Cas effector polypeptide is an RNA-binding CRISPR-Cas effector polypeptide.
  • the CRISPR-Cas effector polypeptide is an RFx Casl3d polypeptide.
  • the CRISPR-Cas effector polypeptide is a dRfxCasl3d polypeptide.
  • the CRISPR-Cas effector polypeptide is a DjCasl3d polypeptide.
  • the CRISPR-Cas effector polypeptide is a PspCasl3b polypeptide, e.g., Prevotella sp. P5-125 Casl3b.
  • the CRISPR-Cas effector polypeptide is a dPspCasl3b polypeptide
  • a Casl3 polypeptide e.g., a Casl3a polypeptide, a Casl3b polypeptide, a Casl3c polypeptide, a Casl3d polypeptide, a Casl3e polypeptide, a Casl3f polypeptide, a Casl3X polypeptide, or a Casl3Y polypeptide
  • HEPN Higher Eukaryotes and Prokaryotes Nucleotide-binding
  • the CRISPR-Cas effector polypeptide is a variant that exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide. For example, where the
  • CRISPR-Cas effector polypeptide (e.g., a Casl3 polypeptide) comprises a HEPN domain
  • the CRISPR- Cas effector polypeptide can comprise a substitution of the Arg and/or the His in the HEPN motif, where such a mutation reduces the catalytic activity of the CRISPR-Cas effector polypeptide.
  • the RNA-binding CRISPR-Cas effector polypeptide binds, but does not cleave, a target RNA.
  • a CRISPR-Cas effector polypeptide is a variant that, when complexed with a guide nucleic acid, binds but does not cleave a target DNA.
  • the CRISPR-Cas effector polypeptide is a Cas7-11 polypeptide (e.g., a DiCas7-l l polypeptide). See, e.g., Ozcan et al. (2021) Nature 597:720.
  • the Cas7-11 polypeptide is a variant that exhibits reduced catalytic activity compared to a wildtype Cas7-ll effector polypeptide.
  • the variant Cas7-l l polypeptide comprises a substitution of one or more of D177, D429, D654, D758, E959, and D998 (where the amino acid numbering is as set forth in FIG. 14H).
  • a variant Cas7-ll can have an Asp at position 177, an Ala at position 429, an Ala at position 654, an Asp at position 758, a Glu at position E959, and an Asp at position 998.
  • a variant Cas7-l l comprises a D429A substitution.
  • the CRISPR-Cas effector polypeptide present in a fusion polypeptide of the present disclosure is itself a fusion polypeptide that comprises: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides other than a CPP and an NLS.
  • a fusion polypeptide of the present disclosure comprises: a) one or more CPPs; b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides other than an NLS and other than a CPP.
  • a fusion polypeptide of the present disclosure comprises: a) one or more CPPs; b) one or more NLSs; and c) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides other than an NLS and other than a CPP.
  • fusion partner a heterologous polypeptide
  • a fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA.
  • the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like).
  • the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like).
  • a transcription activator e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like.
  • the fusion partner is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner (heterologous polypeptide) is a deaminase.
  • a fusion partner has enzymatic activity that modifies a target nucleic acid
  • nuclease activity e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).
  • nuclease activity e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity
  • a fusion partner has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity).
  • a polypeptide e.g., a histone
  • a target nucleic acid e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin
  • proteins (or fragments thereof) that can be used in increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxy
  • TAT Ten
  • proteins (or fragments thereof) that can be used in decrease transcription include but are not limited to: transcriptional repressors such as the Kriippel associated box (KRAB or SKD); K0X1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7/8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC
  • a fusion partner has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA).
  • enzymatic activity that can be provided by the fusion partner include but are not limited to: 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., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-El
  • a fusion partner has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like).
  • a protein associated with the target nucleic acid e.g., ssRNA, dsRNA, ssDNA, dsDNA
  • a histone e.g., an RNA binding protein, a DNA binding protein, and the like.
  • enzymatic activity that modifyies a protein associated with a target nucleic acid
  • enzymatic activity that modifyies a protein associated with a target nucleic acid
  • methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), Vietnamese histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/
  • Suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable fusion CRISPR-Cas effector protein), and a chloroplast transit peptide.
  • DHFR dihydrofolate reductase
  • heterologous polypeptides include, but are not limited to, a polypeptide that directly and/or indirectly provides for increased transcription and/or h anslation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription and/or translation regulator, a translation-regulating protein, etc.).
  • a target nucleic acid e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription and/or translation regulator, a translation-regulating protein, etc.
  • heterologous polypeptides to accomplish increased or decreased transcription include transcription activator and transcription repressor domains.
  • splicing factors e.g., RS domains
  • protein translation components e.g., translation initiation, elongation, and/or release factors; e.g.,
  • a fusion partner can be any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and/or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising; Endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example CPSF, CstF, CFIm and CFIIm); Exonucleases (for example XRN-1 or Exonuclease T); Deadenylases (for example HNT3); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1, UPF2, UPF3, U
  • 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 having nonsense mediated RNA decay 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 modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domain
  • RNA splicing factors that can be used (in whole or as fragments thereof) as heterologous polypeptides for a fusion Casl2L polypeptide have modular organization, with separ ate sequence-specific RNA binding modules and splicing effector domains.
  • members of the Serine/ Arginine-rich (SR) protein family contain N -terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion.
  • RRMs N -terminal RNA recognition motifs
  • ESEs exonic splicing enhancers
  • the hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal Glycine -rich domain.
  • Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites.
  • ss splice site
  • ASF/SF2 can recognize ESEs and promote the use of intron proximal sites
  • hnRNP Al can bind to ESSs and shift splicing towards the use of intron distal sites.
  • One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes.
  • Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions.
  • the long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals.
  • the short isoform Bcl-xS is a pro-apoptotic isoform and 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 ctb-elements that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites).
  • W02010075303 which is hereby incorporated by reference in its entirety.
  • 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 periphery recruitment (e.g., Lamin A, Lamin B, etc.), protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.). Nucleases
  • a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a nuclease; and optionally also includes c) one or more NLSs.
  • Suitable nucleases include, but are not limited to, a homing nuclease polypeptide; a FokI polypeptide; a transcription activator-like effector nuclease (TALEN) polypeptide; a McgaTAL polypeptide; a meganuclease polypeptide; a zinc finger nuclease (ZFN); an ARCUS nuclease; and the like.
  • the meganuclease can be engineered from an LADLIDADG homing endonuclease (LHE).
  • a megaTAL polypeptide can comprise a TALE DNA binding domain and an engineered meganuclease.
  • a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a reverse transcriptase polypeptide; and optionally also includes c) one or more NLSs.
  • the CRISPR-Cas effector polypeptide is catalytically inactive.
  • Suitable reverse transcriptases include, e.g., a murine leukemia virus reverse transcriptase; a Rous sarcoma virus reverse transcriptase; a human immunodeficiency virus type I reverse transcriptase; a Moloney murine leukemia virus reverse transcriptase; and the like. Base editors
  • a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a base editor; and optionally also includes c) one or more NLSs.
  • Suitable base editors include, e.g., an adenosine deaminase; a cytidine deaminase (e.g., an activation- induced cytidine deaminase (AID)); APOBEC3G; and the like); and the like.
  • a suitable adenosine deaminase is any enzyme that is capable of deaminating adenosine in DNA.
  • the deaminase is a TadA deaminase.
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • AGSLMDVLHHPGMNHRVE1TEG1LADECAALLSDFFRMRRQE1KAQKKAQSSTD (SEQ ID NO:25).
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Staphylococcus aureus TadA amino acid sequence:
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Bacillus subtilis TadA amino acid sequence:
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Salmonella typhimurium TadA:
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Shewanella putrefaciens TadA amino acid sequence:
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Haemophilus influenzae F3O31 TadA amino acid sequence:
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Caulobacter crescentus TadA amino acid sequence:
  • a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Geobacter sulfurreducens TadA amino acid sequence:
  • Cytidine deaminases suitable for inclusion in a Cas12L fusion polypeptide include any enzyme that is capable of deaminating cytidine in DNA.
  • the cytidine deaminase is a deaminase from the apolipoprotein B mRNA-cditing complex (APOB EC) family of deaminases.
  • the APOBEC family deaminase is selected from the group consisting of APOBEC 1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase.
  • the cytidine deaminase is an activation induced deaminase (AID).
  • a suitable cytidine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO:34).
  • a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO: 33).
  • a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a transcription factor; and optionally also includes c) one or more NLSs.
  • a transcription factor can include: i) a DNA binding domain; and ii) a transcription activator.
  • a transcription factor can include: i) a DNA binding domain; and ii) a transcription repressor.
  • Suitable transcription factors include polypeptides that include a transcription activator or a transcription repressor domain (e.g., the Kruppel associated box (KRAB or SKD); the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), etc.); zinc-finger-based artificial transcription factors (see, e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificial transcription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics 14:781); and the like.
  • the transcription factor comprises a VP64 polypeptide (transcriptional activation).
  • the transcription factor comprises a Kriippel- associated box (KRAB) polypeptide (transcriptional repression).
  • the transcription factor comprises a Mad mSIN3 interaction domain (SID) polypeptide (transcriptional repression).
  • the transcription factor comprises an ERF repressor domain (ERD) polypeptide (transcriptional repression).
  • the transcription factor is a transcriptional activator, where the transcriptional activator is GAL4-VP16.
  • a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a recombinase; and optionally also includes c) one or more NLSs.
  • Suitable recombinases include, e.g., a Cre recombinase; a Hin recombinase; a Tre recombinase; a FLP recombinase; and the like.
  • a fusion polypeptide of the present disclosure comprises one or more linker polypeptides.
  • the linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used.
  • the linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide.
  • the use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide.
  • the creation of such sequences is routine to those of skill in the art.
  • a variety of different linkers are commercially available and are considered suitable for use.
  • linker polypeptides include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , and (GGGGS)n (SEQ ID NO:36), where n is an integer from 1 to 10), glycine-alanine polymers, serine polymers, and alanine-serine polymers.
  • Exemplary linkers can comprise amino acid sequences including, but not limited to, GGS, GS, GGSG (SEQ ID NO:37), GGSGG (SEQ ID NO:38), GSGSG (SEQ ID NO:39), GSGGG (SEQ ID NO:40), GGGSG (SEQ ID N0:41), GSSSG (SEQ ID NO:42), and the like.
  • the ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.
  • Suitable linkers include, e.g., GSSSSSSGS (SEQ ID NO:43).
  • a suitable spacer peptide includes, e.g., GIHGVPATT (SEQ ID NO:44).
  • a fusion polypeptide of the present disclosure comprises a linker between a CPP and a CRISPR-Cas effector polypeptide present in the fusion polypeptide.
  • the linker is a proteolytically cleavable linker.
  • the proteolytically cleavable linker can include a protease recognition sequence recognized by a protease selected from the group consisting of alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, cndoprotcinasc Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, IgA-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, me
  • the proteolytically cleavable linker can comprise a matrix metalloproteinase cleavage site, e.g., a cleavage site for a MMP selected from collagenase- 1, -2, and -3 (MMP-1, -8, and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and - 11), matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP).
  • MMP-1, -8, and -13 gelatinase A and B
  • MMP-2 and -9 gelatinase A and B
  • MMP-3, -10, and - 11 stromelysin 1, 2, and 3
  • MMP-7 matrilysin
  • MT1-MMP and MT2-MMP membrane metalloproteinases
  • the cleavage sequence of MMP-9 is Pro-X-X-Hy (wherein, X represents an arbitrary residue; Hy, a hydrophobic residue), e.g., Pro-X-X-Hy-(SerZThr), e.g., Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO:45) or Pro-Leu/Gln-Gly-Met-Thr (SEQ ID NO:46).
  • a protease cleavage site is a plasminogen activator cleavage site, e.g., a uPA or a tissue plasminogen activator (tPA) cleavage site.
  • the cleavage site is a furin cleavage site.
  • cleavage sequences of uPA and tPA include sequences comprising Val-Gly-Arg.
  • Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., ENLYTQS (SEQ ID NO:47), where the protease cleaves between the glutamine and the serine.
  • TSV tobacco etch virus
  • protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., DDDDK (SEQ ID NO:48), where cleavage occurs after the lysine residue.
  • enterokinase cleavage site e.g., DDDDK (SEQ ID NO:48)
  • a protease cleavage site that can be included in a proteolytically cleavable linker
  • a thrombin cleavage site e.g., LVPR (SEQ ID NO:49).
  • linkers comprising protease cleavage sites include linkers comprising one or more of the following amino acid sequences: LEVLFQGP (SEQ ID NO:50), cleaved by PreScission protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase; Walker et al. (1994) Biotechnol.
  • a thrombin cleavage site e.g., CGLVPAGSGP (SEQ ID NO:51); SLLKSRMVPNFN (SEQ ID NO:52) or SLLIARRMPNFN (SEQ ID NO:53), cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO:54) or SSYLKASDAPDN (SEQ ID NO:55), cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO:56) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO:57) cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO:58) cleaved by MMP-9; DVDERDVRGFASFL SEQ ID NO:59) cleaved by a thermolysin-like MMP; SLLKSRMVPNFN
  • GGSGQRGRKALE (SEQ ID NO:68) cleaved by tissue-type plasminogen activator(tPA); SLSALLSSDIFN (SEQ ID NO:69) cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ ID NO:70) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO:71 cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:72) cleaved by calpain (calcium activated neutral protease).
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure.
  • the present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure.
  • the nucleic acids and recombinant expression vectors are useful for producing a fusion polypeptide of the present disclosure.
  • the nucleotide sequence encoding the fusion polypeptide is operably linked to one or more transcriptional control elements, e.g., promoters, such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
  • promoters such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
  • Suitable expression vectors are well known and include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus; adeno-associated virus; human immunodeficiency virus, and the like).
  • viral vectors e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus; adeno-associated virus; human immunodeficiency virus, and the like.
  • any of a number of well-known, suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression.
  • the present disclosure provides a genetically modified host cell, where the host cell is genetically modified with a nucleic acid or a recombinant expression vector encoding a fusion polypeptide of the present disclosure.
  • Suitable host cells include eukaryotic cells, such as yeast cells, insect cells, and mammalian cells. In some cases, the host cell is a cell of a mammalian cell line.
  • Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.
  • Suitable mammalian cell lines are likewise well known and include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No.
  • ATCC American Type Culture Collection
  • CCL-2 CHO cells
  • CHO cells e.g., ATCC Nos. CRL9618, CCL61, CRL9096
  • 293 cells e.g., ATCC No. CRL-1573
  • Vero cells e.g., ATCC No. CRL-1573
  • Vero cells e.g., ATCC No. CRL-1658
  • Huh-7 cells BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
  • HEK human embryonic kidney
  • a fusion polypeptide can be produced using a genetically modified host cell as described herein.
  • this disclosure provides methods of producing a fusion polypeptide of the present disclosure. The methods generally involve culturing, in a culture medium, a host cell (an “expression host cell”) that is genetically modified with a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding the fusion polypeptide; and isolating the fusion polypeptide from the genetically modified host cell and/or the culture medium.
  • a host cell an “expression host cell”
  • a nucleic acid e.g., a recombinant expression vector
  • Isolation of the fusion polypeptide from the expression host cell e.g., from a lysate of the expression host cell
  • the culture medium in which the host cell is cultured can be earned out using standard methods of protein purification.
  • a lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • HPLC high performance liquid chromatography
  • exclusion chromatography gel electrophoresis
  • affinity chromatography affinity chromatography
  • the fusion polypeptide can be purified from the culture medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • compositions which are used will comprise at least 80% by weight, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, of the fusion polypeptide, in relation to contaminants related to the method of preparation of the product and its purification.
  • the percentages can be based upon total protein.
  • the fusion polypeptide can be purified using an immobilized binding partner of the affinity tag.
  • the present disclosure provides a composition comprising a fusion polypeptide of the present disclosure.
  • a composition of the present disclosure comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid.
  • a composition of the present disclosure comprises: i) a fusion polypeptide of the present disclosure; ii) a guide nucleic acid; and iii) a donor nucleic acid.
  • a guide RNA can be said to include two segments, a first segment (referred to herein as a “targeting segment”); and a second segment (referred to herein as a “protein-binding segment”).
  • targeting segment it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule.
  • a segment can also mean a region/section of a complex such that a segment may comprise regions of more than one molecule.
  • the “targeting segment” is also referred to herein as a “variable region” of a guide RNA.
  • the “protein-binding segment” is also referred to herein as a “constant region” of a guide RNA.
  • the targeting segment and the protein-binding segment are heterologous to one another.
  • the targeting segment comprises a nucleotide sequence that is complementary to a nucleotide sequence in a eukaryotic target nucleic acid.
  • the first segment (targeting segment) of a guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within a target nucleic acid (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.).
  • the protein-binding segment (or “protein-binding sequence”) interacts with (binds to) a CRISPR/Cas effector polypeptide.
  • the protein-binding segment of a guide RNA includes two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex).
  • Site-specific binding and/or cleavage of a target nucleic acid can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the guide RNA (the guide sequence of the guide RNA) and the target nucleic acid.
  • a guide RNA and a CRISPR/Cas effector polypeptide form a complex (e.g., bind via non- covalent interactions).
  • the guide RNA provides target specificity to the complex by including a targeting segment, which includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid).
  • the CRISPR/Cas effector polypeptide of the complex provides the site-specific activity (e.g., cleavage activity or an activity provided by the CRISPR/Cas effector polypeptide when the CRISPR/Cas effector polypeptide is a CRISPR/Cas effector polypeptide fusion polypeptide, i.e., has a fusion partner).
  • the CRISPR/Cas effector polypeptide is guided to a target nucleic acid sequence (e.g. a target sequence in a chromosomal nucleic acid, e.g., a chromosome; a target sequence in an extrachromosomal nucleic acid, e.g. an episomal nucleic acid, a minicircle, an ssRNA, an ssDNA, etc.; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid; a target sequence in a viral nucleic acid; etc.) by virtue of its association with the guide RNA.
  • a target nucleic acid sequence e.g. a target sequence in a chromosomal nucleic acid, e.g., a chromosome
  • a target sequence in an extrachromosomal nucleic acid e.g. an episomal nucleic acid,
  • the “guide sequence” also referred to as the “targeting sequence” of a guide RNA can be modified so that the guide RNA can target a CRISPR/Cas effector polypeptide to any desired sequence of any desired target nucleic acid, with the exception that the protospacer adjacent motif (PAM) sequence can be taken into account.
  • PAM protospacer adjacent motif
  • a guide RNA can have a targeting segment with a sequence (a guide sequence) that has complementarity with (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like.
  • a eukaryotic cell e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like.
  • a guide RNA includes two separate nucleic acid molecules and is referred to herein as a “dual guide RNA”, a “double -molecule guide RNA”, or a “two-molecule guide RNA” a “dual guide RNA”, or a “dgRNA.”
  • a guide RNA is a single-molecule RNA and is referred to as a “single guide RNA”, or simply “sgRNA.”
  • the targeting segment can have a length of 7 or more nucleotides (nt) (e.g., 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more nucleotides).
  • nt nucleotides
  • the targeting segment can have a length of from 7 to 100 nucleotides (nt) (e.g., from 7 to 80 nt, from 7 to 60 nt, from 7 to 40 nt, from 7 to 30 nt, from 7 to 25 nt, from 7 to 22 nt, from 7 to 20 nt, from 7 to 18 nt, from 8 to 80 nt, from 8 to 60 nt, from 8 to 40 nt, from 8 to 30 nt, from 8 to 25 nt, from 8 to 22 nt, from 8 to 20 nt, from 8 to 18 nt, from 10 to 100 nt, from 10 to 80 nt, from 10 to 60 nt, from 10 to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 10 to 18 nt, from 12 to 100 nt, from 12 to 80 nt, from 12 to 60 nt
  • the guide sequence of a guide RNA can have a length of from 15 nt to 30 nt (e.g., 15 to 25 nt, 15 to 24 nt, 15 to 23 nt, 15 to 22 nt, 15 to 21 nt, 15 to 20 nt, 15 to 19 nt, 15 to 18 nt, 17 to 30 nt, 17 to 25 nt, 17 to 24 nt, 17 to 23 nt, 17 to 22 nt, 17 to 21 nt, 17 to 20 nt, 17 to 19 nt, 17 to 18 nt, 18 to 30 nt, 18 to 25 nt, 18 to 24 nt, 18 to 23 nt, 18 to 22 nt, 18 to 21 nt, 18 to 20 nt, 18 to 19 nt, 19 to 30 nt, 19 to 25 nt, 19 to 24 nt, 19 to 23 nt, 19 to 22 nt, 19 to 30 nt, 19 to 25 nt
  • 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. In some cases, the guide sequence has a length of 24 nt.
  • the guide sequence (also referred to as a “spacer sequence”) has a length of from 15 to 50 nucleotides (e.g., from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt).
  • 15 to 50 nucleotides e.g., from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt.
  • the protein-binding segment of a guide RNA can include two stretches of nucleotides that are complementary to one another and hybridize to form a double stranded RNA duplex (dsRNA duplex).
  • dsRNA duplex double stranded RNA duplex
  • the protein-binding segment includes a dsRNA duplex.
  • the dsRNA duplex region includes a range of from 5-25 base pairs (bp) (e.g., from 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8, 8-25, 8-22, 8-18, 8-15, 8-12, 12-25, 12-22, 12-18, 12- 15, 13-25, 13-22, 13-18, 13-15, 14-25, 14-22, 14-18, 14-15, 15-25, 15-22, 15-18, 17-25, 17-22, or 17-18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.).
  • bp base pairs
  • the dsRNA duplex region includes a range of from 6-15 base pairs (bp) (e.g., from 6-12, 6-10, or 6-8 bp, e.g., 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the duplex region includes 5 or more bp (e.g., 6 or more, 7 or more, or 8 or more bp). In some cases, the duplex region includes 6 or more bp (e.g., 7 or more, or 8 or more bp). In some cases, not all nucleotides of the duplex region are paired, and therefore the duplex forming region can include a bulge.
  • bp base pairs
  • the term “bulge” herein is used to mean a stretch of nucleotides (which can be one nucleotide) that do not contribute to a double stranded duplex, but which are surround 5’ and 3’ by nucleotides that do contribute, and as such a bulge is considered part of the duplex region.
  • the dsRNA includes 1 or more bulges (e.g., 2 or more, 3 or more, 4 or more bulges).
  • the dsRNA duplex includes 2 or more bulges (e.g., 3 or more, 4 or more bulges).
  • the dsRNA duplex includes 1-5 bulges (e.g., 1-4, 1-3, 2-5, 2-4, or 2-3 bulges).
  • a guide nucleic acid can include one or more of: i) a modified sugar; ii) a modified nucleobase; and iii) one or more non-natural internucleoside linkages.
  • Suitable non-natural internucleoside linkage include, e.g., a phosphorothioate, a phosphor amidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'-alkylene phosphonates, a 5'-alkylene phosphonate, a chiral phosphonate, a phosphinate, a, a 3'-amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, and a boranophosphate.
  • a phosphorothioate e.g., a phosphorothioate, a
  • Suitable modified moieties include, e.g., locked nucleic acid (LNA) sugar moieties, 2'- substituted sugar moieties, 2'-O-methoxyethyl modified sugar moieties, one or more 2'-O-methyl modified sugar moieties, 2'-O-(2-methoxyethyl) modified sugar moieties, 2'-fluoro modified sugar moieties, 2'-dimethylaminooxyethoxy modified sugar moieties, and 2'-dimethylaminoethoxyethoxy modified sugar moieties.
  • LNA locked nucleic acid
  • Suitable modified nucleobases include, e.g., 5 -methylcytosines; 5 -hydroxymethyl cytosines; xanthines; hypoxanthines; 2-aminoadenines; 6-methyl derivatives of adenine; 6-methyl derivatives of guanine; 2-propyl derivatives of adenine; 2-propyl derivatives of guanine; 2-thiouracils; 2- thiothymines; 2-thiocytosines; 5-propynyl uracils; 5-propynyl cytosines; 6-azo uracils; 6-azo cytosines; 6-azo thymines; pseudouracils; 4-thiouracils; an 8-haloadenins; 8-aminoadenines; 8-thioladeninse; 8- thioalkyladcnincs; 8-hydroxyladcnincs; 8-haloguanincs; 8-aminoguanincs; 8-
  • a CRISPR-Cas effector protein in some cases generates site-specific double strand breaks (DSBs) or single strand breaks (SSBs) (e.g., when the CRISPR-Cas effector protein is a nickase variant) within double-stranded DNA (dsDNA) target nucleic acids, which are repaired either by non-homologous end joining (NHEJ) or homology-directed recombination (HDR).
  • NHEJ non-homologous end joining
  • HDR homology-directed recombination
  • a composition of the present disclosure comprises: i) a fusion polypeptide of the present disclosure; ii) a guide nucleic acid; and iii) a donor nucleic acid.
  • a donor nucleic acid comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid.
  • contacting a target DNA occurs under conditions that are permissive for nonhomologous end joining or homology-directed repair.
  • a subject method includes contacting the target DNA with a donor polynucleotide (e.g., by introducing the donor polynucleotide into a cell), wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA.
  • the method does not comprise contacting a cell with a donor polynucleotide, and the target DNA is modified such that nucleotides within the target DNA are deleted.
  • CRISPR-Cas effector guide RNA and a fusion polypeptide of the present disclosure are co-administered (e.g., contacted with a target nucleic acid, administered to cells, etc.) with a donor polynucleotide sequence that includes at least a segment with homology to the target DNA sequence
  • the subject methods may be used to add, i.e. insert or replace, nucleic acid material to a target DNA sequence (e.g.
  • a nucleic acid e.g., one that encodes for a protein, an siRNA, an miRNA, etc.
  • a tag e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.
  • a regulatory sequence e.g.
  • a nucleic acid sequence e.g., introduce a mutation, remove a disease causing mutation by introducing a correct sequence
  • modify a nucleic acid sequence e.g., introduce a mutation, remove a disease causing mutation by introducing a correct sequence
  • a donor polynucleotide (a nucleic acid comprising a donor sequence) can also be provided to the cell.
  • a donor sequence or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site cleaved by the CRISPR-Cas effector protein (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like).
  • the donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology.
  • sufficient homology to a genomic sequence at the target site e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology.
  • Donor polynucleotides can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.
  • the donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease-causing base pair to a non-disease-causing base pair).
  • the donor sequence comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non- homologous sequence at the target region.
  • Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest.
  • the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
  • the donor sequence may comprise certain sequence differences as compared to the genomic sequence, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor sequence at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus).
  • selectable markers e.g., drug resistance genes, fluorescent proteins, enzymes etc.
  • sequence differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence.
  • the donor sequence is provided to the cell as single-stranded DNA. In some cases, the donor sequence is provided to the cell as double-stranded DNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphor amidates, and O-methyl ribose or deoxyribose residues.
  • additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination.
  • a donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
  • donor sequences can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV), as described elsewhere herein for nucleic acids encoding a CRISPR-Cas effector guide RNA and/or a CRISPR-Cas effector fusion polypeptide and/or donor polynucleotide.
  • viruses e.g., adenovirus, AAV
  • a composition of the present disclosure can comprise, in addition to a fusion polypeptide of the present disclosure (and optionally also a CRISPR-Cas guide nucleic acid and optionally also a donor nucleic acid), one or more of: a) a lipid; b) a buffer; c) a nuclease inhibitor; and d) a protease inhibitor.
  • the present disclosure provides a system comprising a fusion polypeptide of the present disclosure.
  • a system of the present disclosure can comprise: a) a fusion polypeptide of the present disclosure and a CRISPR-Cas guide RNA; b) a fusion polypeptide of the present disclosure and a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas guide RNA; c) a fusion polypeptide of the present disclosure, a CRISPR-Cas guide RNA, and a donor nucleic acid; or d) a fusion polypeptide of the present disclosure, a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas guide RNA, and a donor nucleic acid.
  • the present disclosure provides methods of modifying a target nucleic acid and/or modifying a polypeptide associated with a target nucleic acid.
  • a method of the present disclosure comprises contacting a target nucleic acid with an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid.
  • a method of the present disclosure comprises contacting a target nucleic acid with: a) an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid; and b) a donor nucleic acid.
  • a method of the present disclosure comprises contacting a eukaryotic cell comprising a target nucleic acid with an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid.
  • a method of the present disclosure comprises contacting a eukaryotic cell comprising a target nucleic acid with: a) an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid; and b) a donor nucleic acid.
  • Suitable eukaryotic cells including mammalian cells, plant cells, insect cells, arachnid cells, protozoan cells, fish cells, fungal cells, yeast cells, amphibian cells, reptile cells, and avian cells.
  • a CRISPR-Cas effector-CPP fusion polypeptide of the present disclosure can be used to (i) modify (e.g., cleave, e.g., nick; methylate; deaminate; etc.) target nucleic acid (DNA or RNA; single stranded or double stranded); (ii) modulate transcription of a target nucleic acid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid (e.g., for purposes of isolation, labeling, imaging, tracking, etc.); (v) modify a polypeptide (e.g., a histone) associated with a target nucleic acid; and the like.
  • modify e.g., cleave, e.g., nick; methylate; deaminate; etc.
  • target nucleic acid DNA or RNA; single stranded or double stranded
  • modulate transcription of a target nucleic acid e
  • a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a CRISPR-Cas effector polypeptide of the present disclosure; and b) one or more (e.g., two) CRISPR-Cas effector guide RNAs.
  • a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a CRISPR-Cas effector polypeptide of the present disclosure; b) a CRISPR-Cas effector guide RNA; and c) a donor nucleic acid (e.g., a donor template).
  • the contacting step is carried out in a cell in vitro.
  • the contacting step is carried out in a cell in vivo.
  • the contacting step is carried out in a cell ex vivo.
  • Modifications of a target nucleic acid that can be accomplished using a method of the present disclosure include, e.g., non-homologous end joining (NHEJ), microhomology- mediated end joining (MMEJ), and homology-directed repair (HDR). Such modifications can result in in gene knockout, DNA fragment insertion, deletion, replacement, or other modification.
  • Modifications of a target nucleic acid that can be accomplished using a method of the present disclosure include base editing (e.g., modification of a cytidine or an adenosine).
  • Modifications of a target nucleic acid that can be accomplished using a method of the present disclosure include any modification that can be carried out by a fusion partner, as described above (e.g., reverse transcription, base editing, etc.).
  • the present disclosure provides (but is not limited to) methods of cleaving a target nucleic acid; methods of editing a target nucleic acid; methods of modulating transcription from a target nucleic acid; methods of isolating a target nucleic acid, methods of binding a target nucleic acid, methods of imaging a target nucleic acid, methods of modifying a target nucleic acid, and the like.
  • a method of modifying a target nucleic acid provides for treatment of a disease or disorder in an individual.
  • the present disclosure provides a method of treating a disease or disorder in an individual, the method comprising administering to the individual: a) a fusion polypeptide of the present disclosure; or b) a fusion polypeptide of the present disclosure and a guide nucleic acid; or c) a fusion polypeptide of the present disclosure, a guide nucleic acid, and a donor nucleic acid.
  • Target nucleic acids and target cells of interest are provided.
  • a target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the CRISPR-Cas effector guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted).
  • a chromosome genomic DNA
  • derived from a chromosome derived from a chromosome
  • chromosomal DNA plasmid
  • viral extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.
  • a target nucleic acid can be DNA or RNA.
  • a target nucleic acid can be double stranded
  • a target nucleic acid is single stranded.
  • a target nucleic acid is a single stranded RNA (ssRNA).
  • a target ssRNA e.g., a target cell ssRNA, a viral ssRNA, etc.
  • a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA).
  • ssDNA single stranded DNA
  • a target nucleic acid is single stranded.
  • a target nucleic acid can be genomic DNA (e.g., nuclear DNA).
  • a target nucleic acid can be mitochondrial DNA.
  • a target nucleic acid can be mitochondrial RNA.
  • a target nucleic acid can be extrachromosomal DNA.
  • a target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo.
  • Suitable target cells include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • a fungal cell e.g., a yeast cell
  • an animal cell e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.
  • a cell of an insect e.g., a mosquito; a bee; an agricultural pest; etc.
  • a cell of an arachnid e.g., a spider; a tick; etc.
  • a cell from a vertebrate animal e.g., a fish, an amphibian, a reptile, a bird, a mammal
  • a cell from a mammal e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g.,
  • a stem cell e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).
  • ES embryonic stem
  • iPS induced pluripotent stem
  • a germ cell e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.
  • a somatic cell
  • Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture.
  • primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage.
  • the primary cell lines are maintained for fewer than 10 passages in vitro.
  • Target cells can be unicellular organisms and/or can be grown in culture. If the cells are primary cells, they may be harvest from an individual by any convenient method.
  • leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy.
  • the subject methods may be employed to induce target nucleic acid cleavage, target nucleic acid modification, and/or to bind target nucleic acids (e.g., for visualization, for collecting and/or analyzing, etc.) in mitotic or post-mitotic cells in vivo and/or ex vivo and/or in vitro (e.g., to disrupt production of a protein encoded by a targeted mRNA, to cleave or otherwise modify target DNA, to genetically modify a target cell, and the like).
  • a mitotic and/or post-mitotic cell of interest in the disclosed methods may include a cell from any organism (e.g.
  • a bacterial cell e.g., a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g.
  • fruit fly cnidarian, echinoderm, nematode, etc.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a cell from a rodent, a cell from a human, etc.
  • a subject CRISPR-Cas effector protein (and/or nucleic acid encoding the protein such as DNA and/or RNA), and/or CRISPR-Cas effector guide RNA (and/or a DNA encoding the guide RNA), and/or donor template, and/or RNP can be introduced into an individual (i.e., the target cell can be in vivo) (e.g., a mammal, a rat, a mouse, a pig, a primate, a non-human primate, a human, etc.).
  • an administration can be for the purpose of treating and/or preventing a disease, e.g., by editing the genome of targeted cells.
  • Plant cells include cells of a monocotyledon, and cells of a dicotyledon.
  • the cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like.
  • Plant cells include cells of agricultural crops such as wheat, corn, rice, sorghum, millet, soybean, etc.
  • Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.
  • Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosscs, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Ch
  • seaweeds e.g. kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like.
  • the cell is a cell that does not originate from a natural organism (e.g.,
  • a cell can be an in vitro cell (e.g., established cultured cell line).
  • a cell can be an ex vivo cell (cultured cell from an individual).
  • a cell can be and in vivo cell (e.g., a cell in an individual).
  • a cell can be an isolated cell.
  • a cell can be a cell inside of an organism.
  • a cell can be an organism.
  • a cell can be a cell in a cell culture (e.g., in vitro cell culture).
  • a cell can be one of a collection of cells.
  • a cell can be a eukaryotic cell or derived from a eukaryotic cell.
  • a cell can be a plant cell or derived from a plant cell.
  • a cell can be an animal cell or derived from an animal cell.
  • a cell can be an invertebrate cell or derived from an invertebrate cell.
  • a cell can be a vertebrate cell or derived from a vertebrate cell.
  • a cell can be a mammalian cell or derived from a mammalian cell.
  • a cell can be a rodent cell or derived from a rodent cell.
  • a cell can be a human cell or derived from a human cell.
  • a cell can be a fungal cell or derived from a fungal cell.
  • a cell can be an insect cell.
  • a cell can be an arthropod cell.
  • a cell can be a protozoan cell.
  • a cell can be a helminth cell.
  • Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • germ cell e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.
  • a somatic cell e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell,
  • Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogenic cells, and post-
  • the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.
  • the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage.
  • the immune cell is a cytotoxic T cell.
  • the immune cell is a helper T cell.
  • the immune cell is a regulatory T cell (Treg).
  • the cell is a stem cell.
  • Stem cells include adult stem cells.
  • Adult stem cells are also referred to as somatic stem cells.
  • ра ⁇ stem cells are resident in differentiated tissue, but retain the properties of selfrenewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found.
  • somatic stem cells include muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.
  • Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc.
  • the stem cell is a human stem cell.
  • the stem cell is a rodent (e.g., a mouse; a rat) stem cell.
  • the stem cell is a non-human primate stem cell.
  • Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1 , OLFM4, CDH17, and PPARGC1 A.
  • stem cell markers e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1 , OLFM4, CDH17, and PPARGC1 A.
  • the stem cell is a hematopoietic stem cell (HSC).
  • HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34 + and CD3 . HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.
  • the stem cell is a neural stem cell (NSC).
  • NSCs neural stem cells
  • a neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively.
  • Methods of obtaining NSCs are known in the art.
  • the stem cell is a mesenchymal stem cell (MSC).
  • MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.
  • a cell is in some cases a plant cell.
  • a plant cell can be a cell of a monocotyledon.
  • a cell can be a cell of a dicotyledon.
  • the cell is a plant cell.
  • the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue- cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like.
  • a major agricultural plant e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts
  • the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns,
  • a cell is in some cases an arthropod cell.
  • the cell can be a cell of a suborder, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemi
  • a cell is in some cases an insect cell.
  • the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.
  • a composition comprising: a) an engineered CRISPR-Cas effector polypeptide or a nucleic acid comprising a first nucleotide sequence encoding the engineered CRISPR- Cas effector polypeptide; and b) a guide nucleic acid or a nucleic acid comprising a second nucleotide sequence encoding the guide nucleic acid; wherein the engineered CRISPR-Cas effector polypeptide comprises one or more heterologous polypeptides that facilitate cellular uptake of a nucleic acid/protein complex comprising the engineered Cas9 polypeptide and the guide nucleic acid into a eukaryotic cell; optionally wherein the one or more heterologous polypeptides facilitate release of the nucleic acid/protein complex from an endosome within the eukaryotic cell.
  • a composition comprising an engineered CRISPR-Cas effector polypeptide or a nucleic acid comprising a first nucleotide sequence encoding the engineered CRISPR-Cas effector polypeptide; wherein the engineered CRISPR-Cas effector polypeptide comprises one or more heterologous polypeptides that replace one or more native amino acids of a non-engineered CRISPR-Cas effector polypeptide; wherein the engineered CRISPR-Cas effector polypeptide is less immunogenic to a human subject compared to the immunogenicity of the non-engineered CRISPR-Cas effector polypeptide to the human subject.
  • a composition comprising an engineered CRISPR-Cas effector polypeptide or a nucleic acid comprising a first sequence encoding the engineered CRISPR-Cas effector polypeptide; wherein the engineered CRISPR-Cas effector polypeptide comprises one or more heterologous polypeptides inserted (i) at the N-terminus of the engineered CRISPR-Cas effector polypeptide, (ii) at the C-terminus of the engineered CRISPR-Cas effector polypeptide or (iii) internally within the engineered CRISPR-Cas effector polypeptide.
  • Aspect 4 The composition of aspect 2 or 3, wherein the composition further comprises a guide nucleic acid or a nucleic acid comprising a second nucleotide sequence encoding the guide nucleic acid.
  • Aspect 5 The composition of aspect 1 or 4, wherein the guide nucleic acid is a singlemolecule guide nucleic acid.
  • Aspect 6 The composition of any one of aspects 1-5, wherein the composition further comprises a donor nucleic acid.
  • Aspect 7 The composition of any one of aspects 1-6, wherein the engineered Cas9 polypeptide is a fusion polypeptide of a CRISPR-Cas effector polypeptide and the one or more heterologous polypeptides.
  • Aspect 8 The composition of any one of aspects 1-7, wherein the one or more heterologous polypeptides comprise one or more cell penetrating polypeptides (CPPs), optionally wherein the one or more CPPs comprises any one of the amino acid sequences set out in Table 1, or a variant thereof.
  • CPPs cell penetrating polypeptides
  • Aspect 9 The composition of aspect 8, wherein the CPP is an A22p polypeptide.
  • Aspect 10 The composition of aspect 9, wherein the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof [00192] Aspect 11. The composition of aspect 9, wherein the CPP is a Bac7 polypeptide.
  • Aspect 12 The composition of aspect 11, wherein the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPRPLPFPRPG (SEQ ID NO: 2), or a variant thereof.
  • Aspect 13 The composition of aspect 9, wherein the CPP is a CATat2 polypeptide.
  • Aspect 14 The composition of aspect 13, wherein the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG), or a variant thereof.
  • Aspect 15 The composition of aspect 9, wherein the CPP is a VP22 polypeptide.
  • Aspect 16 The composition of aspect 15, wherein the VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NOG) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof.
  • Aspect 17 The composition of any one of aspects 9-16, wherein the fusion polypeptide comprises two or more copies of the CPP.
  • Aspect 18 The composition of any one of aspects 9-17, wherein the fusion polypeptide comprises a linker between the CPP and the CRISPR-Cas effector polypeptide.
  • Aspect 19 The composition of aspect 18, wherein the linker between the CPP and the CRISPR-Cas effector polypeptide is a proteolytically cleavable.
  • Aspect 20 The composition of any one of aspects 9-19, wherein the fusion polypeptide comprises one or more nuclear localization sequences (NLSs).
  • Aspect 21 The composition of aspect 20, wherein the fusion polypeptide comprises two or more NLSs.
  • Aspect 22 The composition of aspect 20 or 21, wherein an NLS comprises the amino acid sequence K(K/R)X(K/R), where X is any amino acid.
  • Aspect 23 The composition of any one of aspects 20-22, wherein an NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO:8).
  • Aspect 24 The composition of any one of aspects 20-23, wherein at least one NLS or each NLS is at the N-terminus of the CRISPR-Cas effector polypeptide.
  • Aspect 25 The composition of any one of aspects 9-24, wherein the CPP is at the C- terminus of the CRISPR-Cas effector polypeptide.
  • Aspect 26 The composition of any one of aspects 9-25, wherein the CPP is inserted internally within the CRISPR-Cas effector polypeptide.
  • Aspect 27 The composition of any one of aspects 9-26, wherein the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide.
  • Aspect 28 The composition of any one of aspects 9-26, wherein the CRISPR-Cas effector polypeptide is a Type V CRISPR-Cas effector polypeptide.
  • Aspect 29 The composition of any one of aspects 9-26, wherein the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide.
  • Aspect 30 The composition of any one of aspects 9-29, wherein the CRISPR-Cas effector polypeptide is catalytically active.
  • Aspect 31 The composition of any one of aspects 9-29, wherein the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
  • Aspect 32 The composition of any one of aspects 9-31, wherein the fusion polypeptide further comprises at least one additional heterologous polypeptide.
  • Aspect 33 The composition of any one of aspects 9-32, wherein the at least one additional heterologous polypeptide is a deaminase, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.
  • Aspect 34 The composition of any one of aspects 9-33, wherein the at least one additional heterologous polypeptide is a targeting moiety that binds to a target of a eukaryotic cell.
  • Aspect 35 The composition of aspect 34, wherein the targeting moiety is an antibody or binding domain thereof.
  • Aspect 36 The composition of aspect 34, wherein the targeting moiety is or a small molecule.
  • Aspect 37 The composition of any one of aspects 34-36, wherein the target is a cell surface target of the eukaryotic cell.
  • a composition comprising a nucleic acid, wherein the nucleic acid comprises a nucleotide sequence encoding the engineered CRISPR-Cas effector polypeptide of the composition of any one of aspects 1-37.
  • Aspect 39 The composition of aspect 38, wherein the nucleotide sequence is operably linked to a transcriptional control element.
  • Aspect 40 The composition of aspect 39, wherein the transcriptional control element is a promoter.
  • Aspect 41 The composition of any one of aspects 38-40, wherein the nucleic acid is a recombinant expression vector.
  • a composition comprising a cell, wherein the cell comprises the engineered CRISPR-Cas effector polypeptide of the composition of any one of aspects 1-37 or the nucleic acid of the composition of any one of aspects 38-41.
  • Aspect 43 The composition of aspect 42, wherein the cell is a eukaryotic cell.
  • Aspect 44 The composition of aspect 42 or 43, wherein the cell is in vitro.
  • Aspect 45 The composition of aspect 42 or 43, wherein the cell is in vivo.
  • a pharmaceutical composition comprising a therapeutically effective amount of the composition of any one of aspects 1-44.
  • Aspect 47 A method of treating a disease or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of aspect 46.
  • Aspect 48 A method of modifying a target nucleic acid in a eukaryotic cell comprising introducing into the eukaryotic cell the composition of any one of aspects 1-41.
  • Aspect 49 The method of aspect 48, comprising introducing into the cell a donor nucleic acid.
  • Aspect 50 The method of aspect 48 or 49, wherein the cell is in vitro.
  • Aspect 51 The method of aspect 48 or 49, wherein the cell is in vivo.
  • Aspect 52 The method of any one of aspects 48-51, wherein the cell is a mammalian cell.
  • Aspect 53 The method of any one of aspects 48-51 , wherein the cell is a plant cell.
  • a fusion polypeptide comprising: a) a CRISPR-Cas effector polypeptide; and b) a cell penetrating polypeptide (CPP), wherein the CPP is an A22p polypeptide.
  • CPP cell penetrating polypeptide
  • a fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a Bac7 polypeptide.
  • CPP cell penetrating polypeptide
  • a fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a CAT t2 polypeptide.
  • CPP cell penetrating polypeptide
  • a fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a VP22 polypeptide.
  • CPP cell penetrating polypeptide
  • Aspect 5 The fusion polypeptide of aspect 1, wherein the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
  • Aspect 6 The fusion polypeptide of aspect 2, wherein the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof.
  • Aspect 7 The fusion polypeptide of aspect 3, wherein the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG), or a variant thereof.
  • Aspect 8 The fusion polypeptide of aspect 4, wherein the VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO:6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NOG), or a variant thereof.
  • Aspect 9 The fusion polypeptide of any one of aspects 1-8, wherein the fusion polypeptide comprises two or more copies of the CPP.
  • Aspect 10 The fusion polypeptide of any one of aspects 1-9, wherein the fusion polypeptide comprises a linker between the CPP and the CRISPR-Cas effector polypeptide.
  • Aspect 11 The fusion polypeptide of aspect 10, wherein the linker between the CPP and the CRISPR-Cas effector polypeptide is a proteolytically cleavable.
  • Aspect 12 The fusion polypeptide of any one of aspects 1-11, wherein the two or more NLSs comprise the amino acid sequence K(K/R)X(K/R), where X is any amino acid.
  • Aspect 13 The fusion polypeptide of aspect 12, wherein the two or more NLSs comprise the amino acid sequence PKKKRKV (SEQ ID NO: 8).
  • Aspect 14 The fusion polypeptide of any one of aspects 1-13, wherein the two or more NLS are at the N-terminus of the CRISPR-Cas effector polypeptide.
  • Aspect 15 The fusion polypeptide of any one of aspects 1-14, wherein the CPP is at the C-terminus of the CRISPR-Cas effector polypeptide.
  • Aspect 16 The fusion polypeptide of any one of aspects 1-14, wherein the CPP is inserted internally within the CRISPR-Cas effector polypeptide.
  • Aspect 17 The fusion polypeptide of any one of aspects 1-16, wherein the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide.
  • Aspect 18 The fusion polypeptide of any one of aspects 1-16, wherein the CRISPR-Cas effector polypeptide is a Type V CRISPR-Cas effector polypeptide.
  • Aspect 19 The fusion polypeptide of any one of aspects 1-16, wherein the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide.
  • Aspect 20 The fusion polypeptide of any one of aspects 1-19, wherein the CRISPR-Cas effector polypeptide is catalytically active.
  • Aspect 21 The fusion polypeptide of aspects 1-19, wherein the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
  • Aspect 22 The fusion polypeptide of any one of aspects 1-21, wherein the fusion polypeptide further comprises at least one additional heterologous polypeptide.
  • Aspect 23 The fusion polypeptide of aspect 22, wherein the at least one additional heterologous polypeptide is a deaminase, a base editor, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.
  • a composition comprising: a) a fusion polypeptide of any one of aspects 1- 23, or a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
  • Aspect 25 The composition of aspect 24, wherein the guide nucleic acid is a singlemolecule guide nucleic acid.
  • Aspect 26 The composition of aspect 24, further comprising a donor nucleic acid.
  • a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of aspects 1-23.
  • Aspect 28 The nucleic acid of aspect 27, wherein the nucleotide sequence is operably linked to a transcriptional control clement, optionally wherein the transcriptional control element is a promoter.
  • a recombinant expression vector comprising the nucleic acid of aspect 27.
  • Aspect 30 A cell comprising the fusion polypeptide of any one of aspects 1-23.
  • Aspect 31 The cell of aspect 30, wherein the cell is a eukaryotic cell.
  • Aspect 32 The cell of aspect 30 or aspect 31, wherein the cell is in vitro.
  • Aspect 33 The cell of aspect 30 or aspect 31, wherein the cell is in vivo.
  • Aspect 34 A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with: a) a fusion polypeptide of any one of aspects 1-23; and b) a guide nucleic acid.
  • a method of modifying a target nucleic acid in a eukaryotic cell comprising introducing into the eukaryotic cell: a) a fusion polypeptide of any one of aspects 1-23; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
  • Aspect 36 The method of aspect 35, comprising introducing into the cell a donor nucleic acid.
  • Aspect 37 The method of aspect 35 or 36, wherein the cell is in vitro.
  • Aspect 38 The method of aspect 35 or 36, wherein the cell is in vivo.
  • Aspect 39 The method of any one of aspects 35-38, wherein the cell is a mammalian cell, an insect cell, an avian cell, a reptile cell, an amphibian cell, an arachnid cell, a protozoan cell, or a plant cell.
  • Aspect 40 The method of any one of aspects 35-39, wherein the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.
  • Aspect 41 The method of any one of aspects 35-40, wherein said modifying comprises genome editing.
  • a fusion polypeptide comprising:
  • a cell penetrating polypeptide wherein the CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidsl058 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246 and 1247; and xvi) amino acids 1247 and 1248 based on the
  • Aspect 43 The fusion polypeptide of aspect 42, wherein the CPP is an A22p polypeptide, a Bac7 polypeptide, a CATat2 polypeptide, or a VP22 polypeptide.
  • a fusion polypeptide comprising, in order from N-terminus to C-terminus: a) a first nuclear localization sequence (NLS); b) a second NLS; c) a CRISPR-Cas effector polypeptide; d) at least two cell penetrating polypeptides (CPPs), optionally wherein one or more independently selected peptide linkers are interposed between any two of polypeptides (a) through (d), optionally wherein the CPPs are separated by a peptide linker.
  • Aspect 45 The fusion polypeptide of aspect 44, wherein the at least two CPPs are A22p polypeptides.
  • Aspect 46 The fusion polypeptide of aspect 45, wherein the A22p polypeptides comprise the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
  • Aspect 47 The fusion polypeptide of aspect 44, wherein the at least two CPPs are Bac7 polypeptides.
  • Aspect 48 The fusion polypeptide of aspect 47, wherein the Bac7 polypeptides comprise the amino acid sequence RR1RPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof.
  • Aspect 49 The fusion polypeptide of aspect 44, wherein the at least two CPPs are CATat2 polypeptides.
  • Aspect 50 The fusion polypeptide of aspect 49, wherein the CATat2 polypeptides comprise the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO:3), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NO:5),or a variant thereof.
  • Aspect 51 The fusion polypeptide of aspect 44, wherein the at least two CPPs are VP22 polypeptides.
  • Aspect 52 The fusion polypeptide of aspect 51, wherein the VP22 polypeptides comprise the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO:6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • FIG. 1A-1D An assay for testing the delivery and editing efficiency of various fusion polypeptides comprising a CRISPR-Cas effector polypeptide and a CPP is depicted in FIG. 1A-1D.
  • Streptococcus pyogenes Cas9 (Spy Cas9) was used as a model CRISPR-Cas effector polypeptide.
  • One or more cell penetrating polypeptides (CPPs) were fused to the Spy Cas9.
  • CPPs cell penetrating polypeptides
  • NLSs nuclear localization signals
  • neural progenitor cells were modified to include a target nucleic acid encoding the fluorescent protein tdTomato and including a stop cassette.
  • Guide RNAs were targeted to the stop cassette so that, if delivery and editing occurred, the stop cassette would be excised, as depicted schematically in FIG. 1C, and tdTomato would be expressed.
  • the design of the NLS-Cas9- CPP is depicted schematically in FIG. ID.
  • Fusion proteins comprising Spy Cas9 and CATat2 or CAcTat2 as the CPP were generated.
  • the CATat2 peptide was KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4).
  • the CAcTat2 peptide was KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NO:5).
  • the fusion proteins also included 2 NLSs at the N-terminus.
  • the fusion protein comprising 2NLSs, Spy Cas9, and CATat2 is referred to as “2NLS-Cas9-CATat2.”
  • the fusion protein comprising 2NLSs, Spy Cas9, and CAcTat2 is referred to as “2NLS-Cas9-CAcTat2.”
  • the fusion proteins, with guide RNAs targeting the stop cassette (FIG. 1C) were introduced into NPCs.
  • a fusion protein/guide RNA complex is referred to as a ribonucleoprotein (RNP). 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 2.
  • the “2NLS-Cas9-CATat2” fusion protein efficiently entered the cells and edited the target nucleic acid.
  • Fusion proteins comprising Spy Cas9 and VP22 as the CPP were generated.
  • the VP22 peptide was NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO:6).
  • Three different fusion proteins were generated.
  • a second fusion protein termed “VP22-Cas9-2NLS,” included: (i) a single VP22 peptide at the N-terminus of Spy Cas9; (ii) Spy Cas9; and (iii) 2 NLSs at the C-terminus of Spy Cas9.
  • a third fusion protein termed “c-myc-NLS-Cas9- VP22,” included: (i) a c-myc NLS at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) a VP22 peptide at the C-terminus of Spy Cas9.
  • RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 3.
  • Fusion proteins comprising Spy Cas9 and A22p as the CPP were generated.
  • the A22p peptide was HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1).
  • Four different fusion proteins were generated.
  • a second fusion protein termed “A22p-Cas9-2NLS,” included: i) the A22p peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) 2 NLSs at the C-terminus of Spy Cas9.
  • a third fusion protein termed “c-myc NLS-Cas9-A22p,” included: i) a c-myc NLS at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) the A22p peptide at the C-terminus of Spy Cas9.
  • a fourth fusion protein termed “A22p-Cas9-c-myc NLS,” included: i) the A22p peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) a c-myc NLS at the N-terminus of Spy Cas9.
  • RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 4.
  • Fusion proteins comprising Spy Cas9 and Bac7 as the CPP were generated.
  • the Bac7 peptide was RRIRPRPPRLPRPRPRPRPLPFPRPG (SEQ ID NO:2).
  • Four different fusion proteins were generated.
  • a second fusion protein termed “Bac7-Cas9-2NLS,” included: i) the Bac7 peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) 2 NLSs at the C-terminus of Spy Cas9.
  • a third fusion protein termed “c-myc NLS-Cas9- Bac7,” included: i) a c-myc NLS at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) the A2 Bac7 peptide at the C-terminus of Spy Cas9.
  • a fourth fusion protein termed “A22p-Cas9-c-myc NLS,” included: i) the Bac7 peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) a c-myc NLS at the N-terminus of Spy Cas9.
  • RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 5.
  • Various strategies were employed to further modify the Cas9-CPP fusion proteins, to increase delivery efficiency. One strategy was to insert a CPP internally within Cas9. As depicted in FIG.
  • a fusion protein termed “2NLS-Cas9-2NLS” included SpyCas9 with 2 NLSs at the N-terminus of Spy Cas9 and 2 NLSs at the C- terminus of Spy Cas9, but did not include a CPP.
  • RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 6C (nucleofection) and FIG. 6D (direct introduction of RNPs).
  • FIG. 7 Another strategy to increase delivery of RNPs into cells involved increasing the copy number of the CPP fused to the CRISPR-Cas effector protein.
  • This strategy is depicted schematically in FIG. 7. Fusion proteins including A22p peptide as the CPP and Spy Cas9 as the CRISPR-Cas effector protein were used as models. Fusion proteins containing Spy Cas9 and 2, 3, or 5 copies of A22p are shown in FIG. 8A-8C, respectively. Fusion proteins containing Spy Cas9 and 2 or 3 copies of A22p arc shown in FIG. 8D and FIG. 8E, respectively. RNPs were made by combining the fusion proteins with guide RNA, as described above.
  • the fusion protein termed “2NLS- Cas9-A22p3” and comprising: i) 2 NLSs at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) 3 copies of the A22p peptide at the C-terminus of Spy Cas9, exhibited efficient delivery into cells when delivered by direct delivery of an RNP comprising the fusion protein and the guide RNA.
  • Another strategy for increasing the efficiency of delivery of an RNP into a cell involved use of Cas9 variants in which the Cys at position 80 of Spy Cas9 and the Cys at position 574 of Spy Cas9 was substituted with Ala.
  • a fusion protein was made that included: i) 2 NLSs at the N-terminus; ii) Spy Cas9 (C80SA; C574SA); and iii) 3 copies of the A22p peptide at the C-terminus.
  • RNPs were made by combining the fusion proteins with guide RNA, as described above. 25 pmol, 25 pmol, or 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 10 and FIG. 11.
  • Fusion proteins were made with Lb Casl2a and either: i) 2 NLSs at the N-terminus and 2 NLSs at the C-terminus (and no CPP); or ii) NLSs and the CPP Bac7.
  • the target nucleic acid is depicted in FIG. 12A.
  • RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by nucleofection or by direct delivery into the NPCs. The results are shown in FIG. 12B and FIG. 13.

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Abstract

The present disclosure provides fusion polypeptides comprising a CRISPR-Cas effector polypeptide and a cell penetrating polypeptide. The present disclosure provides compositions comprising a fusion polypeptide of the present disclosure and a guide nucleic acid. The present disclosure provides methods of modifying a target nucleic acid in a eukaryotic cell.

Description

MODIFIED CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF
CROSS REFERENCE
[0001] This application claims benefit of U.S. Provisional Patent Application No. 63/359,359 filed July 8, 2022, which application is incorporated herein by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS AN XML FILE
[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 459WO_SEQ_LIST.xml” created on July 3, 2023 and having a size of 124,952 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
INTRODUCTION
[0003] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. Such CRISPR-Cas systems can bind to and modify a targeted nucleic acid. The programmable nature of these CRISPR-Cas effector systems has facilitated their use as a versatile technology for use in, e.g., gene editing. Many therapeutic approaches involve delivery of a nucleic acid vector or messenger RNA encoding a CRISPR- Cas protein, rather than delivery of the CRISPR-Cas protein per se. Delivery of nucleic acid vectors encoding a CRISPR-Cas protein, however, has certain disadvantages, namely possible insertional mutagenesis and the possibility of off-target gene editing.
[0004] There is a need in the art for modified CRISPR-Cas effector polypeptides that are readily delivered as the proteins per se.
SUMMARY
[0005] The present disclosure provides fusion polypeptides comprising a CRISPR-Cas effector polypeptide and a cell penetrating polypeptide. The present disclosure provides compositions comprising a fusion polypeptide of the present disclosure and a guide nucleic acid. The present disclosure provides methods of modifying a target nucleic acid in a eukaryotic cell.
[0006] In some aspects, the present disclosure provides for a fusion polypeptide comprising a CRISPR-Cas effector polypeptide and a cell penetrating polypeptide (CPP), wherein the CPP is an A22p polypeptide. [0007] In some aspects, the present disclosure provides for a fusion polypeptide comprising two or more nuclear localization sequences (NLSs), a CRISPR-Cas effector polypeptide, and a CPP, wherein the CPP is a Bac7 polypeptide.
[0008] In some aspects, the present disclosure provides for a fusion polypeptide comprising two or more NLSs, a CRISPR-Cas effector polypeptide, and a CPP, wherein the CPP is a CATat2 polypeptide.
[0009] In some aspects, the present disclosure provides for a fusion polypeptide comprising two or more NLSs, a CRISPR-Cas effector polypeptide, and a CPP, wherein the CPP is a VP22 polypeptide. [0010] In some embodiments, the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof. In some embodiments, the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO: 2), or a variant thereof. In some embodiments, the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO:3), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NO:5),or a variant thereof. In some embodiments, the VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO: 6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof.
[0011] In some embodiments, the fusion polypeptide comprises two or more copies of the CPP. In some embodiments, the fusion polypeptide comprises a linker between the CPP and the CRISPR-Cas effector polypeptide. In some embodiments, the linker between the CPP and the CRISPR-Cas effector polypeptide is a proteolytically cleavable.
[0012] In some embodiments, the two or more NLSs comprise the amino acid sequence K(K/R)X(K/R), where X is any amino acid. In some embodiments, the two or more NLSs comprise the amino acid sequence PKKKRKV (SEQ ID NO:8). In some embodiments, the two or more NLS are at the N-terminus of the CRISPR-Cas effector polypeptide. In some embodiments, the CPP is at the C- terminus of the CRISPR-Cas effector polypeptide. In some embodiments, the CPP is inserted internally within the CRISPR-Cas effector polypeptide. In some embodiments, the two or more NLSs are inserted at both the N- and C-termini. In some embodiments, the two or more NLSs are inserted at both the N- and C-termini and are inserted internally within the CRISPR-Cas effector polypeptide.
[0013] In some embodiments, the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a Type V CRISPR- Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is catalytically active. In some embodiments, the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide. In some embodiments, the fusion polypeptide further comprises at least one additional heterologous polypeptide. In some embodiments, the at least one additional heterologous polypeptide is a deaminase, a base editor, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.
[0014] In some aspects, the present disclosure provides for a composition comprising: a) a fusion polypeptide, or a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid. In some embodiments, the guide nucleic acid is a single-molecule guide nucleic acid. In some embodiments, the composition further comprises a donor nucleic acid.
[0015] In some aspects, the present disclosure provides a nucleic acid that comprises a nucleotide sequence encoding the fusion polypeptide. In some embodiments, the nucleotide sequence is operably linked to a transcriptional control clement, optionally wherein the transcriptional control element is a promoter. In some embodiments, the nucleic acid is a recombinant expression vector. In some embodiments, the recombinant expression vector comprises the nucleic acid.
[0016] In some embodiments, a cell comprises the fusion polypeptide. In some embodiments, a cell comprises the recombinant expression vector. In some embodiments, the cell (either of the cells above) is a eukaryotic cell. In some embodiments, the cell (either of the cells above) is in vitro. In some embodiments, the cell is in vivo.
[0017] In some aspects, the present disclosure provides for a method of modifying a target nucleic acid comprising contacting the target nucleic acid (e.g., in a eukaryotic cell) with a) a fusion polypeptide; and b) a guide nucleic acid. In some cases, the contacting includes introducing into a cell (e.g., a eukaryotic cell): a) the fusion polypeptide or a nucleic acid encoding the fusion polypeptide; and b) the guide nucleic acid or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid. In some embodiments, the nucleic acid encoding the fusion polypeptide and/or the nucleic acid encoding the guide nucleic acid is a recombinant expression vector. In some embodiments, the method comprises introducing a recombinant expression vector into the eukaryotic cell.
[0018] In some embodiments, the method comprises introducing into the cell a donor nucleic acid. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo. In some embodiments, the cell is a mammalian cell, an insect cell, an avian cell, a reptile cell, an amphibian cell, an arachnid cell, a protozoan cell, or a plant cell. In some embodiments, the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA. In some embodiments, the modifying comprises genome editing. [0019] In some aspects, the present disclosure provides for a fusion polypeptide comprising: a) a CRISPR-Cas effector polypeptide; and b) a CPP, wherein the CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidsl058 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246 and 1247; and xvi) amino acids 1247 and 1248 based on the Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide. In some embodiments, the CPP is an A22p polypeptide, a Bac7 polypeptide, a CATat2 polypeptide, or a VP22 polypeptide.
[0020] In some aspects, the present disclosure provides for a fusion polypeptide comprising, in order from N-terminus to C-terminus: a) a first NLS; b) a second NLS; c) a CRISPR-Cas effector polypeptide; d) at least two CPPs, optionally wherein one or more independently selected peptide linkers are interposed between any two of polypeptides (a) through (d), optionally wherein the CPPs are separated by a peptide linker.
[0021] In some embodiments, the at least two CPPs are A22p polypeptides. In some embodiments, the A22p polypeptides comprise the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof. In some embodiments, the at least two CPPs are Bac7 polypeptides. In some embodiments, the Bac7 polypeptides comprise the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof. In some embodiments, the at least two CPPs are CATat2 polypeptides. In some embodiments, the CATat2 polypeptides comprise the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG), or a variant thereof. In some embodiments, the at least two CPPs are VP22 polypeptides. In some embodiments, the VP22 polypeptides comprise the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NOG) or DAATATRGRSA ASRPTERPRAPARSASRPRRVD (SEQ ID NOG), or a variant thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A-1D schematically depict an assay for testing delivery and editing efficiency. FIG. 1A: (SEQ ID NOG). FIG. IB: (SEQ ID NO:102). FIG. ID: GPNAT (SEQ ID NO:103) - GIHGVPAAT (SEQ ID NO: 104) - SLEVLFQ (SEQ ID NO: 105). [0023] FIG. 2 depicts the effect of CATat2 cell-penetrating peptide (CPP) on delivery of Cas9- guidc RNA ribonuclcoprotcin (RNP) into neural progenitor cells (NPCs), and gene editing in the NPCs, in vitro.
[0024] FIG. 3 depicts the effect of VP22 CPP on delivery of Cas9-guide RNA RNP into NPCs, and gene editing in the NPCs, in vitro.
[0025] FIG. 4 depicts the effect of A22p CPP on delivery of Cas9-guide RNA RNP into NPCs, and gene editing in the NPCs, in vitro.
[0026] FIG. 5 depicts the effect of Bac7 CPP on delivery of Cas9-guide RNA RNP into NPCs, and gene editing in the NPCs, in vitro.
[0027] FIG. 6A-6D depict sites of insertion of a CPP into a Cas9 protein (FIG. 6A); and testing of a Bac7-inserted Cas9 (FIG. 6B-6D). FIG. 6B, Top:(SEQ ID NOs:106); Bottom: GPNAT (SEQ ID NO: 103) - GIHGVPAAT (SEQ ID NO: 104) - SLEVLFQ (SEQ ID NO: 105).
[0028] FIG. 7 schematically depicts CRISPR-Cas fusion polypeptides with variable numbers of C-terminal CPPs. (SEQ ID NO:1)
[0029] FIG. 8A-8E depict various CRISPR-Cas fusion polypeptides with variable numbers of C-terminal A22p CPPs.
[0030] FIG. 9 depicts a comparison of nucleofection delivery of a nucleic acid encoding a CRISPR-Cas fusion polypeptide and direct delivery of a CRISPR-Cas fusion polypeptide.
[0031] FIG. 10 depicts delivery of RNPs comprising various CRISPR-Cas fusion polypeptides with A22p as the CPP.
[0032] FIG. 11 depicts delivery of RNPs comprising various CRISPR-Cas fusion polypeptides with A22p as the CPP.
[0033] FIG. 12A-12B depict RNP delivery of Lb Cast 2a using CPPs.
[0034] FIG. 13 depicts RNP delivery of Lb Casl2a using CPPs.
[0035] FIG. 14A-14J provide amino acid sequences of various CRISPR-Cas effector polypeptides.
DEFINITIONS
[0036] “Heterologous,” as used herein in the context of a polypeptide, refers to an amino acid sequence that is not found in the native polypeptide. For example, a fusion CRISPR-Cas effector polypeptide comprises: a) a CRISPR-Cas effector polypeptide; and b) one or more heterologous polypeptides, where the heterologous polypeptide comprises an amino acid sequence from a protein other than a CRISPR-Cas effector polypeptide. “Heterologous,” as used herein in the context of a nucleic acid, refers to a nucleotide sequence that is not found in the native nucleic acid. As an example, in a guide nucleic acid, a heterologous guide nucleotide sequence (present in a targeting segment) that can hybridize with a target nucleotide sequence (target region) of a target nucleic acid is a nucleotide sequence that is not found in nature in a guide nucleic acid together with a binding segment that can bind to a CRISPR-Cas effector polypeptide of the present disclosure. For example, in some cases, a heterologous target nucleotide sequence (present in a heterologous targeting segment) is from a different source than a binding nucleotide sequence (present in a binding segment) that can bind to a CRISPR-Cas effector polypeptide of the present disclosure. For example, a guide nucleic acid may comprise a guide nucleotide sequence (present in a targeting segment) that can hybridize with a target nucleotide sequence present in a eukaryotic target nucleic acid. A guide nucleic acid of the present disclosure can be generated by human intervention and can comprise a nucleotide sequence not found in a naturally- occurring guide nucleic acid.
[0037] The term “naturally-occurring” as used herein as applied to a nucleic acid, a protein, a cell, or an organism, refers to a nucleic acid, cell, protein, or organism that is found in nature.
[0038] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides or combinations thereof. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
[0039] The terms "polypeptide," "peptide," and "protein", are used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and nongene tically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence.
[0040] Polypeptides as described herein also include polypeptides having various amino acid additions, deletions, or substitutions relative to the native amino acid sequence of a polypeptide of the present disclosure. In some embodiments, polypeptides that are homologs of a polypeptide of the present disclosure contain non-conservative changes of certain amino acids relative to the native sequence of a polypeptide of the present disclosure. In some embodiments, polypeptides that are homologs of a polypeptide of the present disclosure contain conservative changes of certain amino acids relative to the native sequence of a polypeptide of the present disclosure, and thus may be referred to as conservatively modified variants. A conservatively modified variant may include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well-known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). A modification of an amino acid to produce a chemically similar amino acid may be referred to as an analogous amino acid.
[0041] A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).
[0042] The terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, poly adenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.
[0043] The term “transformation” is used interchangeably herein with “genetic modification” and refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (e.g., DNA exogenous to the cell) into the cell. Genetic change (“modification”) can be accomplished either by incorporation of the new nucleic acid into the genome of the host cell, or by transient or stable maintenance of the new nucleic acid as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of new DNA into the genome of the cell. In prokaryotic cells, permanent changes can be introduced into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. 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.
[0044] “Operably linked” refers to a juxtaposition wherein the components so described arc in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. As used herein, the terms “heterologous promoter” and “heterologous control regions” refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a “transcriptional control region heterologous to a coding region” is a transcriptional control region that is not normally associated with the coding region in nature.
[0045] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, c.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
[0046] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, nonhuman primates, ungulates, felines, canines, bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets.
[0047] The use of the terms “a,” “an,” and “the,” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10- 15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments of the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the embodiments of the disclosure.
[0048] As used herein, the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, “about 100” means an amount of from 90- 110. Where about is used in the context of a range, the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, “from about 100 to about 1000” means that the range extends from 90 to 1100.
[0049] The term “and/or” as used herein a phrase such as “A and/or B” is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used herein a phrase such as “A. B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[0050] The terms “isolated” and “purified” as used herein refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). The term “isolated,” when used in reference to an isolated protein, refers to a protein that has been removed from the culture medium of the host cell that expressed the protein. As such an isolated protein is free of extraneous or unwanted compounds (e.g., nucleic acids, native bacterial or other proteins, etc.).
[0051] 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.
[0052] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0054] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell-penetrating polypeptide” includes a plurality of such polypeptides and reference to “the CRISPR-Cas effector polypeptide” includes reference to one or more CRISPR-Cas effector polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[0055] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.
Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.
[0056] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
[0057] The present disclosure provides fusion polypeptides comprising a CRISPR-Cas effector polypeptide and one or more heterologous polypeptides, where at least one of the one or more heterologous polypeptides is one that facilitates cellular uptake and endosomal escape of a complex comprising the fusion polypeptide. The present disclosure provides compositions comprising a fusion polypeptide of the present disclosure and a guide nucleic acid. A heterologous polypeptide that facilitates cellular uptake and endosomal escape of a complex comprising a fusion polypeptide of the present disclosure is referred to herein as a “cell penetrating polypeptide” (CPP).
[0058] A suitable CPP facilitates cellular uptake and endosomal escape of a fusion polypeptide of the present disclosure. Thus, for example, a suitable CPP is one that, when present in a fusion polypeptide comprising a CRISPR-Cas effector polypeptide, provides for an increase in cellular uptake of the fusion polypeptide (and thus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% (or two-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or more than 50-fold, compared to the cellular update of the same CRISPR-Cas effector polypeptide not fused to the CPP, or not fused to the CPP in the same configuration.
[0059] A CPP can be at or near the N-terminus of a CRISPR-Cas effector polypeptide; for example, a CPP can be at the N-terminus of the CRISPR-Cas effector polypeptide, or can be within 10 amino acids (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of the N-terminus of the CRISPR-Cas effector polypeptide. A CPP can be at or near the C-terminus of a CRISPR-Cas effector polypeptide; for example, a CPP can be at the C terminus of the CRISPR-Cas effector polypeptide, or can be within 10 amino acids (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of the C-terminus of the CRISPR-Cas effector polypeptide. A CPP can be inserted internally within a CRISPR-Cas effector polypeptide. A fusion polypeptide can comprise a single CPP. A fusion polypeptide can comprise 2 CPPs. A fusion polypeptide can comprise 3 CPPs. A fusion polypeptide can comprise 4 CPPs. A fusion polypeptide can comprise more than 4 CPPs. In some cases, a fusion polypeptide of the present disclosure comprises: i) a single CPP at or near the N-terminus of the CRISPR-Cas effector polypeptide; and ii) a single CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises: i) 2 CPPs at or near the N-terminus of the CRISPR-Cas effector polypeptide; and ii) a single CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises: i) a single at or near the N-terminus of the CRISPR-Cas effector polypeptide; and ii) 2 CPPs at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises 2 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises 3 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises 4 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide. In some cases, a fusion polypeptide comprises 5 copies of a CPP at or near the C-terminus of the CRISPR-Cas effector polypeptide.
[0060] A CPP can have a length of from about 10 amino acids to about 60 amino acids; e.g., a CPP can have a length of from 10 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids, from 20 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, from 45 amino acids to 50 amino acids, from 50 amino acids to 55 amino acids, or from 55 amino acids to 60 amino acids. In some cases, a CPP has a length of from 10 amino acids to 15 amino acids (e.g., 10, 11, 12, 13, 14, or 15 amino acids) In some cases, a CPP has a length of from 15 amino acids to 20 amino acids (e.g., 15, 16, 17, 18, 19, or 20 amino acids). In some cases, a CPP has a length of from 20 amino acids to 25 amino acids (e.g., 20, 21, 22, 23, 24, or 25 amino acids). In some cases, a CPP has a length of from 25 amino acids to 30 amino acids (e.g., 25, 26, 27, 28, 29, or 30 amino acids). In some cases, a CPP has a length of from 30 amino acids to 35 amino acids (e.g., 30, 31, 32, 33, 34, or 35 amino acids).
[0061] Suitable CPPs include, but are not limited to, an A22p polypeptide, a Bac7 polypeptide, a CATat2 polypeptide, a VP22 polypeptide, an MPG polypeptide, a TP10 polypeptide, a pVEC polypeptide, a pep-1 polypeptide, a Mel-Pl polypeptide, an ATRAM polypeptide, an Aurein 1.2 polypeptide, a buforin II polypeptide, a ZF5.3 polypeptide, an aPP6Rl polypeptide, and the like.
[0062] Exemplary CPPs are provided in Table 1, below.
Table 1
Figure imgf000014_0001
Figure imgf000015_0001
[0063] In some cases, the CPP is an A22p polypeptide, where the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof. A variant of an A22p polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1). In some cases, an A22p polypeptide has a length of 22 amino acids.
[0064] In some cases, the CPP is a Bac7 polypeptide, where the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NOG), or a variant thereof. A variant of a Bac7 polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2). In some cases, a Bac7 polypeptide has a length of 24 amino acids.
[0065] In some cases, the CPP is a CATat2 polypeptide, where the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), or a variant thereof. A variant of a CATat2 polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG). In some cases, a CATat2 polypeptide has a length of 13 amino acids. In some cases, a CATat2 polypeptide has the following amino acid sequence: KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NOG). In some cases, a CATat2 polypeptide has a length of from 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38, 39, or 40 amino acids). In some cases, a CATat2 polypeptide has the following amino acid sequence: KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG). In some cases, a CATat2 polypeptide has a length of from 40 amino acids to 45 amino acids (e.g., 40, 41, 42, 43, 44, or 45 amino acids).
[0066] In some cases, the CPP is a VP22 polypeptide. In some cases, a VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NOG), or a variant thereof. A variant of a VP22 polypeptide can comprise 1, 2, or 3 amino acid substitutions compared to the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID N0:6). In some cases, a VP22 polypeptide has a length of 32 amino acids. In some cases, a VP22 polypeptide comprises the amino acid sequence: DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7); and has a length of from 30 amino acids to 35 amino acids (e.g., 30, 31, 32, 33, 34, or 35 amino acids).
[0067] A fusion polypeptide of the present disclosure can include, in addition to the CRISPR- Cas effector polypeptide and the CPP, a nuclear localization signal (NLS). Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:8); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:9)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 10) or RQRRNELKRSP (SEQ ID NO: 11); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 12); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:13) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:14) and PPKKARED (SEQ ID NO:15) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO:16) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 17) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO:18) and PKQKKRK (SEQ ID NO:19) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO:20) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO:21) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 22) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO:23) of the steroid hormone receptors (human) glucocorticoid. In some cases, an NLS comprises the amino acid sequence K(K/R)X(K/R), where X is any amino acid. In some cases, an NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 8).
[0068] In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of the fusion protein in a detectable amount in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.
[0069] In some cases, a fusion polypeptide of the present disclosure includes (is fused to) a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a fusion polypeptide of the present disclosure includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N- terminus and/or the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C- terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C- terminus.
[0070] In some cases, a fusion polypeptide of the present disclosure comprises a single NLS at the N-terminus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide. In some cases, a fusion polypeptide of the present disclosure comprises a single NLS at the C-terminus of the CRISPR- Cas effector polypeptide present in the fusion polypeptide. In some cases, a fusion polypeptide of the present disclosure comprises two NLSs at the N-terminus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide. In some cases, a fusion polypeptide of the present disclosure comprises 2 NLSs at the C-terminus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide. [0071] The following are non-limiting examples of fusion polypeptides comprising both a CPP and an NLS. As one example, in some cases, a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) a single CPP at the C- terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 2 CPPs at the C-terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 3 CPPs at the C-terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N- terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 4 CPPs at the C-terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) 2 NLSs at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 5 CPPs at the C-terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) a single NLS at the C-terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 2 NLSs at the C-terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 3 NLSs at the C-terminus. As another example, in some cases, a fusion polypeptide of the present disclosure comprises: i) a single CPP at the N-terminus; ii) a CRISPR-Cas effector polypeptide; and iii) 4 NLSs at the C-terminus.
[0072] As noted above, in some cases, a CPP is inserted internally within a CRISPR-Cas effector polypeptide. For example, in some cases, a CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidslO58 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246 and 1247; and xvi) amino acids 1247 and 1248 of a Cas9 polypeptide (e.g., where the amino acid numbering is based on the Streptococcus pyogenes Cas9 (Spy Cas9) amino acid sequence depicted in FIG. 14A). For example, in some cases, a CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidsl058 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246 and 1247; and xvi) amino acids 1247 and 1248 based on the Spy Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide (e.g., in another type II CRISPR-Cas effector polypeptide). In some cases, a CPP is inserted between amino acids 204 and 205 based on the Spy Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide (e.g., in another type 11 CRISPR-Cas effector polypeptide). In some cases, a CPP is inserted between amino acids 205 and 206 based on the Spy Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide (e.g., in another type II CRISPR- Cas effector polypeptide).
CRISPR-Cas effector polypeptides
[0073] As noted above, a fusion polypeptide of the present disclosure comprises at least one CPP and a CRISPR-Cas effector polypeptide, and may further include one or more NLSs. Suitable CRISPR-Cas effector polypeptides include Type II CRISPR-Cas effector polypeptides, Type III CRISPR Cas effector polypeptides, Type V CRISPR Cas effector polypeptides, and Type VI CRISPR-Cas effector polypeptides. Suitable CRISPR-Cas effector polypeptides include fusion polypeptides that comprise: i) a CRISPR-Cas effector polypeptide; and ii) a heterologous polypeptide other than a CPP and an NLS. For example, a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in any one of FIG. 14A-14J. Many CRISPR-Cas effector polypeptides are known to those skilled in the art, and any CRISPR-Cas effector polypeptide can be used in a fusion polypeptide of the present disclosure. The amino acid sequences depicted in FIG. 14A-14J are provided as examples.
[0074] In some cases, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide. In some cases, the type II CRISPR-Cas effector polypeptide is a Cas9 polypeptide, e.g., Staphylococcus aureus Cas9, Streptococcus pyogenes Cas9 (SpCas9), etc. In some cases, the CRISPR- Cas effector polypeptide is a variant of a wild-type SpCas9 and comprises one or more of the following substitutions: A61R, Lil HR, A1322R, D1135L, S1136W, G1218K, E1219Q, N1317R, R1333P, R1335A, and T1337R. In some cases, the CRISPR-Cas effector polypeptide is an SpG polypeptide or a SpRY polypeptide; see, e.g., Walton et al. (2020) Science 368:290, and WO 2019/051097. For example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes DI 135V, R1135Q, and T1137R substitutions, relative to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes DI 135V, R1335Q, T1337R, and G1218R substitutions, relative to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes D1135L, S1136W, G1218K, E1219Q, R1335A, and T1337R substitutions, relative to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes L1111R, A1322R, D1135L, S1136W, G1218K, E1219Q, R1335A, and T1337R substitutions, relative to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes A61R, L1111R, A1322R, D1135L, S1136W, G1218K, E1219Q, N1317R, R1333P, R1335A, and T1337R substitutions, relative to wild-type SpCas9. The amino acid sequence of a wild-type SpCas9 polypeptide is provided in FIG. 14A.
[0075] In some cases, one or more surface Cys residues of a CRISPR-Cas effector polypeptide are substituted. As one example, in some cases, a Cys at position 80 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with an amino acid other than a Cys. For example, in some cases, a Cys at position 80 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with a Ser. As another example, in some cases, a Cys at position 574 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with an amino acid other than a Cys. For example, in some cases, a Cys at position 574 of the SpCas9 amino acid sequence depicted in FIG. 14A, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with a Ser. For example, a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14A, where amino acid 80 is other than a Cys, e.g., where amino acid 80 is a Ser. For example, a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14A, where amino acid 574 is other than a Cys, e.g., where amino acid 574 is a Ser. [0076] In some cases, the CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide, e.g., a Casl2a, a Casl2b, a Casl2c, a Casl2d, or a Casl2c polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type VI CRISPR-Cas effector polypeptide, e.g., a Casl3a polypeptide, a Casl3b polypeptide, a Casl3c polypeptide, or a Casl3d polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Casl4 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Casl4a polypeptide, a Casl4b polypeptide, or a Casl4c polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas7-ll polypeptide; see, e.g., Ozcan et al. (2021) Nature 597:720. In some cases, the CRISPR-Cas effector polypeptide is a CRISPRi polypeptide; see, e.g., Qi et al. (2013) Cell 152:1173; and Jensen et al. (2021) Genome Research doi:10.1101/gr.275607.121. In some cases, the CRISPR-Cas effector polypeptide is a CRISPRa polypeptide; see, e.g., Jensen et al. (2021) Genome Researc/z doi:10.1101/gr.275607.121; and Breinig et al. (2019) Nature Methods 16:51. In some cases, the CRISPR-Cas effector polypeptide is a CRISPRoff polypeptide. See, e.g., Nunez et al. (2021) Cell 184:2503. In some cases, the CRISPR-Cas effector polypeptide is a nickase. In some cases, the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
[0077] As noted above, in some cases, a CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide, such as a Casl3a polypeptide, a Casl3b polypeptide, a Casl3c polypeptide, a Casl3d polypeptide, a Casl3e polypeptide, a Casl3f polypeptide, a Casl3X polypeptide, or a Casl3Y polypeptide. As noted above, in some cases, a CRISPR-Cas effector polypeptide is a Type III CRISPR-Cas effector polypeptide. A suitable Type III CRISPR-Cas effector polypeptide is a Cas7-l l polypeptide (see, e.g., Ozcan et al. (2021) Nature 597:710). In some cases, a CRISPR-Cas effector polypeptide is an RNA-binding CRISPR-Cas effector polypeptide. For example, in some cases, the CRISPR-Cas effector polypeptide is an RFx Casl3d polypeptide. As another example, in some cases, the CRISPR-Cas effector polypeptide is a dRfxCasl3d polypeptide. As another example, in some cases, the CRISPR-Cas effector polypeptide is a DjCasl3d polypeptide. As another example, in some cases, the CRISPR-Cas effector polypeptide is a PspCasl3b polypeptide, e.g., Prevotella sp. P5-125 Casl3b. As another example, in some cases, the CRISPR-Cas effector polypeptide is a dPspCasl3b polypeptide In some cases, a Casl3 polypeptide (e.g., a Casl3a polypeptide, a Casl3b polypeptide, a Casl3c polypeptide, a Casl3d polypeptide, a Casl3e polypeptide, a Casl3f polypeptide, a Casl3X polypeptide, or a Casl3Y polypeptide) comprises two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, each comprising a HEPN motif, where each HEPN motif is RXXXXH, RXXXXXH, or RXXXXXXH, where X is any amino acid.
[0078] In some cases, the CRISPR-Cas effector polypeptide is a variant that exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide. For example, where the
CRISPR-Cas effector polypeptide (e.g., a Casl3 polypeptide) comprises a HEPN domain, the CRISPR- Cas effector polypeptide can comprise a substitution of the Arg and/or the His in the HEPN motif, where such a mutation reduces the catalytic activity of the CRISPR-Cas effector polypeptide. In some cases, the RNA-binding CRISPR-Cas effector polypeptide binds, but does not cleave, a target RNA. In some cases, a CRISPR-Cas effector polypeptide is a variant that, when complexed with a guide nucleic acid, binds but does not cleave a target DNA.
[0079] As another example, in some cases, the CRISPR-Cas effector polypeptide is a Cas7-11 polypeptide (e.g., a DiCas7-l l polypeptide). See, e.g., Ozcan et al. (2021) Nature 597:720. In some cases, the Cas7-11 polypeptide is a variant that exhibits reduced catalytic activity compared to a wildtype Cas7-ll effector polypeptide. For example, in some cases, the variant Cas7-l l polypeptide comprises a substitution of one or more of D177, D429, D654, D758, E959, and D998 (where the amino acid numbering is as set forth in FIG. 14H). D177, D429, D654, D758, E959, and D998 are in bold in FIG. 14H. For example, a variant Cas7-ll can have an Asp at position 177, an Ala at position 429, an Ala at position 654, an Asp at position 758, a Glu at position E959, and an Asp at position 998. In some cases, a variant Cas7-l l comprises a D429A substitution.
Fusion polypeptides comprising a fusion CRISPR-Cas effector polypeptide
[0080] As noted above, in some cases, the CRISPR-Cas effector polypeptide present in a fusion polypeptide of the present disclosure is itself a fusion polypeptide that comprises: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides other than a CPP and an NLS. Thus, e.g., in some cases, a fusion polypeptide of the present disclosure comprises: a) one or more CPPs; b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides other than an NLS and other than a CPP. In some cases, a fusion polypeptide of the present disclosure comprises: a) one or more CPPs; b) one or more NLSs; and c) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides other than an NLS and other than a CPP. The term “heterologous polypeptide” is used interchangeably herein with “fusion partner.”
[0081] In some cases, a fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a 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 transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like). 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 acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like).
[0082] In some cases, the fusion partner (heterologous polypeptide) is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner (heterologous polypeptide) is a deaminase.
[0083] In some cases, a fusion partner has enzymatic activity that modifies a target nucleic acid
(e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).
[0084] In some cases, a fusion partner has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity).
[0085] Examples of proteins (or fragments thereof) that can be used in increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.
[0086] Examples of proteins (or fragments thereof) that can be used in decrease transcription include but are not limited to: transcriptional repressors such as the Kriippel associated box (KRAB or SKD); K0X1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7/8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.
[0087] In some cases, a fusion partner has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to: 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., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like) , DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).
[0088] In some cases, a fusion partner has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifyies a protein associated with a target nucleic acid) that can be provided by the fusion partner include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HBO1/MYST2, HMOF/MYST1, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.
[0089] Additional examples of suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable fusion CRISPR-Cas effector protein), and a chloroplast transit peptide.
[0090] Additional suitable heterologous polypeptides include, but are not limited to, a polypeptide that directly and/or indirectly provides for increased transcription and/or h anslation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription and/or translation regulator, a translation-regulating protein, etc.). Non-limiting examples of heterologous polypeptides to accomplish increased or decreased transcription include transcription activator and transcription repressor domains.
[0091] Non-limiting examples of heterologous polypeptides for use when targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain).
[0092] A fusion partner can be any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and/or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising; Endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example CPSF, CstF, CFIm and CFIIm); Exonucleases (for example XRN-1 or Exonuclease T); Deadenylases (for example HNT3); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1, UPF2, UPF3, UPF3b, RNP SI, Y14, DEK, REF2, and SRml60); proteins and protein domains responsible for stabilizing RNA (for example PABP) ; proteins and protein domains responsible for repressing translation (for example Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (for example Staufen); proteins and protein domains responsible for (e.g., capable ol) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (for example PAP1, GLD-2, and Star- PAP) ; proteins and protein domains responsible for polyuridinylation of RNA (for example CI DI and terminal uridylate transferase) ; proteins and protein domains responsible for RNA localization (for example from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (for example Rrp6); proteins and protein domains responsible for nuclear export of RNA (for example TAP, NXF1, THO, TREX, REF, and Aly) ; proteins and protein domains responsible for repression of RNA splicing (for example PTB, Sam68, and hnRNP Al) ; proteins and protein domains responsible for stimulation of RNA splicing (for example Serine/ Arginine-rich (SR) domains) ; proteins and protein domains responsible for reducing the efficiency of transcription (for example FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (for example 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 having nonsense mediated RNA decay 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 modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of 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 more detail in WO2012068627, which is hereby incorporated by reference in its entirety.
[0093] Some RNA splicing factors that can be used (in whole or as fragments thereof) as heterologous polypeptides for a fusion Casl2L polypeptide have modular organization, with separ ate sequence-specific RNA binding modules and splicing effector domains. For example, members of the Serine/ Arginine-rich (SR) protein family contain N -terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal Glycine -rich domain. Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF/SF2 can recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al can bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoform and 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 ctb-elements that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites). For more examples, see W02010075303, which is hereby incorporated by reference in its entirety.
[0094] 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 periphery recruitment (e.g., Lamin A, Lamin B, etc.), protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.). Nucleases
[0095] In some cases, a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a nuclease; and optionally also includes c) one or more NLSs.
Suitable nucleases include, but are not limited to, a homing nuclease polypeptide; a FokI polypeptide; a transcription activator-like effector nuclease (TALEN) polypeptide; a McgaTAL polypeptide; a meganuclease polypeptide; a zinc finger nuclease (ZFN); an ARCUS nuclease; and the like. The meganuclease can be engineered from an LADLIDADG homing endonuclease (LHE). A megaTAL polypeptide can comprise a TALE DNA binding domain and an engineered meganuclease. See, e.g., WO 2004/067736 (homing endonuclease); Urnov et al. (2005) Nature 435:646 (ZFN); Mussolino et al. (2011) Nude. Adds Res. 39:9283 (TALE nuclease); Boissel et al. (2013) Nud. Acids Res. 42:2591 (MegaTAL).
Reverse transcriptases
[0096] In some cases, a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a reverse transcriptase polypeptide; and optionally also includes c) one or more NLSs. In some cases, the CRISPR-Cas effector polypeptide is catalytically inactive. Suitable reverse transcriptases include, e.g., a murine leukemia virus reverse transcriptase; a Rous sarcoma virus reverse transcriptase; a human immunodeficiency virus type I reverse transcriptase; a Moloney murine leukemia virus reverse transcriptase; and the like. Base editors
[0097] In some cases, a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a base editor; and optionally also includes c) one or more NLSs.
Suitable base editors include, e.g., an adenosine deaminase; a cytidine deaminase (e.g., an activation- induced cytidine deaminase (AID)); APOBEC3G; and the like); and the like. A suitable adenosine deaminase is any enzyme that is capable of deaminating adenosine in DNA. In some cases, the deaminase is a TadA deaminase.
[0098] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO:24)
[0099] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGA
AGSLMDVLHHPGMNHRVE1TEG1LADECAALLSDFFRMRRQE1KAQKKAQSSTD (SEQ ID NO:25).
[00100] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Staphylococcus aureus TadA amino acid sequence:
MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIER AAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFN HRAIVDKGVLKEACSTLLTTFFK NLRANKKSTN: (SEQ ID NO:26)
[00101] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Bacillus subtilis TadA amino acid sequence:
MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML VIDEACK ALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQA EVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE (SEQ ID NO:27) [00102] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Salmonella typhimurium TadA:
MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGR HDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGA AGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV (SEQ ID NO:28)
[00103] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Shewanella putrefaciens TadA amino acid sequence:
MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAG KKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQ VEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO:29)
[00104] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Haemophilus influenzae F3O31 TadA amino acid sequence:
MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAE IIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDY KMNHTLEITSGVLAEECSQKLS TFFQKRREEKKIEKALLKSLSDK (SEQ ID NO:30)
[00105] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Caulobacter crescentus TadA amino acid sequence:
MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAE IAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFA QPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO:31)
[00106] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Geobacter sulfurreducens TadA amino acid sequence:
MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAE MIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSA DPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP (SEQ ID NO:32) [00107] Cytidine deaminases suitable for inclusion in a Cas12L fusion polypeptide include any enzyme that is capable of deaminating cytidine in DNA. [00108] In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA-cditing complex (APOB EC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC 1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is an activation induced deaminase (AID).
[00109] In some cases, a suitable cytidine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
[00110] MDSLLMNRRKFLYQFKNVRWAKGRRETYLC Y VVKRRDS ATSFSLDFGYLRNK
NGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFC EDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRIL LPLYEVDDLRDAFRTLGL (SEQ ID NO:33)
[00111] In some cases, a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO:34).
[00112] In some cases, a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO: 33).
Transcription factors
[00113] In some cases, a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a transcription factor; and optionally also includes c) one or more NLSs. A transcription factor can include: i) a DNA binding domain; and ii) a transcription activator. A transcription factor can include: i) a DNA binding domain; and ii) a transcription repressor. Suitable transcription factors include polypeptides that include a transcription activator or a transcription repressor domain (e.g., the Kruppel associated box (KRAB or SKD); the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), etc.); zinc-finger-based artificial transcription factors (see, e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificial transcription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics 14:781); and the like. In some cases, the transcription factor comprises a VP64 polypeptide (transcriptional activation). In some cases, the transcription factor comprises a Kriippel- associated box (KRAB) polypeptide (transcriptional repression). In some cases, the transcription factor comprises a Mad mSIN3 interaction domain (SID) polypeptide (transcriptional repression). In some cases, the transcription factor comprises an ERF repressor domain (ERD) polypeptide (transcriptional repression). For example, in some cases, the transcription factor is a transcriptional activator, where the transcriptional activator is GAL4-VP16.
Recombinases
[00114] In some cases, a fusion polypeptide of the present disclosure comprises: a) a CPP; and b) a fusion CRISPR-Cas effector polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous polypeptides (one or more “fusion partners”), where at least one of the one or more heterologous polypeptides is a recombinase; and optionally also includes c) one or more NLSs. Suitable recombinases include, e.g., a Cre recombinase; a Hin recombinase; a Tre recombinase; a FLP recombinase; and the like.
Linkers
[00115] In some cases, a fusion polypeptide of the present disclosure comprises one or more linker polypeptides. The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use.
[00116] Examples of linker polypeptides include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, and (GGGGS)n (SEQ ID NO:36), where n is an integer from 1 to 10), glycine-alanine polymers, serine polymers, and alanine-serine polymers. Exemplary linkers can comprise amino acid sequences including, but not limited to, GGS, GS, GGSG (SEQ ID NO:37), GGSGG (SEQ ID NO:38), GSGSG (SEQ ID NO:39), GSGGG (SEQ ID NO:40), GGGSG (SEQ ID N0:41), GSSSG (SEQ ID NO:42), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. Suitable linkers include, e.g., GSSSSSSGS (SEQ ID NO:43). A suitable spacer peptide includes, e.g., GIHGVPATT (SEQ ID NO:44).
[00117] In some cases, a fusion polypeptide of the present disclosure comprises a linker between a CPP and a CRISPR-Cas effector polypeptide present in the fusion polypeptide. In some cases, the linker is a proteolytically cleavable linker.
[00118] The proteolytically cleavable linker can include a protease recognition sequence recognized by a protease selected from the group consisting of alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, cndoprotcinasc Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, IgA-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E, picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrcin, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB, and Xaa-pro aminopeptidase.
[00119] For example, the proteolytically cleavable linker can comprise a matrix metalloproteinase cleavage site, e.g., a cleavage site for a MMP selected from collagenase- 1, -2, and -3 (MMP-1, -8, and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and - 11), matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy (wherein, X represents an arbitrary residue; Hy, a hydrophobic residue), e.g., Pro-X-X-Hy-(SerZThr), e.g., Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO:45) or Pro-Leu/Gln-Gly-Met-Thr (SEQ ID NO:46). Another example of a protease cleavage site is a plasminogen activator cleavage site, e.g., a uPA or a tissue plasminogen activator (tPA) cleavage site. In some cases, the cleavage site is a furin cleavage site. Specific examples of cleavage sequences of uPA and tPA include sequences comprising Val-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., ENLYTQS (SEQ ID NO:47), where the protease cleaves between the glutamine and the serine. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., DDDDK (SEQ ID NO:48), where cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., LVPR (SEQ ID NO:49). Additional suitable linkers comprising protease cleavage sites include linkers comprising one or more of the following amino acid sequences: LEVLFQGP (SEQ ID NO:50), cleaved by PreScission protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase; Walker et al. (1994) Biotechnol. 12:601); a thrombin cleavage site, e.g., CGLVPAGSGP (SEQ ID NO:51); SLLKSRMVPNFN (SEQ ID NO:52) or SLLIARRMPNFN (SEQ ID NO:53), cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO:54) or SSYLKASDAPDN (SEQ ID NO:55), cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO:56) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO:57) cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO:58) cleaved by MMP-9; DVDERDVRGFASFL SEQ ID NO:59) cleaved by a thermolysin-like MMP; SLPLGLWAPNFN (SEQ ID NO:60) cleaved by matrix metalloproteinase 2(MMP-2); SLL1FRSWANFN (SEQ ID NO:61) cleaved by cathespin L; SGVVIATVIVIT (SEQ ID NO:62) cleaved by cathepsin D; SLGPQGIWGQFN (SEQ ID NOG) cleaved by matrix metalloproteinase l(MMP-l); KKSPGRVVGGSV (SEQ ID NO:64) cleaved by urokinasetype plasminogen activator; PQGLLGAPGILG (SEQ ID NO:65) cleaved by membrane type 1 matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO:66) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin- 1 ; GPQGLAGQRG1V (SEQ ID NO:67) cleaved by matrix metalloproteinase 13 (collagenase-3);
GGSGQRGRKALE (SEQ ID NO:68) cleaved by tissue-type plasminogen activator(tPA); SLSALLSSDIFN (SEQ ID NO:69) cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ ID NO:70) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO:71 cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:72) cleaved by calpain (calcium activated neutral protease).
NUCLEIC ACIDS, EXPRESSION VECTORS, AND MODIFIED HOST CELLS
[00120] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure. The present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure. The nucleic acids and recombinant expression vectors are useful for producing a fusion polypeptide of the present disclosure. In some cases, the nucleotide sequence encoding the fusion polypeptide is operably linked to one or more transcriptional control elements, e.g., promoters, such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
[00121] Suitable expression vectors are well known and include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus; adeno-associated virus; human immunodeficiency virus, and the like). Depending on the host/vector system utilized, any of a number of well-known, suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression.
[00122] The present disclosure provides a genetically modified host cell, where the host cell is genetically modified with a nucleic acid or a recombinant expression vector encoding a fusion polypeptide of the present disclosure. Suitable host cells include eukaryotic cells, such as yeast cells, insect cells, and mammalian cells. In some cases, the host cell is a cell of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines are likewise well known and include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. [00123] A fusion polypeptide can be produced using a genetically modified host cell as described herein. Thus, this disclosure provides methods of producing a fusion polypeptide of the present disclosure. The methods generally involve culturing, in a culture medium, a host cell (an “expression host cell”) that is genetically modified with a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding the fusion polypeptide; and isolating the fusion polypeptide from the genetically modified host cell and/or the culture medium.
[00124] Isolation of the fusion polypeptide from the expression host cell (e.g., from a lysate of the expression host cell) and/or the culture medium in which the host cell is cultured, can be earned out using standard methods of protein purification.
[00125] For example, a lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Alternatively, where the fusion polypeptide is secreted from the expression host cell into the culture medium, the fusion polypeptide can be purified from the culture medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some cases, the compositions which are used will comprise at least 80% by weight, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, of the fusion polypeptide, in relation to contaminants related to the method of preparation of the product and its purification. The percentages can be based upon total protein.
[00126] In some cases, e.g., where the fusion polypeptide comprises an affinity tag, the fusion polypeptide can be purified using an immobilized binding partner of the affinity tag. COMPOSITIONS AND SYSTEMS
[00127] The present disclosure provides a composition comprising a fusion polypeptide of the present disclosure. In some cases, a composition of the present disclosure comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid. In some cases, a composition of the present disclosure comprises: i) a fusion polypeptide of the present disclosure; ii) a guide nucleic acid; and iii) a donor nucleic acid.
Guide nucleic acids
[00128] Guide nucleic acids that form a complex with a CRISPR-Cas effector polypeptide ar e well known in the art. A guide RNA (can be said to include two segments, a first segment (referred to herein as a “targeting segment”); and a second segment (referred to herein as a “protein-binding segment”). By “segment” it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule. A segment can also mean a region/section of a complex such that a segment may comprise regions of more than one molecule. The “targeting segment” is also referred to herein as a “variable region” of a guide RNA. The “protein-binding segment” is also referred to herein as a “constant region” of a guide RNA.
[00129] In many instances, the targeting segment and the protein-binding segment are heterologous to one another. In some cases, the targeting segment comprises a nucleotide sequence that is complementary to a nucleotide sequence in a eukaryotic target nucleic acid.
[00130] The first segment (targeting segment) of a guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within a target nucleic acid (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.). The protein-binding segment (or “protein-binding sequence”) interacts with (binds to) a CRISPR/Cas effector polypeptide. The protein-binding segment of a guide RNA includes two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex). Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the guide RNA (the guide sequence of the guide RNA) and the target nucleic acid.
[00131] A guide RNA and a CRISPR/Cas effector polypeptide form a complex (e.g., bind via non- covalent interactions). The guide RNA provides target specificity to the complex by including a targeting segment, which includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid). The CRISPR/Cas effector polypeptide of the complex provides the site-specific activity (e.g., cleavage activity or an activity provided by the CRISPR/Cas effector polypeptide when the CRISPR/Cas effector polypeptide is a CRISPR/Cas effector polypeptide fusion polypeptide, i.e., has a fusion partner). In other words, the CRISPR/Cas effector polypeptide is guided to a target nucleic acid sequence (e.g. a target sequence in a chromosomal nucleic acid, e.g., a chromosome; a target sequence in an extrachromosomal nucleic acid, e.g. an episomal nucleic acid, a minicircle, an ssRNA, an ssDNA, etc.; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid; a target sequence in a viral nucleic acid; etc.) by virtue of its association with the guide RNA.
[00132] The “guide sequence” also referred to as the “targeting sequence” of a guide RNA can be modified so that the guide RNA can target a CRISPR/Cas effector polypeptide to any desired sequence of any desired target nucleic acid, with the exception that the protospacer adjacent motif (PAM) sequence can be taken into account. Thus, for example, a guide RNA can have a targeting segment with a sequence (a guide sequence) that has complementarity with (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like.
[00133] In some cases, a guide RNA includes two separate nucleic acid molecules and is referred to herein as a “dual guide RNA”, a “double -molecule guide RNA”, or a “two-molecule guide RNA” a “dual guide RNA”, or a “dgRNA.” In some cases, a guide RNA is a single-molecule RNA and is referred to as a “single guide RNA”, or simply “sgRNA.”
[00134] The targeting segment can have a length of 7 or more nucleotides (nt) (e.g., 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more nucleotides). In some cases, the targeting segment can have a length of from 7 to 100 nucleotides (nt) (e.g., from 7 to 80 nt, from 7 to 60 nt, from 7 to 40 nt, from 7 to 30 nt, from 7 to 25 nt, from 7 to 22 nt, from 7 to 20 nt, from 7 to 18 nt, from 8 to 80 nt, from 8 to 60 nt, from 8 to 40 nt, from 8 to 30 nt, from 8 to 25 nt, from 8 to 22 nt, from 8 to 20 nt, from 8 to 18 nt, from 10 to 100 nt, from 10 to 80 nt, from 10 to 60 nt, from 10 to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 10 to 18 nt, from 12 to 100 nt, from 12 to 80 nt, from 12 to 60 nt, from 12 to 40 nt, from 12 to 30 nt, from 12 to 25 nt, from 12 to 22 nt, from 12 to 20 nt, from 12 to 18 nt, from 14 to 100 nt, from 14 to 80 nt, from 14 to 60 nt, from 14 to 40 nt, from 14 to 30 nt, from 14 to 25 nt, from 14 to 22 nt, from 14 to 20 nt, from 14 to 18 nt, from 16 to 100 nt, from 16 to 80 nt, from 16 to 60 nt, from 16 to 40 nt, from 16 to 30 nt, from 16 to 25 nt, from 16 to 22 nt, from 16 to 20 nt, from 16 to 18 nt, from 18 to 100 nt, from 18 to 80 nt, from 18 to 60 nt, from 18 to 40 nt, from 18 to 30 nt, from 18 to 25 nt, from 18 to 22 nt, or from 18 to 20 nt).
[00135] The guide sequence of a guide RNA can have a length of from 15 nt to 30 nt (e.g., 15 to 25 nt, 15 to 24 nt, 15 to 23 nt, 15 to 22 nt, 15 to 21 nt, 15 to 20 nt, 15 to 19 nt, 15 to 18 nt, 17 to 30 nt, 17 to 25 nt, 17 to 24 nt, 17 to 23 nt, 17 to 22 nt, 17 to 21 nt, 17 to 20 nt, 17 to 19 nt, 17 to 18 nt, 18 to 30 nt, 18 to 25 nt, 18 to 24 nt, 18 to 23 nt, 18 to 22 nt, 18 to 21 nt, 18 to 20 nt, 18 to 19 nt, 19 to 30 nt, 19 to 25 nt, 19 to 24 nt, 19 to 23 nt, 19 to 22 nt, 19 to 21 nt, 19 to 20 nt, 20 to 30 nt, 20 to 25 nt, 20 to 24 nt, 20 to 23 nt, 20 to 22 nt, 20 to 21 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt). 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. In some cases, the guide sequence has a length of 24 nt.
[00136] In some cases, the guide sequence (also referred to as a “spacer sequence”) has a length of from 15 to 50 nucleotides (e.g., from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt).
[00137] The protein-binding segment of a guide RNA can include two stretches of nucleotides that are complementary to one another and hybridize to form a double stranded RNA duplex (dsRNA duplex). Thus, in some cases, the protein-binding segment includes a dsRNA duplex.
[00138] In some cases, the dsRNA duplex region includes a range of from 5-25 base pairs (bp) (e.g., from 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8, 8-25, 8-22, 8-18, 8-15, 8-12, 12-25, 12-22, 12-18, 12- 15, 13-25, 13-22, 13-18, 13-15, 14-25, 14-22, 14-18, 14-15, 15-25, 15-22, 15-18, 17-25, 17-22, or 17-18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the dsRNA duplex region includes a range of from 6-15 base pairs (bp) (e.g., from 6-12, 6-10, or 6-8 bp, e.g., 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the duplex region includes 5 or more bp (e.g., 6 or more, 7 or more, or 8 or more bp). In some cases, the duplex region includes 6 or more bp (e.g., 7 or more, or 8 or more bp). In some cases, not all nucleotides of the duplex region are paired, and therefore the duplex forming region can include a bulge. The term “bulge” herein is used to mean a stretch of nucleotides (which can be one nucleotide) that do not contribute to a double stranded duplex, but which are surround 5’ and 3’ by nucleotides that do contribute, and as such a bulge is considered part of the duplex region. In some cases, the dsRNA includes 1 or more bulges (e.g., 2 or more, 3 or more, 4 or more bulges). In some cases, the dsRNA duplex includes 2 or more bulges (e.g., 3 or more, 4 or more bulges). In some cases, the dsRNA duplex includes 1-5 bulges (e.g., 1-4, 1-3, 2-5, 2-4, or 2-3 bulges).
[00139] A guide nucleic acid can include one or more of: i) a modified sugar; ii) a modified nucleobase; and iii) one or more non-natural internucleoside linkages.
[00140] Suitable non-natural internucleoside linkage include, e.g., a phosphorothioate, a phosphor amidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'-alkylene phosphonates, a 5'-alkylene phosphonate, a chiral phosphonate, a phosphinate, a, a 3'-amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, and a boranophosphate.
[00141] Suitable modified moieties include, e.g., locked nucleic acid (LNA) sugar moieties, 2'- substituted sugar moieties, 2'-O-methoxyethyl modified sugar moieties, one or more 2'-O-methyl modified sugar moieties, 2'-O-(2-methoxyethyl) modified sugar moieties, 2'-fluoro modified sugar moieties, 2'-dimethylaminooxyethoxy modified sugar moieties, and 2'-dimethylaminoethoxyethoxy modified sugar moieties.
[00142] Suitable modified nucleobases include, e.g., 5 -methylcytosines; 5 -hydroxymethyl cytosines; xanthines; hypoxanthines; 2-aminoadenines; 6-methyl derivatives of adenine; 6-methyl derivatives of guanine; 2-propyl derivatives of adenine; 2-propyl derivatives of guanine; 2-thiouracils; 2- thiothymines; 2-thiocytosines; 5-propynyl uracils; 5-propynyl cytosines; 6-azo uracils; 6-azo cytosines; 6-azo thymines; pseudouracils; 4-thiouracils; an 8-haloadenins; 8-aminoadenines; 8-thioladeninse; 8- thioalkyladcnincs; 8-hydroxyladcnincs; 8-haloguanincs; 8-aminoguanincs; 8-thiolguanincs; 8- thioalkylguanines; 8-hydroxylguanines; 5-halouracils; 5 -bromouracils; 5-trifluoromethyluracils; 5- halocytosines; 5 -bromocytosines; 5 -trifluoromethylcytosines; 5-substituted uracils; 5-substituted cytosines; 7-methylguanines; 7-methyladenines; 2-F-adenines; 2-amino-adenines; 8-azaguanines; 8- azaadenines; 7-deazaguanines; 7-deazaadenines; 3-deazaguanines; 3 -deazaadenines; tricyclic pyrimidines; phenoxazine cytidines; phenothiazine cytidines; substituted phenoxazine cytidines; carbazole cytidines; pyridoindolc cytidines; 7-dcazaguanosincs; 2-aminopyridincs; 2-pyridoncs; 5- substituted pyrimidines; 6-azapyrimidines; N-2, N-6 or 0-6 substituted purines; 2-aminopropyladenines; 5-propynyluracils; and 5-propynylcytosines.
Donor nucleic acid
[00143] Guided by a CRISPR-Cas effector guide RNA, a CRISPR-Cas effector protein in some cases generates site-specific double strand breaks (DSBs) or single strand breaks (SSBs) (e.g., when the CRISPR-Cas effector protein is a nickase variant) within double-stranded DNA (dsDNA) target nucleic acids, which are repaired either by non-homologous end joining (NHEJ) or homology-directed recombination (HDR).
[00144] In some cases, a composition of the present disclosure comprises: i) a fusion polypeptide of the present disclosure; ii) a guide nucleic acid; and iii) a donor nucleic acid. A donor nucleic acid comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid.
[00145] In some cases, contacting a target DNA (with a fusion polypeptide of the present disclosure and a CRISPR-Cas effector guide RNA) occurs under conditions that are permissive for nonhomologous end joining or homology-directed repair. Thus, in some cases, a subject method includes contacting the target DNA with a donor polynucleotide (e.g., by introducing the donor polynucleotide into a cell), wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. In some cases, the method does not comprise contacting a cell with a donor polynucleotide, and the target DNA is modified such that nucleotides within the target DNA are deleted.
[00146] In some cases, CRISPR-Cas effector guide RNA and a fusion polypeptide of the present disclosure are co-administered (e.g., contacted with a target nucleic acid, administered to cells, etc.) with a donor polynucleotide sequence that includes at least a segment with homology to the target DNA sequence, the subject methods may be used to add, i.e. insert or replace, nucleic acid material to a target DNA sequence (e.g. to “knock in” a nucleic acid, e.g., one that encodes for a protein, an siRNA, an miRNA, etc.), to add a tag (e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g. promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), to modify a nucleic acid sequence (e.g., introduce a mutation, remove a disease causing mutation by introducing a correct sequence), and the like.
[00147] In applications in which it is desirable to insert a polynucleotide sequence into the genome where a target sequence is cleaved, a donor polynucleotide (a nucleic acid comprising a donor sequence) can also be provided to the cell. By a “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site cleaved by the CRISPR-Cas effector protein (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) can support homology-directed repair. Donor polynucleotides can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.
[00148] The donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease-causing base pair to a non-disease-causing base pair). In some embodiments, the donor sequence comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non- homologous sequence at the target region. Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
[00149] The donor sequence may comprise certain sequence differences as compared to the genomic sequence, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor sequence at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). In some cases, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). Alternatively, these sequences differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence.
[00150] In some cases, the donor sequence is provided to the cell as single-stranded DNA. In some cases, the donor sequence is provided to the cell as double-stranded DNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphor amidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the termini of a linear donor sequence, additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination. A donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor sequences can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV), as described elsewhere herein for nucleic acids encoding a CRISPR-Cas effector guide RNA and/or a CRISPR-Cas effector fusion polypeptide and/or donor polynucleotide. [00151] A composition of the present disclosure can comprise, in addition to a fusion polypeptide of the present disclosure (and optionally also a CRISPR-Cas guide nucleic acid and optionally also a donor nucleic acid), one or more of: a) a lipid; b) a buffer; c) a nuclease inhibitor; and d) a protease inhibitor.
[00152] The present disclosure provides a system comprising a fusion polypeptide of the present disclosure. A system of the present disclosure can comprise: a) a fusion polypeptide of the present disclosure and a CRISPR-Cas guide RNA; b) a fusion polypeptide of the present disclosure and a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas guide RNA; c) a fusion polypeptide of the present disclosure, a CRISPR-Cas guide RNA, and a donor nucleic acid; or d) a fusion polypeptide of the present disclosure, a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas guide RNA, and a donor nucleic acid.
METHODS OF MODIFYING A TARGET NUCLEIC ACID
[00153] The present disclosure provides methods of modifying a target nucleic acid and/or modifying a polypeptide associated with a target nucleic acid.
[00154] In some cases, a method of the present disclosure comprises contacting a target nucleic acid with an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid. In some cases, a method of the present disclosure comprises contacting a target nucleic acid with: a) an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid; and b) a donor nucleic acid.
[00155] In some cases, a method of the present disclosure comprises contacting a eukaryotic cell comprising a target nucleic acid with an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid. In some cases, a method of the present disclosure comprises contacting a eukaryotic cell comprising a target nucleic acid with: a) an RNP, where the RNP comprises: i) a fusion polypeptide of the present disclosure; and ii) a guide nucleic acid; and b) a donor nucleic acid. Suitable eukaryotic cells including mammalian cells, plant cells, insect cells, arachnid cells, protozoan cells, fish cells, fungal cells, yeast cells, amphibian cells, reptile cells, and avian cells.
[00156] For example, a CRISPR-Cas effector-CPP fusion polypeptide of the present disclosure can be used to (i) modify (e.g., cleave, e.g., nick; methylate; deaminate; etc.) target nucleic acid (DNA or RNA; single stranded or double stranded); (ii) modulate transcription of a target nucleic acid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid (e.g., for purposes of isolation, labeling, imaging, tracking, etc.); (v) modify a polypeptide (e.g., a histone) associated with a target nucleic acid; and the like. Thus, the present disclosure provides a method of modifying a target nucleic acid. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a CRISPR-Cas effector polypeptide of the present disclosure; and b) one or more (e.g., two) CRISPR-Cas effector guide RNAs. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a CRISPR-Cas effector polypeptide of the present 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 carried out in a cell in vitro. In some cases, the contacting step is carried out in a cell in vivo. In some cases, the contacting step is carried out in a cell ex vivo. Modifications of a target nucleic acid that can be accomplished using a method of the present disclosure include, e.g., non-homologous end joining (NHEJ), microhomology- mediated end joining (MMEJ), and homology-directed repair (HDR). Such modifications can result in in gene knockout, DNA fragment insertion, deletion, replacement, or other modification. Modifications of a target nucleic acid that can be accomplished using a method of the present disclosure include base editing (e.g., modification of a cytidine or an adenosine). Modifications of a target nucleic acid that can be accomplished using a method of the present disclosure include any modification that can be carried out by a fusion partner, as described above (e.g., reverse transcription, base editing, etc.).
[00157] For example, the present disclosure provides (but is not limited to) methods of cleaving a target nucleic acid; methods of editing a target nucleic acid; methods of modulating transcription from a target nucleic acid; methods of isolating a target nucleic acid, methods of binding a target nucleic acid, methods of imaging a target nucleic acid, methods of modifying a target nucleic acid, and the like. [00158] In some cases, a method of modifying a target nucleic acid provides for treatment of a disease or disorder in an individual. Thus, the present disclosure provides a method of treating a disease or disorder in an individual, the method comprising administering to the individual: a) a fusion polypeptide of the present disclosure; or b) a fusion polypeptide of the present disclosure and a guide nucleic acid; or c) a fusion polypeptide of the present disclosure, a guide nucleic acid, and a donor nucleic acid.
Target nucleic acids and target cells of interest
[00159] A CRISPR-Cas effector polypeptide of the present disclosure, or a CRISPR-Cas effector fusion polypeptide of the present disclosure, when bound to a CRISPR-Cas effector guide RNA, can bind to a target nucleic acid, and in some cases, can bind to and modify a target nucleic acid. A target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the CRISPR-Cas effector guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted).
[00160] A target nucleic acid can be DNA or RNA. A target nucleic acid can be double stranded
(e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is single stranded. In some cases, a target nucleic acid is a single stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and microRNA (miRNA). In some cases, a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some cases, a target nucleic acid is single stranded. A target nucleic acid can be genomic DNA (e.g., nuclear DNA). A target nucleic acid can be mitochondrial DNA. A target nucleic acid can be mitochondrial RNA. A target nucleic acid can be extrachromosomal DNA.
[00161] A target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo. Suitable target cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell from a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell from a mammal (e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).
[00162] Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in vitro. Target cells can be unicellular organisms and/or can be grown in culture. If the cells are primary cells, they may be harvest from an individual by any convenient method. For example, leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy. [00163] In some of the above applications, the subject methods may be employed to induce target nucleic acid cleavage, target nucleic acid modification, and/or to bind target nucleic acids (e.g., for visualization, for collecting and/or analyzing, etc.) in mitotic or post-mitotic cells in vivo and/or ex vivo and/or in vitro (e.g., to disrupt production of a protein encoded by a targeted mRNA, to cleave or otherwise modify target DNA, to genetically modify a target cell, and the like). Because the guide RNA provides specificity by hybridizing to target nucleic acid, a mitotic and/or post-mitotic cell of interest in the disclosed methods may include a cell from any organism (e.g. a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal, a cell from a rodent, a cell from a human, etc.). In some cases, a subject CRISPR-Cas effector protein (and/or nucleic acid encoding the protein such as DNA and/or RNA), and/or CRISPR-Cas effector guide RNA (and/or a DNA encoding the guide RNA), and/or donor template, and/or RNP can be introduced into an individual (i.e., the target cell can be in vivo) (e.g., a mammal, a rat, a mouse, a pig, a primate, a non-human primate, a human, etc.). In some case, such an administration can be for the purpose of treating and/or preventing a disease, e.g., by editing the genome of targeted cells.
[00164] Plant cells include cells of a monocotyledon, and cells of a dicotyledon. The cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like. Plant cells include cells of agricultural crops such as wheat, corn, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.
[00165] Additional examples of target cells are listed above in the section titled “Modified cells.” Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosscs, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).
[00166] A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). A cell can be and in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a fungal cell or derived from a fungal cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.
[00167] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.
[00168] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogenic cells, and post-natal stem cells. [00169] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).
[00170] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.
[00171] Adult stem cells are resident in differentiated tissue, but retain the properties of selfrenewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.
[00172] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.
[00173] Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1 , OLFM4, CDH17, and PPARGC1 A.
[00174] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3 . HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.
[00175] In other cases, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.
[00176] In other cases, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.
[00177] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon.
[00178] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue- cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (Chinese mustard), gallon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, Jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (russian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled/ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams, yu choy, yuca (cassava), and the like.
[00179] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a suborder, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola , Hymenoptera , Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera , Mecoptera , Siphonaptera, Diptera, Trichoptera, or Lepidoptera.
[00180] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.
Examples of Non-Limiting Aspects of the Disclosure SPECTS SET A [00181] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below.
[00182] Aspect 1. A composition comprising: a) an engineered CRISPR-Cas effector polypeptide or a nucleic acid comprising a first nucleotide sequence encoding the engineered CRISPR- Cas effector polypeptide; and b) a guide nucleic acid or a nucleic acid comprising a second nucleotide sequence encoding the guide nucleic acid; wherein the engineered CRISPR-Cas effector polypeptide comprises one or more heterologous polypeptides that facilitate cellular uptake of a nucleic acid/protein complex comprising the engineered Cas9 polypeptide and the guide nucleic acid into a eukaryotic cell; optionally wherein the one or more heterologous polypeptides facilitate release of the nucleic acid/protein complex from an endosome within the eukaryotic cell.
[00183] Aspect 2. A composition comprising an engineered CRISPR-Cas effector polypeptide or a nucleic acid comprising a first nucleotide sequence encoding the engineered CRISPR-Cas effector polypeptide; wherein the engineered CRISPR-Cas effector polypeptide comprises one or more heterologous polypeptides that replace one or more native amino acids of a non-engineered CRISPR-Cas effector polypeptide; wherein the engineered CRISPR-Cas effector polypeptide is less immunogenic to a human subject compared to the immunogenicity of the non-engineered CRISPR-Cas effector polypeptide to the human subject.
[00184] Aspect 3. A composition comprising an engineered CRISPR-Cas effector polypeptide or a nucleic acid comprising a first sequence encoding the engineered CRISPR-Cas effector polypeptide; wherein the engineered CRISPR-Cas effector polypeptide comprises one or more heterologous polypeptides inserted (i) at the N-terminus of the engineered CRISPR-Cas effector polypeptide, (ii) at the C-terminus of the engineered CRISPR-Cas effector polypeptide or (iii) internally within the engineered CRISPR-Cas effector polypeptide.
[00185] Aspect 4. The composition of aspect 2 or 3, wherein the composition further comprises a guide nucleic acid or a nucleic acid comprising a second nucleotide sequence encoding the guide nucleic acid.
[00186] Aspect 5. The composition of aspect 1 or 4, wherein the guide nucleic acid is a singlemolecule guide nucleic acid. [00187] Aspect 6. The composition of any one of aspects 1-5, wherein the composition further comprises a donor nucleic acid.
[00188] Aspect 7. The composition of any one of aspects 1-6, wherein the engineered Cas9 polypeptide is a fusion polypeptide of a CRISPR-Cas effector polypeptide and the one or more heterologous polypeptides.
[00189] Aspect 8. The composition of any one of aspects 1-7, wherein the one or more heterologous polypeptides comprise one or more cell penetrating polypeptides (CPPs), optionally wherein the one or more CPPs comprises any one of the amino acid sequences set out in Table 1, or a variant thereof.
[00190] Aspect 9. The composition of aspect 8, wherein the CPP is an A22p polypeptide.
[00191] Aspect 10. The composition of aspect 9, wherein the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof [00192] Aspect 11. The composition of aspect 9, wherein the CPP is a Bac7 polypeptide.
[00193] Aspect 12. The composition of aspect 11, wherein the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO: 2), or a variant thereof.
[00194] Aspect 13. The composition of aspect 9, wherein the CPP is a CATat2 polypeptide.
[00195] Aspect 14. The composition of aspect 13, wherein the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG), or a variant thereof.
[00196] Aspect 15. The composition of aspect 9, wherein the CPP is a VP22 polypeptide.
[00197] Aspect 16. The composition of aspect 15, wherein the VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NOG) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof.
[00198] Aspect 17. The composition of any one of aspects 9-16, wherein the fusion polypeptide comprises two or more copies of the CPP.
[00199] Aspect 18. The composition of any one of aspects 9-17, wherein the fusion polypeptide comprises a linker between the CPP and the CRISPR-Cas effector polypeptide.
[00200] Aspect 19. The composition of aspect 18, wherein the linker between the CPP and the CRISPR-Cas effector polypeptide is a proteolytically cleavable.
[00201] Aspect 20. The composition of any one of aspects 9-19, wherein the fusion polypeptide comprises one or more nuclear localization sequences (NLSs). [00202] Aspect 21. The composition of aspect 20, wherein the fusion polypeptide comprises two or more NLSs.
[00203] Aspect 22. The composition of aspect 20 or 21, wherein an NLS comprises the amino acid sequence K(K/R)X(K/R), where X is any amino acid.
[00204] Aspect 23. The composition of any one of aspects 20-22, wherein an NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO:8).
[00205] Aspect 24. The composition of any one of aspects 20-23, wherein at least one NLS or each NLS is at the N-terminus of the CRISPR-Cas effector polypeptide.
[00206] Aspect 25. The composition of any one of aspects 9-24, wherein the CPP is at the C- terminus of the CRISPR-Cas effector polypeptide.
[00207] Aspect 26. The composition of any one of aspects 9-25, wherein the CPP is inserted internally within the CRISPR-Cas effector polypeptide.
[00208] Aspect 27. The composition of any one of aspects 9-26, wherein the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide.
[00209] Aspect 28. The composition of any one of aspects 9-26, wherein the CRISPR-Cas effector polypeptide is a Type V CRISPR-Cas effector polypeptide.
[00210] Aspect 29. The composition of any one of aspects 9-26, wherein the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide.
[00211] Aspect 30. The composition of any one of aspects 9-29, wherein the CRISPR-Cas effector polypeptide is catalytically active.
[00212] Aspect 31. The composition of any one of aspects 9-29, wherein the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
[00213] Aspect 32. The composition of any one of aspects 9-31, wherein the fusion polypeptide further comprises at least one additional heterologous polypeptide.
[00214] Aspect 33. The composition of any one of aspects 9-32, wherein the at least one additional heterologous polypeptide is a deaminase, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.
[00215] Aspect 34. The composition of any one of aspects 9-33, wherein the at least one additional heterologous polypeptide is a targeting moiety that binds to a target of a eukaryotic cell.
[00216] Aspect 35. The composition of aspect 34, wherein the targeting moiety is an antibody or binding domain thereof.
[00217] Aspect 36. The composition of aspect 34, wherein the targeting moiety is or a small molecule. [00218] Aspect 37. The composition of any one of aspects 34-36, wherein the target is a cell surface target of the eukaryotic cell.
[00219] Aspect 38. A composition comprising a nucleic acid, wherein the nucleic acid comprises a nucleotide sequence encoding the engineered CRISPR-Cas effector polypeptide of the composition of any one of aspects 1-37.
[00220] Aspect 39. The composition of aspect 38, wherein the nucleotide sequence is operably linked to a transcriptional control element.
[00221] Aspect 40. The composition of aspect 39, wherein the transcriptional control element is a promoter.
[00222] Aspect 41. The composition of any one of aspects 38-40, wherein the nucleic acid is a recombinant expression vector.
[00223] Aspect 42. A composition comprising a cell, wherein the cell comprises the engineered CRISPR-Cas effector polypeptide of the composition of any one of aspects 1-37 or the nucleic acid of the composition of any one of aspects 38-41.
[00224] Aspect 43. The composition of aspect 42, wherein the cell is a eukaryotic cell.
[00225] Aspect 44. The composition of aspect 42 or 43, wherein the cell is in vitro.
[00226] Aspect 45. The composition of aspect 42 or 43, wherein the cell is in vivo.
[00227] Aspect 46. A pharmaceutical composition comprising a therapeutically effective amount of the composition of any one of aspects 1-44.
[00228] Aspect 47. A method of treating a disease or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of aspect 46.
[00229] Aspect 48. A method of modifying a target nucleic acid in a eukaryotic cell comprising introducing into the eukaryotic cell the composition of any one of aspects 1-41.
[00230] Aspect 49. The method of aspect 48, comprising introducing into the cell a donor nucleic acid.
[00231] Aspect 50. The method of aspect 48 or 49, wherein the cell is in vitro.
[00232] Aspect 51. The method of aspect 48 or 49, wherein the cell is in vivo.
[00233] Aspect 52. The method of any one of aspects 48-51, wherein the cell is a mammalian cell.
[00234] Aspect 53. The method of any one of aspects 48-51 , wherein the cell is a plant cell. SPECTS SET B
[00235] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below.
[00236] Aspect 1. A fusion polypeptide comprising: a) a CRISPR-Cas effector polypeptide; and b) a cell penetrating polypeptide (CPP), wherein the CPP is an A22p polypeptide.
[00237] Aspect 2. A fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a Bac7 polypeptide.
[00238] Aspect 3. A fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a CAT t2 polypeptide.
[00239] Aspect 4. A fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a VP22 polypeptide.
[00240] Aspect 5. The fusion polypeptide of aspect 1, wherein the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
[00241] Aspect 6. The fusion polypeptide of aspect 2, wherein the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof.
[00242] Aspect 7. The fusion polypeptide of aspect 3, wherein the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG), or a variant thereof.
[00243] Aspect 8. The fusion polypeptide of aspect 4, wherein the VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO:6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NOG), or a variant thereof.
[00244] Aspect 9. The fusion polypeptide of any one of aspects 1-8, wherein the fusion polypeptide comprises two or more copies of the CPP.
[00245] Aspect 10. The fusion polypeptide of any one of aspects 1-9, wherein the fusion polypeptide comprises a linker between the CPP and the CRISPR-Cas effector polypeptide.
[00246] Aspect 11. The fusion polypeptide of aspect 10, wherein the linker between the CPP and the CRISPR-Cas effector polypeptide is a proteolytically cleavable. [00247] Aspect 12. The fusion polypeptide of any one of aspects 1-11, wherein the two or more NLSs comprise the amino acid sequence K(K/R)X(K/R), where X is any amino acid.
[00248] Aspect 13. The fusion polypeptide of aspect 12, wherein the two or more NLSs comprise the amino acid sequence PKKKRKV (SEQ ID NO: 8).
[00249] Aspect 14. The fusion polypeptide of any one of aspects 1-13, wherein the two or more NLS are at the N-terminus of the CRISPR-Cas effector polypeptide.
[00250] Aspect 15. The fusion polypeptide of any one of aspects 1-14, wherein the CPP is at the C-terminus of the CRISPR-Cas effector polypeptide.
[00251] Aspect 16. The fusion polypeptide of any one of aspects 1-14, wherein the CPP is inserted internally within the CRISPR-Cas effector polypeptide.
[00252] Aspect 17. The fusion polypeptide of any one of aspects 1-16, wherein the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide.
[00253] Aspect 18. The fusion polypeptide of any one of aspects 1-16, wherein the CRISPR-Cas effector polypeptide is a Type V CRISPR-Cas effector polypeptide.
[00254] Aspect 19. The fusion polypeptide of any one of aspects 1-16, wherein the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide.
[00255] Aspect 20. The fusion polypeptide of any one of aspects 1-19, wherein the CRISPR-Cas effector polypeptide is catalytically active.
[00256] Aspect 21. The fusion polypeptide of aspects 1-19, wherein the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
[00257] Aspect 22. The fusion polypeptide of any one of aspects 1-21, wherein the fusion polypeptide further comprises at least one additional heterologous polypeptide.
[00258] Aspect 23. The fusion polypeptide of aspect 22, wherein the at least one additional heterologous polypeptide is a deaminase, a base editor, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.
[00259] Aspect 24. A composition comprising: a) a fusion polypeptide of any one of aspects 1- 23, or a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
[00260] Aspect 25. The composition of aspect 24, wherein the guide nucleic acid is a singlemolecule guide nucleic acid.
[00261] Aspect 26. The composition of aspect 24, further comprising a donor nucleic acid.
[00262] Aspect 27. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of aspects 1-23. [00263] Aspect 28. The nucleic acid of aspect 27, wherein the nucleotide sequence is operably linked to a transcriptional control clement, optionally wherein the transcriptional control element is a promoter.
[00264] Aspect 29. A recombinant expression vector comprising the nucleic acid of aspect 27.
[00265] Aspect 30. A cell comprising the fusion polypeptide of any one of aspects 1-23.
[00266] Aspect 31. The cell of aspect 30, wherein the cell is a eukaryotic cell.
[00267] Aspect 32. The cell of aspect 30 or aspect 31, wherein the cell is in vitro.
[00268] Aspect 33. The cell of aspect 30 or aspect 31, wherein the cell is in vivo.
[00269] Aspect 34. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with: a) a fusion polypeptide of any one of aspects 1-23; and b) a guide nucleic acid.
[00270] Aspect 35. A method of modifying a target nucleic acid in a eukaryotic cell, the method comprising introducing into the eukaryotic cell: a) a fusion polypeptide of any one of aspects 1-23; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
[00271] Aspect 36. The method of aspect 35, comprising introducing into the cell a donor nucleic acid.
[00272] Aspect 37. The method of aspect 35 or 36, wherein the cell is in vitro.
[00273] Aspect 38. The method of aspect 35 or 36, wherein the cell is in vivo.
[00274] Aspect 39. The method of any one of aspects 35-38, wherein the cell is a mammalian cell, an insect cell, an avian cell, a reptile cell, an amphibian cell, an arachnid cell, a protozoan cell, or a plant cell.
[00275] Aspect 40. The method of any one of aspects 35-39, wherein the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.
[00276] Aspect 41. The method of any one of aspects 35-40, wherein said modifying comprises genome editing.
[00277] Aspect 42. A fusion polypeptide comprising:
[00278] a) a CRISPR-Cas effector polypeptide; and
[00279] b) a cell penetrating polypeptide (CPP), wherein the CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidsl058 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246 and 1247; and xvi) amino acids 1247 and 1248 based on the Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide.
[00280] Aspect 43. The fusion polypeptide of aspect 42, wherein the CPP is an A22p polypeptide, a Bac7 polypeptide, a CATat2 polypeptide, or a VP22 polypeptide.
[00281] Aspect 44. A fusion polypeptide comprising, in order from N-terminus to C-terminus: a) a first nuclear localization sequence (NLS); b) a second NLS; c) a CRISPR-Cas effector polypeptide; d) at least two cell penetrating polypeptides (CPPs), optionally wherein one or more independently selected peptide linkers are interposed between any two of polypeptides (a) through (d), optionally wherein the CPPs are separated by a peptide linker.
[00282] Aspect 45. The fusion polypeptide of aspect 44, wherein the at least two CPPs are A22p polypeptides.
[00283] Aspect 46. The fusion polypeptide of aspect 45, wherein the A22p polypeptides comprise the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
[00284] Aspect 47. The fusion polypeptide of aspect 44, wherein the at least two CPPs are Bac7 polypeptides.
[00285] Aspect 48. The fusion polypeptide of aspect 47, wherein the Bac7 polypeptides comprise the amino acid sequence RR1RPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof.
[00286] Aspect 49. The fusion polypeptide of aspect 44, wherein the at least two CPPs are CATat2 polypeptides.
[00287] Aspect 50. The fusion polypeptide of aspect 49, wherein the CATat2 polypeptides comprise the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO:3), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NO:5),or a variant thereof.
[00288] Aspect 51. The fusion polypeptide of aspect 44, wherein the at least two CPPs are VP22 polypeptides.
[00289] Aspect 52. The fusion polypeptide of aspect 51, wherein the VP22 polypeptides comprise the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO:6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof. EXAMPLES
[00290] The following examples arc put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
Example 1
[00291] An assay for testing the delivery and editing efficiency of various fusion polypeptides comprising a CRISPR-Cas effector polypeptide and a CPP is depicted in FIG. 1A-1D. Streptococcus pyogenes Cas9 (Spy Cas9) was used as a model CRISPR-Cas effector polypeptide. One or more cell penetrating polypeptides (CPPs) were fused to the Spy Cas9. In some cases, one or more nuclear localization signals (NLSs) were also fused to the Spy Cas9, as shown schematically in FIG. 1 A. As shown schematically in FIG. IB, neural progenitor cells (NPCs) were modified to include a target nucleic acid encoding the fluorescent protein tdTomato and including a stop cassette. Guide RNAs were targeted to the stop cassette so that, if delivery and editing occurred, the stop cassette would be excised, as depicted schematically in FIG. 1C, and tdTomato would be expressed. The design of the NLS-Cas9- CPP is depicted schematically in FIG. ID.
[00292] Fusion proteins comprising Spy Cas9 and CATat2 or CAcTat2 as the CPP were generated. The CATat2 peptide was KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4). The CAcTat2 peptide was KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NO:5). The fusion proteins also included 2 NLSs at the N-terminus. The fusion protein comprising 2NLSs, Spy Cas9, and CATat2 is referred to as “2NLS-Cas9-CATat2.” The fusion protein comprising 2NLSs, Spy Cas9, and CAcTat2 is referred to as “2NLS-Cas9-CAcTat2.” The fusion proteins, with guide RNAs targeting the stop cassette (FIG. 1C) were introduced into NPCs. A fusion protein/guide RNA complex is referred to as a ribonucleoprotein (RNP). 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 2. As shown in FIG. 2, the “2NLS-Cas9-CATat2” fusion protein efficiently entered the cells and edited the target nucleic acid.
[00293] Fusion proteins comprising Spy Cas9 and VP22 as the CPP were generated. The VP22 peptide was NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO:6). Three different fusion proteins were generated. One fusion protein, termed “2NLS-Cas9-VP22,” included (i) 2 NLS at the N- terminus of Spy Cas9; (ii) Spy Cas9; and (iii) a single copy of VP22. A second fusion protein, termed “VP22-Cas9-2NLS,” included: (i) a single VP22 peptide at the N-terminus of Spy Cas9; (ii) Spy Cas9; and (iii) 2 NLSs at the C-terminus of Spy Cas9. A third fusion protein, termed “c-myc-NLS-Cas9- VP22,” included: (i) a c-myc NLS at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) a VP22 peptide at the C-terminus of Spy Cas9. RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 3.
[00294] Fusion proteins comprising Spy Cas9 and A22p as the CPP were generated. The A22p peptide was HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1). Four different fusion proteins were generated. One fusion protein, termed “2NLS-Cas9-A22p” included: i) 2 NLSs at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) the A22p peptide at the C-terminus of Spy Cas9. A second fusion protein, termed “A22p-Cas9-2NLS,” included: i) the A22p peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) 2 NLSs at the C-terminus of Spy Cas9. A third fusion protein, termed “c-myc NLS-Cas9-A22p,” included: i) a c-myc NLS at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) the A22p peptide at the C-terminus of Spy Cas9. A fourth fusion protein, termed “A22p-Cas9-c-myc NLS,” included: i) the A22p peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) a c-myc NLS at the N-terminus of Spy Cas9. RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 4.
[00295] Fusion proteins comprising Spy Cas9 and Bac7 as the CPP were generated. The Bac7 peptide was RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2). Four different fusion proteins were generated. One fusion protein, termed “2NLS-Cas9-Bac7” included: i) 2 NLSs at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) the Bac7 peptide at the C-terminus of Spy Cas9. A second fusion protein, termed “Bac7-Cas9-2NLS,” included: i) the Bac7 peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) 2 NLSs at the C-terminus of Spy Cas9. A third fusion protein, termed “c-myc NLS-Cas9- Bac7,” included: i) a c-myc NLS at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) the A2 Bac7 peptide at the C-terminus of Spy Cas9. A fourth fusion protein, termed “A22p-Cas9-c-myc NLS,” included: i) the Bac7 peptide at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) a c-myc NLS at the N-terminus of Spy Cas9. RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 5. [00296] Various strategies were employed to further modify the Cas9-CPP fusion proteins, to increase delivery efficiency. One strategy was to insert a CPP internally within Cas9. As depicted in FIG. 6A, sites of insertion into Spy Cas9 included at amino acids 198, 205, 257, 384, 532, 1052, 1059, 1068, and 1247. To test this strategy, a Bac7 peptide was inserted into Spy Cas9 at amino acid 205. The Bac7 insert is depicted in FIG. 6B. The following fusion proteins were generated. A fusion protein termed “2NLS-Cas9-2NLS” included SpyCas9 with 2 NLSs at the N-terminus of Spy Cas9 and 2 NLSs at the C- terminus of Spy Cas9, but did not include a CPP. A fusion protein termed “2NLS-Cas9-Bac7” included: i) 2 NLSs at the N-terminus of Spy Cas9; ii) Spy Cas9; and ii) the Bac7 peptide. A fusion protein termed “2NLS-Cas9(Bac7ins)-2NLS,” included: i) 2 NLSs at the N-terminus of Spy Cas9; ii) Spy Cas9 with the Bac7 peptide inserted at amino acid 205 of Spy Cas9; and 2 NLSs at the C-terminus of Spy Cas9. Expression constructs encoding the fusion proteins were also generated. RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 6C (nucleofection) and FIG. 6D (direct introduction of RNPs).
[00297] Another strategy to increase delivery of RNPs into cells involved increasing the copy number of the CPP fused to the CRISPR-Cas effector protein. This strategy is depicted schematically in FIG. 7. Fusion proteins including A22p peptide as the CPP and Spy Cas9 as the CRISPR-Cas effector protein were used as models. Fusion proteins containing Spy Cas9 and 2, 3, or 5 copies of A22p are shown in FIG. 8A-8C, respectively. Fusion proteins containing Spy Cas9 and 2 or 3 copies of A22p arc shown in FIG. 8D and FIG. 8E, respectively. RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by nucleofection or by direct delivery into the NPCs. The results are shown in FIG. 9. As shown in FIG. 9, the fusion protein termed “2NLS- Cas9-A22p3” and comprising: i) 2 NLSs at the N-terminus of Spy Cas9; ii) Spy Cas9; and iii) 3 copies of the A22p peptide at the C-terminus of Spy Cas9, exhibited efficient delivery into cells when delivered by direct delivery of an RNP comprising the fusion protein and the guide RNA.
[00298] Another strategy for increasing the efficiency of delivery of an RNP into a cell involved use of Cas9 variants in which the Cys at position 80 of Spy Cas9 and the Cys at position 574 of Spy Cas9 was substituted with Ala. A fusion protein was made that included: i) 2 NLSs at the N-terminus; ii) Spy Cas9 (C80SA; C574SA); and iii) 3 copies of the A22p peptide at the C-terminus. RNPs were made by combining the fusion proteins with guide RNA, as described above. 25 pmol, 25 pmol, or 100 pmol RNP was introduced by direct delivery into the NPCs. The results are shown in FIG. 10 and FIG. 11. [00299] Fusion proteins were made with Lb Casl2a and either: i) 2 NLSs at the N-terminus and 2 NLSs at the C-terminus (and no CPP); or ii) NLSs and the CPP Bac7. The target nucleic acid is depicted in FIG. 12A. RNPs were made by combining the fusion proteins with guide RNA, as described above. 100 pmol RNP was introduced by nucleofection or by direct delivery into the NPCs. The results are shown in FIG. 12B and FIG. 13.
[00300] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:
1. A fusion polypeptide comprising: a) a CRISPR-Cas effector polypeptide; and b) a cell penetrating polypeptide (CPP), wherein the CPP is an A22p polypeptide.
2. A fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a Bac7 polypeptide.
3. A fusion polypeptide comprising: a) two or more nuclear' localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a CATat2 polypeptide.
4. A fusion polypeptide comprising: a) two or more nuclear localization sequences; b) a CRISPR-Cas effector polypeptide; and c) a cell penetrating polypeptide (CPP), wherein the CPP is a VP22 polypeptide.
5. The fusion polypeptide of claim 1 , wherein the A22p polypeptide comprises the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1), or a variant thereof.
6. The fusion polypeptide of claim 2, wherein the Bac7 polypeptide comprises the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof.
7. The fusion polypeptide of claim 3, wherein the CATat2 polypeptide comprises the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NOG), or a variant thereof.
8. The fusion polypeptide of claim 4, wherein the VP22 polypeptide comprises the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO: 6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof.
9. The fusion polypeptide of any one of claims 1-8, wherein the fusion polypeptide comprises two or more copies of the CPP.
10. The fusion polypeptide of any one of claims 1-9, wherein the fusion polypeptide comprises a linker between the CPP and the CRISPR-Cas effector polypeptide.
11. The fusion polypeptide of claim 10, wherein the linker between the CPP and the CRISPR-Cas effector polypeptide is a proteolytically cleavable.
12. The fusion polypeptide of any one of claims 1-11, wherein the two or more NLSs comprise the amino acid sequence K(K/R)X(K/R), where X is any amino acid.
13. The fusion polypeptide of claim 12, wherein the two or more NLSs comprise the amino acid sequence PKKKRKV (SEQ ID NO: 8).
14. The fusion polypeptide of any one of claims 1-13, wherein the two or more NLS are at the N- terminus of the CRISPR-Cas effector polypeptide.
15. The fusion polypeptide of any one of claims 1-14, wherein the CPP is at the C-terminus of the CRISPR-Cas effector polypeptide.
16. The fusion polypeptide of any one of claims 1-14, wherein the CPP is inserted internally within the CRISPR-Cas effector polypeptide.
17. The fusion polypeptide of any one of claims 1-16, wherein the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide.
18. The fusion polypeptide of any one of claims 1-16, wherein the CRISPR-Cas effector polypeptide is a Type V CRISPR-Cas effector polypeptide.
19. The fusion polypeptide of any one of claims 1-16, wherein the CRISPR-Cas effector polypeptide is a Type VI CRISPR-Cas effector polypeptide.
20. The fusion polypeptide of any one of claims 1-19, wherein the CRISPR-Cas effector polypeptide is catalytically active.
21. The fusion polypeptide of claims 1-19, wherein the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.
22. The fusion polypeptide of any one of claims 1-21, wherein the fusion polypeptide further comprises at least one additional heterologous polypeptide.
23. The fusion polypeptide of claim 22, wherein the at least one additional heterologous polypeptide is a deaminase, a base editor, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.
24. A composition comprising: a) a fusion polypeptide of any one of claims 1-23, or a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
25. The composition of claim 24, wherein the guide nucleic acid is a single-molecule guide nucleic acid.
26. The composition of claim 24, further comprising a donor nucleic acid.
27. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of claims 1-23.
28. The nucleic acid of claim 27, wherein the nucleotide sequence is operably linked to a transcriptional control element, optionally wherein the transcriptional control element is a promoter.
29. A recombinant expression vector comprising the nucleic acid of claim 27.
30. A cell comprising the fusion polypeptide of any one of claims 1-23.
31. The cell of claim 30, wherein the cell is a eukaryotic cell.
32. The cell of claim 30 or claim 31, wherein the cell is in vitro.
33. The cell of claim 30 or claim 31, wherein the cell is in vivo.
34. A method of modifying a tar get nucleic acid, the method comprising contacting the target nucleic acid with: a) a fusion polypeptide of any one of claims 1-23; and b) a guide nucleic acid.
35. A method of modifying a target nucleic acid in a eukaryotic cell, the method comprising introducing into the eukaryotic cell: a) a fusion polypeptide of any one of claims 1-23; and b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.
36. The method of claim 35, comprising introducing into the cell a donor nucleic acid.
37. The method of claim 35 or 36, wherein the cell is in vitro.
38. The method of claim 35 or 36, wherein the cell is in vivo.
39. The method of any one of claims 35-38, wherein the cell is a mammalian cell, an insect cell, an avian cell, a reptile cell, an amphibian cell, an arachnid cell, a protozoan cell, or a plant cell.
40. The method of any one of claims 35-39, wherein the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.
41. The method of any one of claims 35-40, wherein said modifying comprises genome editing.
42. A fusion polypeptide comprising: a) a CRISPR-Cas effector polypeptide; and b) a cell penetrating polypeptide (CPP), wherein the CPP is inserted between one or more of: i) amino acids 197 and 198; ii) amino acids 198 and 199; iii) amino acids 204 and 205; iv) amino acids 205 and 206; v) amino acids 256 and 257; vi) amino acids 257 and 258; vii) amino acids 383 and 384; viii) amino acids 384 and 385; ix) amino acids 531 and 532; x) amino acids 532 and 533; xi) amino acids 1051 and 1052; xii) amino acids 1052 and 1053; xiii) amino acidslO58 and 1059; xiv) amino acids 1059 and 1060; xv) amino acids 1067 and 1068; xvi) amino acids 1068 and 1069; xv) amino acids 1246 and 1247; and xvi) amino acids 1247 and 1248 based on the Cas9 amino acid sequence depicted in FIG. 14A, or corresponding positions in another CRISPR-Cas effector polypeptide.
43. The fusion polypeptide of claim 42, wherein the CPP is an A22p polypeptide, a Bac7 polypeptide, a CATat2 polypeptide, or a VP22 polypeptide.
44. A fusion polypeptide comprising, in order from N-terminus to C-terminus: a) a first nuclear localization sequence (NLS); b) a second NLS; c) a CRISPR-Cas effector polypeptide; d) at least two cell penetrating polypeptides (CPPs), optionally wherein one or more independently selected peptide linkers are interposed between any two of polypeptides (a) through (d), optionally wherein the CPPs are separated by a peptide linker.
45. The fusion polypeptide of claim 44, wherein the at least two CPPs are A22p polypeptides.
46. The fusion polypeptide of claim 45, wherein the A22p polypeptides comprise the amino acid sequence HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO:1 ), or a variant thereof.
47. The fusion polypeptide of claim 44, wherein the at least two CPPs arc Bac7 polypeptides.
48. The fusion polypeptide of claim 47, wherein the Bac7 polypeptides comprise the amino acid sequence RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO:2), or a variant thereof.
49. The fusion polypeptide of claim 44, wherein the at least two CPPs are CATat2 polypeptides.
50. The fusion polypeptide of claim 49, wherein the CATat2 polypeptides comprise the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NOG), KWKLFKKYGRKKRRQRRRPPQGGSGRKKRRQRRRPPQ (SEQ ID NO:4), or KWKLFKKYGRKKRRQRRRPPQGGSCYGRKKRRQRRRCGPKR (SEQ ID NO:5),or a variant thereof.
51. The fusion polypeptide of claim 44, wherein the at least two CPPs are VP22 polypeptides.
52. The fusion polypeptide of claim 51, wherein the VP22 polypeptides comprise the amino acid sequence NAATATRGRSAASRPTQRPRAPARSASRPRRP (SEQ ID NO: 6) or DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:7), or a variant thereof.
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