Modified crispr-cas effector polypeptides and methods of use thereof

Abstract

Aspects of the present disclosure relate to fusion polypeptides comprising a CRISPR-Cas effector polypeptide and a heterologous polypeptide (e.g., a targeting moiety, an endosomal escape peptide, and / or a pH responsive polypeptide domain). In some aspects, the present disclosure provides compositions comprising a fusion polypeptide of the present disclosure and a guide nucleic acid. In some aspects, the present disclosure also provides fusion polynucleotides (e.g., an oligonucleotide or a short interference RNA) and a heterologous polypeptide (e.g., a targeting moiety, an endosomal escape peptide, and / or a pH responsive polypeptide domain). The present disclosure provides methods of modifying a target nucleic acid in a cell, such as a eukaryotic cell.

Description

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 619,705 filed on January 10, 2024, and U.S. Provisional Patent Application No. 63 / 727,069 filed on December 2, 2024, each of which is incorporated by reference in its entirety. REFERENCE TO A SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 8, 2025, is named 60555-702_601_SL. xml and is 132,814,069 bytes in size. BACKGROUND

[0003] Precise delivery of molecular and cellular therapeutic payloads (e.g., gene-manipulating nucleic acid payloads) unlocks a wide range of therapeutic applications. The growing understanding of payload delivery promotes the application of various molecular and cellular therapeutics to selectively function on hitherto “undruggable” cells, proteins, transcripts, and genes, thus potentially broadening therapeutic targets. For example, several molecular and cellular therapeutics have been approved for clinical use such as the antisense oligonucleotide (ASO) mipomersen for familial hypercholesterolemia, and the splice-switching oligonucleotide (SSO) eteplirsen for Duchenne muscular dystrophy. Molecular and cellular therapeutics also include siRNA delivery, with a recent shift toward mRNA delivery. The shift to mRNA payloads opens a myriad of therapeutic applications. These range from expression of antigens of choice as prophylactic viral and bacterial vaccines as well as therapeutic cancer vaccine applications, supplementation of missing proteins for enzyme replacement therapies, and expression of gene editing machinery, such as CRISPR, to gene edit aberrant natively expressed proteins.

[0004] In the world of delivering molecular and cellular therapeutic payloads (e.g., RNA-based therapeutics) into cells, most, if not all, roads can eventually lead to the endosomal escape abyss. Delivery of molecular and cellular therapeutics often involves internalization via endocytosis followed by trafficking through various membrane-bound vesicular compartments. For example, endocytosed molecular and cellular therapeutics can be transferred to early endosomes, which can mature into late endosomes and eventually into lysosomes. The development of delivery modalities, such as LNPs, has addressed some of the challenges related to inefficient delivery. However, despite recent developments in the field, a commonly overlooked aspect regards the very limited release of the payloads into the cytoplasm. For efficient delivery, in some situations, the payloads would need to be released into the cytosol before the maturation of late endosomes to lysosomes where the majority of the foreign materials are either degraded enzymatically or recycled outside of the target cells, with only a very limited amount of payload released into the cytoplasm. The release of the payload prior to lysosomal maturation can be a crucial stage for efficient delivery and is known as endosomal escape. This process can be inefficient and is considered a bottleneck. For example, large, hydrophilic, negatively-charged properties often prevent molecular and cellular therapeutic payloads (e.g., nucleic acid payloads) from passively diffusing across the lipid bilayers. Thus, escaping from the endosome and releasing payloads into the cytoplasm in a non-toxic manner is a critical technical problem, resulting in inefficient access of therapeutic payloads to the sites of action in the nucleus or cytosol of cells. This problem has meant that large payload doses are often administered to attain therapeutic effects, risking drug-related toxicities. One approach to overcoming this challenge has been the use of complex delivery systems such as cationic lipid or polymer nanoparticles, however these approaches often suffer from toxicity and biodistribution problems associated with the delivery system itself. Therefore, there remains a need for the development of alternative strategies to enhance the delivery of molecular and cellular therapeutic payloads to their intracellular targets.

[0005] 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. There is a need in the art for modified CRISPR-Cas effector polypeptides that are readily delivered as the proteins per se. SUMMARY

[0006] The compositions and methods described herein can overcome the drawbacks of the currently available approaches for payload delivery to sites of action in the nucleus and / or cytosol of cells. Provided herein, are compositions, methods, compositions, and kits useful for delivering therapeutic payloads to intracellular targets.

[0007] Provided herein, in some aspects, are compositions comprising a fusion polypeptide comprising: (a) an effector polypeptide; and (b) a targeting moiety that specifically binds to an extracellular domain of a receptor expressed by a target cell, wherein the targeting moiety is not an antibody or an antigen binding domain of an antibody. In some embodiments, the fusion polypeptide further comprises a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain is capable of binding to an endosomal escape peptide at pH 7.4, and wherein when the fusion polypeptide is present at a pH lower than 7.0, the pH responsive polypeptide domain contains one or more amino acids that are protonated. In some embodiments, the fusion polypeptide further comprises an endosomal escape peptide, wherein the endosomal escape peptide is capable of disrupting a membrane of a membrane-bound organelle.

[0008] Provided herein, in some aspects, are compositions comprising a fusion polypeptide comprising: (a) an effector polypeptide; and(b) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the fusion polypeptide further comprises a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain is capable of binding to an endosomal escape peptide at pH 7.4, and wherein when the fusion polypeptide is present at a pH lower than 7.0, the pH responsive polypeptide domain contains one or more amino acids that are protonated. In some embodiments, the fusion polypeptide further comprises an endosomal escape peptide, wherein the endosomal escape peptide is capable of disrupting a membrane of a membrane-bound organelle.

[0009] Provided herein, in some aspects, are compositions comprising a fusion polypeptide comprising: (a) an effector polypeptide; and (b) a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain is capable of binding to an endosomal escape peptide at pH 7.4, and wherein when the fusion polypeptide is present at a pH lower than 7.0, the pH responsive polypeptide domain contains one or more amino acids that are protonated.

[0010] Provided herein, in some aspects, are compositions comprising a fusion polypeptide comprising: (a) an effector polypeptide; and(b) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 336-349. In some embodiments, the fusion polypeptide further comprises a targeting moiety that binds specifically to an extracellular domain of a receptor expressed by a target cell. In some embodiments, the targeting moiety is not an antibody or an antigen binding domain of an antibody.

[0011] Provided herein, in some aspects, are compositions comprising a fusion polypeptide comprising: (a) an effector polypeptide; and(b) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 152-335.

[0012] Provided herein, in some aspects, are compositions comprising a protein complex, the protein complex comprising: (a) the fusion polypeptide of the composition of claim 7 or 8, and (b) an endosomal escape peptide, wherein the endosomal escape peptide is bound to the fusion polypeptide, and wherein the endosomal escape peptide is capable of disrupting a membrane of a membranebound organelle.

[0013] In some embodiments, the fusion polypeptide further comprises a targeting moiety that binds specifically to an extracellular domain of a receptor expressed by a target cell. In some embodiments, the endosomal escape peptide is bound to a pH responsive polypeptide domain. In some embodiments, the targeting moiety bindsto an extracellular domain of transferrin receptor protein 1 (TfRl), IGF1R, IGF2R, Dnmt3a, orNephrin. In some embodiments, the targeting moiety comprises a polypeptide sequence with less than 150 amino acids. In some embodiments, the targeting moiety comprises a polypeptide sequence with less than 100 amino acids. In some embodiments, the targeting moiety comprises a polypeptide sequence with more than 50 amino acids. In some embodiments, the targeting moiety comprises a polypeptide sequence with more than 60 amino acids. In some embodiments, the targeting moiety comprises a polypeptide sequence with more than 70 amino acids. In some embodiments, the targeting moiety comprises a polypeptide sequence with 80 amino acids or more. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the pH responsive polypeptide domain contains one or more amino acids that are protonated at a pH of 6.8 or less, and wherein the one or more amino acids that are protonated at a pH of 6.8 or less are not protonated at a pH of 7.4. In some embodiments, the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 6.6 or less, and wherein the one or more amino acids that are protonated at a pH of 6.6 or less are not protonated at a pH of 7.4. In some embodiments, the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 6 or less, and wherein the one or more amino acids that are protonated at a pH of 6 or less are not protonated at a pH of 7.4. In some embodiments, the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 5.5 or less, and wherein the one or more amino acids that are protonated at a pH of 5.5 or less are not protonated at a pH of 7.4. In some embodiments, the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 5 or less, and wherein the one or more amino acids that are protonated at a pH of 5 or less are not protonated at a pH of 7.4. In some embodiments, the pH responsive polypeptide domain contains one or more amino acids that are protonated when the fusion polypeptide is present within an endosome. In some embodiments, the pH responsive polypeptide domain contains one or more amino acids that are protonated when the fusion polypeptide is present within a lysosome. In some embodiments, the pH responsive polypeptide domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 336-349. In some embodiments, at a pH of 6.8 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 6.6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, ata pH of 5.5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 6.8 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200,500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 6.6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200,500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 5.5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least 2, 3,4, 5, 6, 7, 8, 9, 10, 15,20,25,30,40,50,60, 70, 80, 90, 100, 200, 500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200,500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, the endosomal escape peptide is released from the protein complex when the protein complex is present within an endosome. In some embodiments, the endosomal escape peptide is released from the protein complex when the protein complex is present within a lysosome. In some embodiments, the endosomal escape peptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 152-335. In some embodiments, the pH responsive polypeptide domain is capable of binding to two or more endosomal escape peptides at pH 7.4. In some embodiments, the protein complex comprises two or more copies of the endosomal escape peptide. In some embodiments, the fusion polypeptide further comprises a linker linking the effector polypeptide and one or more other peptide sequences of the fusion polypeptide. In some embodiments, the linker is a proteolytically cleavable linker. In some embodiments, the fusion polypeptide further comprises one or more nuclear localization sequences (NLSs). In some embodiments, the one or more NLSs comprise the amino acid sequence K(K / R)X(K / R), and where X is any amino acid. In some embodiments, the one or more NLSs comprise the amino acid sequence PKKKRKV. In some embodiments, the one or more NLSs are at the N-terminus of the effector polypeptide. In some embodiments, the effector polypeptide is a CRISPR-Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide, a Type V CRISPR-Cas effector polypeptide, or a Type VI CRISPR-Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a SpyCas9 or a variant thereof. In some embodiments, the CRISPR-Cas effector polypeptide is GeoCas9 or a variant thereof. 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 CRISPR-Cas effector polypeptide is catalytically inactive. 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] Provided herein, in some aspects, are compositions comprising: (a) a fusion polypeptide of the compositions disclosed herein, or a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide and / or endosomal escape 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 compositions further comprise a donor nucleic acid.

[0015] Provided herein, in some aspects, are nucleic acids comprising a nucleotide sequence encoding the fusion polypeptide of the compositions disclosed herein. In some embodiments, the nucleotide sequence is operably linked to a transcriptional control element, optionally wherein the transcriptional control element is a promoter.

[0016] Provided herein, in some aspects, are recombinant expression vectors comprising the nucleic acids disclosed herein.

[0017] Provided herein, in some aspects, are cells comprising the compositions disclosed herein, or the nucleic acids disclosed herein, or the recombinant expression vector disclosed herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.

[0018] Provided herein, in some aspects, are methods of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with: (a) the compositions disclosed herein; and (b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.

[0019] Provided herein, in some aspects, are methods of modifying a target nucleic acid in a eukaryotic cell, the method comprising introducing into the eukaryotic cell: (a) the compositions disclosed herein; and (b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid. In some embodiments, the methods further comprise 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. INCORPORATION BY REFERENCE

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: FIG. 1 shows exemplary configurations of the fusion polypeptides and fusion polynucleotides of the present disclosure. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaningas 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.

[0025] It may 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 “an endosomal escape peptide” 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.

[0026] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0027] The publications discussed herein do not constitute an admission of the art, but is used for helping with the understanding of the technology described. Compositions

[0028] The present disclosure provides compositions. Compositions provided herein can comprise a polymer (such as a polynucleotide, a polypeptide, etc.), a fusion polymer (such as a fusion polypeptide, a fusion polynucleotide, etc.), or any combination thereof. In some embodiments, the fusion polymer comprises a polymer and one or more heterologous polypeptides. Polymer

[0029] Disclosed herein in some embodiments are compositions comprising polymers. A “polymer” as disclosed herein can refer to any class of natural or engineered substances composed of large molecules (e.g., macromolecules), which are multiples of monomers (e.g., simpler chemical units). In some embodiments, a polymer can comprise a polynucleotide, a polypeptide, or a combination thereof. In some embodiments, a polymer can be a natural polymer, an engineered polymer, or any combination thereof. In some embodiments, an engineered polymer can comprise an engineered polynucleotide, an engineered polypeptide, or a combination thereof. In some embodiments, an “engineered polymer”, “engineered polynucleotide”, or “engineered polypeptide” as referred to herein can comprise a composition that is not present in nature. In some embodiments, a composition that is not present in nature can comprise a composition that was produced artificially by humans. In some embodiments an engineered polymer can comprise a sequence that is not present in nature, a combination of components that are not present together in nature, a composition that has been altered from a composition that is otherwise present in nature, or a combination thereof. Polypeptide

[0030] Disclosed herein in some embodiments are polymers comprising polypeptides. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably, and can refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically 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. In some embodiments a protein or polypeptide can refer to a polymeric form of amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. In some embodiments a polypeptide can comprise any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. In some embodiments a polypeptide can comprise short chains, such as a peptide, an oligopeptide or an oligomers. In some embodiments a polypeptide can comprise longer chains, such as a protein, of which there are many types. In some embodiments a polypeptide can comprise a biologically active fragment, a substantially homologous polypeptide, an oligopeptide, a homodimer, a heterodimer, a variant of a polypeptide, a modified polypeptide, a derivative, an analog, or a fusion protein, among others. In some embodiments a polypeptide can comprise a natural peptide, a recombinant peptide, or a combination thereof. In some embodiments, a polypeptide can comprise an engineered polypeptide. An engineered polypeptide can refer to a synthetically constructed polypeptide. For example, a polypeptide can be synthesized by chemical synthesis, enzymatic synthesis, or any combination thereof. In some embodiments, the polypeptide is a synthetic polypeptide.

[0031] Polypeptides (e.g., proteins) can exist in different forms based on the number and arrangement of their subunits. These forms can be a single subunit (monomer), two subunits (dimer), or multiple subunits (multimeric proteins). In some cases, the polypeptide(s) disclosed herein can comprise one or more subunits (e.g., the polypeptide can be monomeric, dimeric, or multimeric). Incorporation of a modifications in the polypeptide can modulate the activity and / or functionality of the polypeptide. For example, incorporation of an amino acid at a particular position of the polypeptide can modulate the folding or the function of the polypeptide. In some cases, incorporation of the modifications can occur within one or more subunits of a multimeric polypeptide. In some cases, incorporation of the modifications at a particular position of the polypeptide can render the polypeptide functional, wherein the particular amino acid and modified amino acid have at least one difference in their chemical structures. Such difference can modulate the folding or the function of the polypeptide when the particular amino acid or the modified amino acid is incorporated at the particular position of the polypeptide. In other cases, incorporation of the modified amino acid at the particular position of the polypeptide can render the polypeptide non-functional. In some cases, incorporation of a particular amino acid in place of the modified amino acid at a particular position of the polypeptide can render the polypeptide non-functional, wherein the particular amino acid and modified amino acid have at least one difference in their chemical structures. Similar considerations apply to any polypeptides described herein (for example, an engineered component). The identity of the modified amino acid have, the particular amino acid, and / or the particular position of the amino acid can depend on the identity of the polypeptide.

[0032] 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. Amino acid modifications

[0033] In some embodiments, the polypeptides disclosed herein can comprise any type of amino acid molecule (e.g., standard amino acids, such as the 20 amino acids commonly found in proteins and used by the genetic code during protein synthesis, nonstandard amino acids, such as chemically modified standard amino acids, amino acids that occur in living organisms but are typically not found in proteins, etc.). In some embodiments, the polypeptide comprises one or more nonstandard amino acid, such as hydroxyproline, hydroxylysine, gamma-carboxyglutamate, desmosine, isodesmosine, citrulline, ornithine, beta-alanine, and selenocysteine. In some embodiments, the polypeptide comprises one or more amino acid modifications. Amino acid modifications can refer to post-translational modifications, such as modifications that can alter the structure and / or function of a polypeptide by changing the properties (e.g., chemical) of an amino acid. Post-translational modifications can occur on the amino acid side chains of a polypeptide, at a C-terminus of the polypeptide, at an N-terminus of the polypeptide, or any combination thereof. Amino acid modifications can include, but are not limited to, acetylation, oxidation, hydroxylation, methylation, phosphorylation, amidation, carboxylation, and dehydroalanine. Amino acid modifications can impact a polypeptide's function, stability, and / or cellular localization.

[0034] In some cases, the polypeptides disclosed herein comprise an amino acid side chain modification. Amino acid side chain modifications can refer to phosphorylation, glycosylation, oxidative modification, proteolytic cleavage, alkene modifications, chemical modifiers, or any combination thereof. For example, a carbohydrate molecule can be incorporated into an amino acid side chain to improve stability and promote folding. In some cases, the polypeptides disclosed herein comprise a C-terminus modification. Modifications in the C-terminus of a polypeptide can include, but are not limited to, amidation (e.g., adding an amide group), prenylation (e.g., adding a lipid anchor), glycosylation (e.g., adding a sugar molecule), phosphorylation, methylation, and the addition of a GPI (glycosylphosphatidylinositol) anchor. For example, a GPI anchor can be incorporated into a C-terminus of a polypeptide to promote cellular localization of the polypeptide to specific cellular membranes. Modifications in the N-terminus of a polypeptide can include, but are not limited to, removal of the initiator methionine, addition of small chemical groups (e.g., acetylation, propionylation, methylation, myristoylation, palmitoylation, and / or ubiquitylation), and / or attachment of lipid anchors, such as myristoyl or palmitoyl groups. For example, an acetyl group can be added to an N-terminal amino group of a polypeptide to impact protein stability and / or protein-protein interactions. Endosomal Escape Peptide

[0035] In some aspects, provided herein are polypeptides that can facilitate release of payloads (e.g., molecular and cellular therapeutics such as the fusion polypeptides disclosed herein or the effector polypeptides of the fusion polypeptides disclosed herein) into the cytoplasm. For example, endocytosed molecular and cellular therapeutics (such as payloads internalized into a cell via endocytosis) can be transferred to early endosomes, which can mature into late endosomes and eventually into lysosomes. For efficient delivery, the payloads must be released into the cytosol before the maturation of late endosomes to lysosomes where majority of the payload can be either degraded enzymatically or recycled outside of the target cells, with only a very limited amount of payload released into the cytoplasm (e.g., endosomal escape). In some embodiments, the polypeptide that can facilitate release of payloads into cytoplasm is an endosomal escape peptide. An endosomal escape peptide (EEP) can refer to a peptide that can disrupt the endosomal membrane. For example, the EEP can disrupt the endosomal membrane thus, promoting the escape of a payload (e.g., a fusion polypeptide disclosed herein) from the endosome and releasing the payload into the cytoplasm, resulting in efficient access of therapeutic payloadsto the sites of action in the nucleus or cytosol of cells. For example, an ASO delivered to a cell and internalized by endocytosis, can be released from the endosome by the EEP which disrupts the endosomal membrane. An EEP can be used to deliver molecules that are otherwise unable to pass through the cell membrane by themselves, such as proteins, and nucleic acids.

[0036] In some embodiments, the endosomal escape peptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 152-335. In some embodiments, the endosomal escape peptide comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast80%, atleast85%, atleast90%, atleast91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 152-335. In some embodiments, the endosomal escape peptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 152-335. In some embodiments, the endosomal escape peptide comprises an amino acid sequence to any one of SEQ ID NOs: 152-335. In some embodiments, the endosomal escape peptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 152335. In some embodiments, the endosomal escape peptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 152-335. TABLE 4 lists the sequences of SEQ ID NOs: 152-335. Effector polypeptides

[0037] "Effector polypeptides" can refer to a polypeptide (e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas, etc.) that directly interact with other molecules (e.g., RNA, DNA, polypeptides, etc.) to regulate gene expression (e.g., by inhibiting translation, cleaving DNA, causing degradation of a target mRNA, etc.), essentially acting as "effectors" to influence cellular processes. In some embodiments, the polypeptide can be configured for polypeptide-mediated gene expression alteration. Gene expression alteration can include, but is not limited to, changing the level of activity of a gene, such as turning it up (e.g., increasing expression) or down (e.g., decreasing expression) through various mechanisms. Gene expression alterationscan also refer to changes in gene regulation mechanisms, such as translation, transcription, epigenetics, post-transcription, post-translation, or any combination thereof. For example, in some cases, the polypeptide can be configured for polypeptide-mediated translation disruption, such as CRISPR-associated (Cas) proteins which can be used to activate or repress translation by binding to the 5' untranslated regions (UTRs) of messenger RNAs (mRNAs). Gene expression can be altered in a number of ways including, epigenetic alterations, DNA methylation, genome editing, site-directed mutagenesis, or any combination thereof. For example, in some cases, the polypeptide can be configured for polypeptide-mediated DNA cleavage, such as ZFNs which can lead to insertions, deletions, inversions, or gene disruption following DNA cleavage. In some embodiments, the effector polypeptide is an enzyme.

[0038] In some embodiments, the effector polypeptide comprises a nuclease. In some embodiments, the polypeptide comprises dTALEs (e.g., transcription activator-like effectors that are highly target specific and can distinguish 1-2 nucleotide differences in a target sequence), TALENs (e.g., a type of transcription activator-like effector (TALE) nuclease that can target any desired sequence within a genome, without restrictions on PAM sites), or ZFNs (e.g., a restriction enzyme that cuts DNA at a specific sequence in a genome). In some embodiments, the effector polypeptide is an endonuclease. In some embodiments, the effector polypeptide is a transposon-associated RNA-guided endonucleases. In some embodiments, the polypeptide comprises TnpB (e.g., a transposon-encoded protein that can be programmed to cleave DNA in the presence of a transposon-adjacent motif (TAM)), Fanzor (e.g., an RNA-guided DNA-cutting enzyme that can be reprogrammed to target specific sites in the genome), or Cas (e.g., CRISPR / Cas). In some embodiments, the polypeptide comprises a Cas enzyme.

[0039] Cas enzymes along with their associated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (RNAs) appear to be a pervasive (-45% of bacteria, -84% of archaea) component of prokaryotic immune systems, serving to protect such microorganisms against non-self nucleic acids, such as infectious viruses and plasmids by CRISPR-RNA guided nucleic acid cleavage. While the deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse, containing a wide variety of nucleic acid-interacting domains. While CRISPR DNA elements have been observed as early as 1987, the programmable endonuclease cleavage ability of CRISPR / Cas complexes has only been recognized relatively recently, leading to the use of recombinant CRISPR / Cas systems in diverse DNA manipulation and gene editing applications. Owing to the utility of these enzymes, they are being repurposed for a wide variety of biotechnology, gene editing, and therapeutic applications. In some embodiments, the engineered polypeptide can comprise at least one CRISPR-Cas effector polypeptide. CRISPR-Cas effector polypeptides include, but are not limited to, Type I CRISPR-Cas effector polypeptides, Type II CRISPR-Cas effector polypeptides, Type III CRISPR Cas effector polypeptides, Type IV CRISPR-Cas effector polypeptides, Type V CRISPR Cas effector polypeptides, and Type VI CRISPR-Cas effector polypeptides. In some embodiments, the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide, a Type V CRISPR-Cas effector polypeptide, or a Type VI CRISPR-Cas effector polypeptide.

[0040] In some cases, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide. Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g. Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are DNA nucleases. Type II effectors generally exhibit a structure comprising a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain. The RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand. 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, LI 111R, A1322R, DI 135L, SI 136W, G1218K, E1219Q, N1317R, R1333P, R1335A, andT1337R. 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, RI 135Q, and T1137R substitutions, relative to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes DI 135 V, R1335Q, T1337R, and G1218R substitutions, relative to wild-type SpCas9. As another example, a suitable CRISPR-Cas effector polypeptide is an SpCas9 polypeptide includes DI 135L, SI 136W, 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 Lill 1R, Al 322R, D113 5L, S113 6W, G1218K, El 219Q, RI 33 5A, and T133 7R sub stitutions, relative to wildtype 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. In some cases, the CRISPR-Cas effector polypeptide is a SpyCas9 or a variant thereof.

[0041] 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 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, 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, or a corresponding Cys in another CRISPR-Cas effector polypeptide is substituted with an amino acid other than a Cys.

[0042] 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 Casl2e polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type VI CRISPR-Cas effector polypeptide, e.g., a Cast 3a polypeptide, a Cas 13b polypeptide, a Casl3cpolypeptide, 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 Cas7-11 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 lensen 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., lensen etal. (2021) Genome Research doi:10.1101 / gr.275607.121; and Breinig etal. (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.

[0043] 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 Cas7-11 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, Q.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.

[0044] 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 Cast3 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.

[0045] As another example, in some cases, the CRISPR-Cas effector polypeptide is a Cas7-11 polypeptide (e.g., a DiCas7-ll polypeptide). See, e.g., Ozcan etal. (2021) Nature 597:720. In some cases, the Cas7-11 polypeptide is a variant that exhibits reduced catalytic activity compared to a wild-type Cas7-11 effector polypeptide. For example, in some cases, the variant Cas7-11 polypeptide comprises a substitution of one or moreof D177, D429,D654, D758, E959, andD998.For example, a variant Cas7-11 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 Cas7-11 comprises a D429A substitution.

[0046] In some embodiments, as an alternative to SpyCas9, other Cas9 enzymes, like GeoCas9 that offer a number of features over SpyCas9 can be employed. Because of the lineage of GeoCas9 from thermophiles, there is no exposure to GeoCas9 in humans and therefore no preexisting antibodies exist in humans to GeoCas9. This expands the possible reach of an RNP therapeutic in terms of patient numbers. In addition, GeoCas9 RNPs have a higher melting temp than SpyCas9 RNPs (72 vs 40°C) leading to a more stable RNP complex that does not release guide RNA after complexation. This can be beneficial for circulation in blood where the guide RNA can disassemble and be degraded by nucleases. GeoCas9 RNPs are not only more stable in mouse and human plasma, but also at lower pH ranges and have an expanded window of endosomal escape, allowing GeoCas9 to escape from early and late endosomes. Unlike GeoCas9, SpyCas9 can have significant loss in activity after exposure to pH 6.0 and below.

[0047] In some embodiments, the CRISPR-Cas effector polypeptide is a GeoCas9 or variant thereof. In some embodiments, the CRISPR-Cas effector polypeptide comprises at least 40%, at least 45%, at least 50%, atleast 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast90%, atleast91%, atleast92%, atleast93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to GeoCas9. In some embodiments, the CRISPR-Cas effector polypeptide comprises at most 40%, at most 45%, at most 50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to a GeoCas9. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to a GeoCas9. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has atleast 1, at least 2, at least 3, atleast 4, atleast 5, at least 10, atleast 11, atleast 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to a GeoCas9. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to a GeoCas9. In some embodiments, the CRISPR-Cas effector polypeptide is a GeoCas9.

[0048] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 111. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, atleast 60%, atleast65%, atleast70%, at least 75%, at least 80%, atleast 85%, at least 90%, atleast 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-11. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to any one of SEQ ID NOs: 1-11. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to any one of SEQ ID NOs: 111. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, atleast25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 1-11. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has atmost 1, at most 2, at most 3, at most 4, atmost 5, at most 10, atmost 11, at most 12, atmost 13, atmost 14, atmost 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 1-11. TABLE 1 below lists the sequences of SEQ ID NOs: 111

[0049] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, atleast 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to SEQ ID NO: 1. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 1. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has atleast 1, at least 2, atleast 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 1 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, atmost 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 1

[0050] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to SEQ ID NO: 2. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 2. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 2 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 2

[0051] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to SEQ ID NO: 3. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 3. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 3 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, atmost 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 3

[0052] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 4. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 4. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 4 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, atmost 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 4

[0053] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, atleast 60%, atleast 65%, at least70%, atleast75%, atleast80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, atmost 91%, at most 92%, at most 93%, atmost 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 5. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 5. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has atleast 1, at least 2, atleast 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 5 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 5

[0054] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to SEQ ID NO: 6. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 6. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 6 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, atmost 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 6

[0055] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, atleast 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, atmost 91%, at most 92%, at most 93%, atmost 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 7. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 7. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has atleast 1, at least 2, atleast 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 7. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 7

[0056] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to SEQ ID NO: 8. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 8. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 8 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, atmost 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 8

[0057] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, atleast 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, atmost 91%, at most 92%, at most 93%, atmost 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 9. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 9. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has atleast 1, at least 2, atleast 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 9 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 9

[0058] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, atleast45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to SEQ ID NO: 10. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 10. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 10 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, atmost 5, at most 10, atmost 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, atmost 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 10

[0059] In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the CRISPR-Cas effector polypeptide comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, atmost 91%, at most 92%, at most 93%, atmost 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 11. In some embodiments, the CRISPR-Cas effector polypeptide comprises 100% sequence identity to SEQ ID NO: 11. In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at leasts, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 11 In some embodiments, the CRISPR-Cas effector polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 11 Table 1. Exemplary polypeptide amino acid sequences SEQ ID NO. Amino acid sequence Organism 1 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK NLSDAILLSDILRVNTEnKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIE RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF ANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNY WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN YHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW DPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFE KNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAF KYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Streptococcus pyogenes Cas9 2 MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQK LSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEK YVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQ SFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEEL RSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKK KPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIE NAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNL SLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFIL SPWKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKR NRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLN NPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSK ISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVD Staphylococcus aureus Cas9 TRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNK GYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEI ETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKD DKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKL IMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAH LDITDDYPNSRNKWKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKEN YYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNND LLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNL YEVKSKKHPQIIKKG 3 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFK SAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDN GIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAK YLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKE NKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHT KNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRY NSIDEFYREVENQGYKLTFENISESYIDSWNQGKLYLFQIYNKDFSAYS KGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPA KEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANK FNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIG NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQWHEI AKLVIEYNAIWFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVT GFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAA KGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGH GECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVN GNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNL VIKNEEYFEFVQNRNN Francisella tularensis Casl2a 4 TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELK PIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQAT YRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVT TTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKF KENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQT QIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIP LFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNE LNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAK EKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPT TLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLE MEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNG AILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDA AKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEP KKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPS SQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDF AKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRM AHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALL PNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYL KEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNRE KERVAARQAWSWGTIKDLKQGYLSQYIHEIVDLMIHYQAVWLENLN FGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQ LTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHES RKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFE KNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGI Acidaminococcu s sp. BV3L6 Casl2a VFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDL KLQNGISNQDWLAYIQELRN 5 MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKG VKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEI NLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTA FTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHE VQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKI KGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVL EVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFG EWNVIRDI<WNAEYDDIHLI<I<I<AVVTEI<YEDDRRI<SFI<I<IGSFSLEQLQ EYADADLSWEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKND AVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLK VDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATIL RYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLP KVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDS ISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVD KLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLS GGAELFMRRASLKKEELWHPANSPIANKNPDNPKKTTTLSYDVYKDK RFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNL LYIVWDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQ NWTSIENIKELKAGYISQWHKICELVEKYDAVIALEDLNSGFKNSRVK VEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKS MSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMY VPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFD WEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSL MLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADAN GAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH Lachnospiraceae bacterium ND2006 (LbCasl2a) 6 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQEN LYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDEL LQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAG NKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPL MRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWN QRVGQEYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLE SKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRN TRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMF ATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKV ENGVARE VDDVT VPISM SEQLDNLLPRDPNEPIALYFRDYGAEQHFTGE FGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPY AAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSV DLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQL LKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRER SWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAV YESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDWGGNSIEQIE YLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKK LADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDR PPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTG APGIRCRRVPARCTQEHNPEPFPWWLNKFWEHTLDACPLRADDLIPTG EGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWG EVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFA QEI<LSEEEAELLVEADEAREI<SVVLMRDPSGIINRGNWTRQI<EFWSMV NQRIEGYLVKQIRSRVPLQDSACENTGDI AacCasl2b 7 MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPR RLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQL RVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIE ENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIF SKQREFGNMSCTEEFENEYmWASQRPVASKDDIEKKVGFCTFEPKEKR API<ATYTFQSFIAWEHINI<LRLISPSGARGLTDEERRLLYEQAFQI<NI<IT YHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVD KVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPN GeoCas9 LANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACER AGYTFTGPKKKQKTMLLPNIPPIANPWMRALTQARKWNAIIKKYGSP VSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPT GHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDS YTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKK RDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQ KVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAVIVACTrPSDIAKV TAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNL GNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQT WKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQE PLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEK DGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYP NDLIRIELPREI<TVI<TAAGEEINVI<DVFVYYI<TIDSANGGLELISHDHRF SLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKPGKTIRP LQSTRD 8 MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKE KIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDD FLETEEWLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQ ENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKI IEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEI LGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEF WNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDK IVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFK KHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKM EKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDF SRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNY VLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKE RDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDI KISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYV NKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQI SASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIK DINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFAT SVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEK DFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDD ETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCR IIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLF LNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLN DKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYK KIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIK LSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDL SENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNN STYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELE SYNSDYIKNLIIELLTKIENTNDTL Leptotrichia shahii (WP_018451595 .1) Casl3a 9 MTTTMKISIEFLEPFRMTKWQESTRRNKNNKEFVRGQAFARWHRNKK DNTKGRPYITGTLLRSAVIRSAENLLTLSDGKISEKTCCPGKFDTEDKDR LLQLRQRSTLRWTDKNPCPDNAETYCPFCELLGRSGNDGKKAEKKDW RFRIHFGNLSLPGKPDFDGPKAIGSQRVLNRVDFKSGKAHDFFKAYEVD HTRFPRFEGEITIDNKVSAEARKLLCDSLKFTDRLCGALCVIRFDEYTPA ADSGKQTENVQAEPNANLAEKTAEQIISILDDNKKTEYTRLLADAIRSLR RSSKLVAGLPKDHDGKDDHYLWDIGKKKKDENSVTIRQILTTSADTKE LKNAGKWREFCEKLGEALYLKSKDMSGGLKITRRILGDAEFHGKPDRL EKSRSVSIGSVLKETWCGELVAKTPFFFGAIDEDAKQTDLQVLLTPDN KYRLPRSAVRGILRRDLQTYFDSPCNAELGGRPCMCKTCRIMRGITVMD ARSEYNAPPEIRHRTRINPFTGTVAEGALFNMEVAPEGIVFPFQLRYRGS EDGLPDALKTVLKWWAEGQAFMSGAASTGKGRFRMENAKYETLDLS DENQRNDYLKNWGWRDEKGLEELKKRLNSGLPEPGNYRDPKWHEINV SIEMASPFINGDPIRAAVDKRGTDWTFVKYKAEGEEAKPVCAYKAESF RGVIRSAVARIHMEDGVPLTELTHSDCECLLCQIFGSEYEAGKIRFEDLV FESDPEPVTFDHVAIDRFTGGAADKKKFDDSPLPGSPARPLMLKGSFWI RRDVLEDEEYCKALGKALADVNNGLYPLGGKSAIGYGQVKSLGIKGD DiCas7-ll DKRISRLMNPAFDETDVAVPEKPKTDAEVRIEAEKVYYPHYFVEPHKK VEREEKPCGHQKFHEGRLTGKIRCKLITKTPLIVPDTSNDDFFRPADKEA RKEKDEYHKSYAFFRLHKQIMIPGSELRGMVSSVYETVTNSCFRIFDET KRLSWRMDADHQNVLQDFLPGRVTADGKHIQKFSETARVPFYDKTQK HFDILDEQEIAGEKPVRMWVKRFIKRLSLVDPAKHPQKKQDNKWKRRK EGIATFIEQKNGSYYFNWTNNGCTSFHLWHKPDNFDQEKLEGIQNGEK LDCWVRDSRYQKAFQEIPENDPDGWECKEGYLHWGPSKVEFSDKKG DVINNFQGTLPSVPNDWKTIRTNDFKNRKRKNEPVFCCEDDKGNYYTM AKYCETFFFDLKENEEYEIPEKARIKYKELLRVYNNNPQAVPESVFQSR VARENVEKLKSGDLVYFKHNEKYVEDIVPVRISRTVDDRMIGKRMSAD LRPCHGDWVEDGDLSALNAYPEKRLLLRHPKGLCPACRLFGTGSYKGR VRFGFASLENDPEWLIPGKNPGDPFHGGPVMLSLLERPRPTWSIPGSDN KFKVPGRKFYVHHHAWKTIKDGNHPTTGKAIEQSPNNRTVEALAGGNS FSFEIAFENLKEWELGLLIHSLQLEKGLAHKLGMAKSMGFGSVEIDVES VRLRKDWKQWRNGNSEIPNWLGKGFAKLKEWFRDELDFIENLKKLLW FPEGDQAPRVCYPMLRKKDDPNGNSGYEELKDGEFKKEDRQKKLTTP WTPWA 10 IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVN EGEAFSAEMADKNAGYKIGNAKFSHPKGYAWANNPLYTGPVQQDML GLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNIS GLDKDIIGFGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFD NFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLRHWVVHNN EEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAANVN YIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIR KNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSE KDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTRE YKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKF DNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIA DARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKHGMRN FIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQ NGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIE DTGRENAEREI<FI<I<IISLYLTVIYHILI<NIVNINARYVIGFHCVERDAQLY KEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKE SIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTK WNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDIAEVNSYFQL YHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGY CIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS RfxCasl3d 11 NQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENNENLWFHPV MSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYK QNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLNDGCEFLTS TEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYG KKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLS RLPIFSSYNAQSEERRIIIRSFGINSII<LPI<DRIHSEI<SNI<SVAMDMLNEVI< RCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKL FDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAET MRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNK VEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGS KKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLIS GNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRG FKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGD DYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVEFYERYL IERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLP VELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDF QTFYQWNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASR TERYRKQASNKIRSNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRR YRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSF TFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDI MEEFNKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSILKI LLNNKNINKEQSDILRKIRNAFDHNNYPDKGWEIKALPEIAMSIKKAFG EYAIMK PspCasl3b

[0060] In some embodiments, the CRISPR-Cas effector polypeptide is catalytically inactive. 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 CRISPR-Cas effector polypeptide exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide exhibits at most at most 5%, at most 10%, at most 15%, 20%, at most 25%, at most30%, atmost35%, atmost40%, atmost45%, atmost50%, atmost55%, atmost60%, atmost 65%, at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide. Engineered guide ribonucleic acid

[0061] 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 polypeptide of the present disclosure (e.g., a fusion polypeptide); and ii) a guide nucleic acid. In some cases, a composition of the present disclosure comprises: i) a polypeptide of the present disclosure (e.g., a fusion polypeptide); ii) a guide nucleic acid (e.g., an engineered guide ribonucleic acid structure); and iii) a donor nucleic acid.

[0062] In one aspect, the present disclosure provides a composition comprising a CRISPR-Cas effector polypeptide and an engineered guide ribonucleic acid structure. The engineered guide ribonucleic acid structure may comprise a guide ribonucleic acid sequence. The guide ribonucleic acid sequence may be configured to hybridize to a target deoxyribonucleic acid sequence. The engineered guide ribonucleic acid structure may comprise a tracr ribonucleic acid sequence. The tracr ribonucleic acid sequence may be configured to bind to the CRISPR-Cas effector polypeptide. The engineered guide ribonucleic acid structure may lack a tracr ribonucleic acid sequence. For example, the engineered guide ribonucleic acid structure may function as a single molecule capable of binding to a target DNA without the need for a separate tracrRNA component, such as a singleguide RNA structure characteristic of a type V CRISPR system (e.g., Casl2a (Cpf 1) which utilizes a single guide RNA structure that lacks a tracrRNA component). In some embodiments, the composition comprises an engineered guide ribonucleic acid structure or a nucleic acid encoding the engineered guide ribonucleic acid structure. In some embodiments, the engineered guide ribonucleic acid structure is configured to form a complex with the CRISPR-Cas effector polypeptide. In some embodiments, the engineered guide ribonucleic acid structure comprises a guide ribonucleic acid sequence configured to hybridize to a target nucleic acid sequence; and a tracr ribonucleic acid sequence. In some embodiments, the engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides; or a single ribonucleic acid polynucleotide comprising the guide ribonucleic acid sequence and the tracr ribonucleic acid sequence. In some embodiments, the tracr ribonucleic acid sequence is configured to bind to the CRISPR-Cas effector polypeptide. In some embodiments, the guide ribonucleic acid sequence is complementary to a eukaryotic, a fungal, a plant, a mammalian, or a human genomic sequence. In some embodiments, the engineered guide ribonucleic acid structure comprises a ribonucleic acid sequence comprising a stem and a loop, wherein said stem comprises at least 12 pairs of ribonucleotides. In some embodiments, the engineered guide ribonucleic acid structure further comprises a second stem and a second loop, wherein the second stem comprises at least 5 pairs of ribonucleotides. In some embodiments, the engineered guide ribonucleic acid structure further comprises a ribonucleic acid structure comprising at least two hairpins.

[0063] Guide nucleic acids (e.g., an engineered guide ribonucleic acid structure) that form a complex with a CRISPR-Cas effector polypeptide are well known in the art. A guide RNA (e.g., an engineered guide ribonucleic acid structure) 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 “proteinbinding 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] The “guide sequence” also referred to as the “targeting sequence” or “spacer 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.

[0068] 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.”

[0069] 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 3 0 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).

[0070] 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 3 0 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.

[0071] In some cases, the guide 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).

[0072] 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.

[0073] 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, 1215, 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).

[0074] 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.

[0075] Suitable non-natural internucleoside linkage include, e.g., a phosphorothioate, a phosphoramidate, 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.

[0076] Suitable modified moieties include, e.g., locked nucleic acid (LNA) sugar moieties, 2'-substituted sugar moieties, 2'-O-meth oxy ethyl modified sugar moieties, one or more 2'-O-methyl modified sugar moieties, 2'-O-(2-methoxy ethyl) modified sugar moieties, 2'-fluoro modified sugar moieties, 2'-dimethylaminooxyethoxy modified sugar moieties, and 2'-dimethylaminoethoxyethoxy modified sugar moieties.

[0077] 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-thioalkyladenines; 8-hydroxyladenines; 8-haloguanines; 8-aminoguanines; 8-thiolguanines; 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-aminoadenines; 8-azaguanines; 8-azaadenines; 7-deazaguanines; 7-deazaadenines; 3-deazaguanines; 3-deazaadenines; tricyclic pyrimidines; phenoxazine cytidines; phenothiazine cytidines; substituted phenoxazine cytidines; carbazole cytidines; pyridoindole cytidines; 7-deazaguanosines; 2-aminopyridines; 2-pyridones; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N-6 or 0-6 substituted purines; 2-aminopropyladenines; 5-propynyluracils; and 5-propynylcytosines.

[0078] In some embodiments, the engineered guide ribonucleic acid structure comprises at least one modification. In some embodiments, the at least one modification includes any of the modifications disclosed herein, such as the nucleic acid modifications described herein. In some embodiments, the at least one modification includes 2'-O-methoxyethoxy (2'-0Me), 2'-O-(2-methoxyethyl) (2'-O-moe), 2'-fluoro (2'-F), locked nucleic acid (LNA), pseudouridine (y), phosphorothioate (PS) bond, phosphorodiamidate morpholino oligomer (PMO), and / or 2'-phosphorylation (2'-P). In some embodiments, the engineered guide ribonucleic acid structure comprises (i) a 5' end modification, (ii) a 3' end modification, or (iii) a 5' end modification and a 3' end modification.

[0079] In some embodiments, the engineered guide ribonucleic acid structure comprises a ribonucleic acid sequence comprising at least four hairpins comprising a stem and a loop. In some embodiments, the engineered guide ribonucleic acid structure comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following non-degenerate nucleotide sequence: NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 12).

[0080] 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).

[0081] 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.

[0082] 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.

[0083] 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 maybe 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.

[0084] 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.

[0085] 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.

[0086] 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 atthe cleavage site or in some cases may be used for other purposes (e.g., to signify expression atthe 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.

[0087] 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, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the termini of a linear donor sequence, additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination. A donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. 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.

[0088] 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.

[0089] 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. Fusion polypeptides

[0090] In some aspects, provided herein are fusion polypeptides. In some embodiments, the fusion polypeptide comprises: i) an effector polypeptide (e.g., any effector polypeptide can be used in a fusion polypeptide of the present disclosure); and ii) one or more heterologous polypeptide (e.g., a targeting moiety that binds to an extracellular domain of a receptor); and optionally also includes a) an endosomal escape peptide (e.g., an EEP that can be used in a fusion polypeptide of the present disclosure); and / or b) one or more nuclear localization signals (NLSs). In some embodiments, the fusion polypeptide comprises: i) a CRISPR-Cas effector polypeptide (e.g., any CRISPR-Cas effector polypeptide can be used in a fusion polypeptide of the present disclosure); and ii) one or more heterologous polypeptide, e.g., a targeting moiety that binds to an extracellular domain of a receptor such as a transferrin protein 1 (TfRl, CD71) ); and optionally also includes one or more nuclear localization signals (NLSs).

[0091] The term “heterologous polypeptide” is often used interchangeably herein with “fusion partner.” “Heterologous,” as used herein in the context of a polypeptide, can refer 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. The term “naturally-occurring” as used herein as applied to a nucleic acid, a protein, a cell, or an organism, can refer to a nucleic acid, cell, protein, or organism that is found in nature.

[0092] In some cases, a fusion partner of an effector polypeptide provided herein 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).

[0093] In some cases, a fusion partner of an effector polypeptide provided herein 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).

[0094] In some cases, a fusion partner of an effector polypeptide provided herein 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).

[0095] Examples of proteins (or fragments thereof) that can be used in increase transcription include butare notlimited 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, M0Z / MYST3, M0RF / MYST4, SRC1, ACTR, Pl60, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.

[0096] 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 suchasHhaIDNAm5c-methyltransferase(M.HhaI), 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.

[0097] In some cases, a fusion partner of an effector polypeptide provided herein 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 3 a (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 APOBEC 1), 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).

[0098] In some cases, a fusion partner of an effector polypeptide provided herein has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the 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 KMT1 A), 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, andthe 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.

[0099] 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.

[00100] Additional suitable heterologous polypeptides include, but are not limited to, a polypeptide that directly and / or indirectly provides for increased transcription and / or translation 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.

[00101] 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 such as eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, such as 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).

[00102] 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 consisting of: 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 of) 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 PAPI, 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 consisting of: 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.

[00103] 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 separate 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 c<n-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 WO2010075303, which is hereby incorporated by reference in its entirety.

[00104] 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.).

[00105] In some cases, the one or more heterologous polypeptides comprise a nuclease. Suitable nucleases include, but are not limited to, a homing nuclease polypeptide; a FokI polypeptide; a transcription activator-like effector nuclease (TALEN) polypeptide; a MegaTAL polypeptide; a meganuclease polypeptide; a zinc finger nuclease (ZFN); an ARCUS nuclease; and the like. The meganuclease can be engineered from an LADLIDADG homing endonuclease (LHE). A megaTAL polypeptide can comprise a TALE DNA binding domain and an engineered meganuclease. See, e.g., WO 2004 / 067736 (homing endonuclease); Urnovet al. (2005) Nature 435:646 (ZFN); Mussolino et al. (2011)Nucle. Acids Res. 39:9283 (TALE nuclease); Boissel etal. (2013) Nucl. Acids Res. 42:2591 (MegaTAL).

[00106] 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. In some cases, the one or more heterologous polypeptides comprise a reverse transcriptase polypeptide. 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.

[00107] In some cases, the one or more heterologous polypeptides comprise a base editor. Suitable base editors include, e.g., an adenosine deaminase; a cytidine deaminase (e.g., an activation-induced cytidine deaminase (AID)); APOBEC3G; and the like); and the like. A suitable adenosine deaminase is any enzyme that is capable of deaminating adenosine in DNA. In some cases, the deaminase is a TadA deaminase.

[00108] In some cases, the one or more heterologous polypeptides comprise a transcription factor. In some cases, the one or more heterologous polypeptides are capable of binding to a transcription factor. 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. DrugDeliv. 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 Krtippel-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.

[00109] In some cases, the one or more heterologous polypeptides comprise a recombinase. Suitable recombinases include, e.g., a Cre recombinase; a Hin recombinase; a Tre recombinase; a FLP recombinase; and the like. Nuclear localization signal

[00110] A fusion polypeptide of the present disclosure can include, in addition to the CRISPR-Cas effector polypeptide, a nuclear localization signal (NLS). In some cases, the one or more heterologous polypeptides comprise a nuclear localization signal (NLS). 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.

[00111] 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) an N- terminus and / or a C-terminus of the fusion polypeptide. 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 moreNLSs (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.

[00112] In some cases, a fusion polypeptide of the present disclosure comprises a single NLS at an N-terminus of the CRISPR-Cas effector polypeptide present in the fusion polypeptide. In some cases, the fusion polypeptide of the present disclosure comprises a single NLS at a 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. Non-limiting examples of NLSs include NLS sequences in Table 2. Table 2. Exemplary NLS amino acid sequences SEQ ID NO. Amino acid sequence NLS source 13 PKKKRKV SV40 virus large T-antigen 14 KRPAATKKAGQAKKKK nucleoplasmin (e.g., the nucleoplasmin bipartite) 15 PAAKRVKLD c-myc 16 RQRRNELKRSP c-myc 17 NQ SSNFGPMKGGNFGGRSSGPYGGGGQ Y FAKPRNQGGY hRNPAl M9 18 RMRIZFKNKGKDTAELRRRRVEVSVELRK AKKDEQILKRRNV IBB domain from importin-alpha 19 VSRKRPRP myoma T protein 20 PPKKARED myoma T protein 21 PQPKKKPL human p53 22 SALIKKKKKMAP mouse c-abl IV 23 DRLRR influenza virus NS1 24 PKQKKRK influenza virus NS1 25 RKLKKKIKKL Hepatitis virus delta antigen 26 REKKKFLKRR mouse Mxl protein 27 KRKGDEVDGVDEVAKKKSKK human poly(ADP-ribose) polymerase 28 RKCLQAGMNLEARKTKK steroid hormone receptors (human) glucocorticoid K(K / R)X(K / R), where X is any amino acid synthetic

[00113] In some embodiments, the NLS comprises an amino acid sequence having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1328. In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast50%, atleast55%, atleast60%, atleast65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, atleast93%, atleast94%, atleast95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 13-28. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most45%, atmost 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 13-28. In some embodiments, the NLS comprises 100% sequence identity to any one of SEQ ID NOs: 13-28 In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 13-28. In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 13-28.

[00114] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, atleast 90%, atleast 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost91%, at most 92%, atmost93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 13. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 13. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 13 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 13

[00115] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 14. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 14. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 14 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 14

[00116] In some embodiments, theNLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 15. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 15. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 15 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 15

[00117] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 16. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 16. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 16 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 16

[00118] In some embodiments, theNLS comprises an amino acid sequence having at least 40%, at least45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 17. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 17. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 17 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 17

[00119] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost91%, at most 92%, atmost93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 18. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 18. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 18 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 18

[00120] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, atleast 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 19. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 19. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 19 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 19

[00121] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least45%, atleast50%, atleast55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 20. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 20. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 20 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 20

[00122] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or atmost 99% sequence identity to SEQ ID NO: 21. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 21. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 21 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 21

[00123] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 22. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 22. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 22. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 22 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 22

[00124] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast50%, atleast55%, atleast60%, atleast65%, atleast70%, atleast75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 23. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 23. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 23 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 23

[00125] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 24. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 24. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 24 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 24

[00126] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 25. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost91%, at most 92%, atmost93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 25. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 25. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 25 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 25

[00127] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 26. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 26. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 26. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 26 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 26

[00128] In some embodiments, the NLS comprises an amino acid sequence having at least 40%, at least 45%, atleast 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 27. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 27. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 27. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 27. In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 27.

[00129] In some embodiments, theNLS comprises an amino acid sequence having at least 40%, at least 45%, atleast50%, atleast55%, atleast60%, atleast65%, atleast70%, atleast75%, at least 80%, atleast85%, atleast90%, atleast91%, atleast92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence having at most 40%, at most 45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost 90%, atmost91%, at most 92%, atmost 93%, atmost 94%, atmost95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 28. In some embodiments, the NLS comprises 100% sequence identity to SEQ ID NO: 28. In some embodiments, the NLS comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5 or more amino acid substitutions or mutations relative to SEQ ID NO: 28 In some embodiments, the NLS comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5 amino acid substitutions or mutations relative to SEQ ID NO: 28

[00130] In some embodiments, the one or more NLSs comprise the amino acid sequence K(K / R)X(K / R), where X is any amino acid. Targeting moiety

[00131] A fusion polypeptide of the present disclosure can include, in addition to the effector polypeptide, a targeting moiety. In some embodiments the fusion polypeptide comprises an effector polypeptide, and a targeting moiety (e.g., a CRISPR-Cas effector polypeptide covalently linked to a targeting moiety and coupled to an EEP, see for example FIG. 1). In some embodiments an N-terminus of the effector polypeptide is covalently linked to a C-terminus of the targeting moiety. In some embodiments a C-terminus of the effector polypeptide is covalently linked to an N-terminus of the targeting moiety. In some embodiments the fusion polypeptide comprises an effector polypeptide, a targeting moiety, and an EEP (e.g., a CRISPR-Cas effector polypeptide covalently linked to a targeting moiety and an EEP, see for example FIG. 1). In some embodiments an N-terminus of the effector polypeptide is linked to a C-terminus of the targeting moiety and a C-terminus of the effector polypeptide is linked to an N-terminus of the EEP. In some embodiments an N-terminus of the effector polypeptide is linked to a C-terminus of the EEP and a C-terminus of the effector polypeptide is linked to an N-terminus of the targeting moiety. In some embodiments an N-terminus of the effector polypeptide is linked to a C-terminus of the targeting moiety and an N-terminus of the targeting moiety is linked to a C-terminus of the EEP. In some embodiments a C-terminus of the effector polypeptide is linked to an N-terminus of the targeting moiety and a C-terminus of the targeting moiety is linked to an N-terminus of the EEP.

[00132] In some embodiments the fusion polypeptide comprises a CRISPR-Cas effector polypeptide, an endosomal escape polypeptide, and one or more heterologous polypeptides. In some cases, the fusion polypeptide comprises a CRISPR-Cas effector polypeptide and a targeting moiety. In some embodiments the fusion polypeptide comprises a CRISPR-Cas effector polypeptide and one or more heterologous polypeptides. In some embodiments at least one of the one or more heterologous polypeptides is a targeting moiety. A targeting moiety can be used to direct a polypeptide to a target cell or tissue. A targeting moiety in a fusion polypeptide disclosed herein can be but is not limited to a lipophilic moiety, a small molecule, a peptide (e.g., a polypeptide), an RNA molecule, a nanoparticle, an antibody, a single-domain antibody, a miniprotein, or an antigen binding fragment thereof. A targeting moiety in a fusion polypeptide disclosed herein can be specific to an antigen or receptor on the target cell or tissue (e.g., Asialoglycoprotein receptor (ASGPR)). In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein specifically binds to a target protein. In some embodiments, the target protein is a receptor expressed by a target cell. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to an extracellular domain of a receptor expressed by a target cell and facilitates cellular uptake. Nonlimiting examples of a target protein can include IGF1R, IGF2R, TfRl, Nephrin, Nephi, Megalin, cKIT, GLUT4, GLUT1, Neuropilin, FOL1, FOL3, CCR5, CXCR1-4, EGFR, VEGFR1, VEGFR2, PDGFRA, ADRB3, NTRK2, GRIA1, GRIN1, GABRA1, and GABBR1.

[00133] A targeting moiety in a fusion polypeptide disclosed herein can be a lipophilic moiety, lipophilic moiety can comprise one or more fatty acid groups or salts thereof. A lipophilic be a lipid. Lipids are fatty acids and their derivatives which are insoluble in water but soluble in organic solvents. In some embodiments, a lipophilic moiety can be unsaturated. Alternatively, or in addition to, a lipophilic moiety can be monosaturated. Alternatively, or in addition to, a lipophilic moiety can be poly saturated. In some embodiments, a double bond of an unsaturated lipophilic moiety can be in a cis conformation. Alternatively, or in addition to, a double bond of an unsaturated lipophilic moiety can be in a trans conformation. Non-limiting examples of lipophilic moieties can be a triglyceride, a phospholipid, a sterol, an oil, a wax, a hormone, a vitamin, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis- 0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03- (oleoyl)cholenic acid, dimeth oxytrityl, or phenoxazine.

[00134] A targeting moiety in a fusion polypeptide disclosed herein can be a small molecule. A small molecule can be a sugar, an amino acid, a phenolic compound, an alkaloid, a sterol, a lipid, a fatty acid, or other small chemical compound. A chemical compound can be a molecule that has a molecular weight of less than 1000 Daltons. Alternatively, or in addition to, a small molecule is a molecule with a size on the order of 1 nm.

[00135] A targeting moiety in a fusion polypeptide disclosed herein can be a sugar or sugar moiety. A sugar can be a monosaccharide. Alternatively, a sugar can be a disaccharide. Alternatively, a sugar can be a polysaccharide. Non-limiting examples of sugars include glucose, dextrose, fructose, galactose, a sugar alcohol, a pentose, xylose, ribose, sucrose, cellulose, starch, lactose, maltose, trehalose, lactulose, cellobiose, chitobiose, glycogen, or chitin. A small molecule can be an amino sugar such as but not limited to N-acetyl Galactosamine (GalNAc), N-acetylglucosamine or sialic acid.

[00136] A targeting moiety in a fusion polypeptide disclosed herein can be an RNA molecule. An RNA molecule can comprise an aptamer, a ribozyme, or a hairpin RNA.

[00137] A targeting moiety in a fusion polypeptide disclosed herein can be an antibody or an antigen-binding fragment thereof. An antibody, also known as an immunoglobulin, is a blood protein produced to counteract a specific antigen. Antibodies can be Y-shaped proteins which comprise variable binding sites that are specific to particular epitopes. An antibody can be a monoclonal antibody. Alternatively, an antibody can be a polyclonal antibody. An antibody can be a singledomain antibody. An antibody can be an antibody fragment. An antibody can be an agonist. Alternatively, an antibody can be an antagonist. Alternatively, an antibody can be an allosteric modulator (e.g., a positive allosteric modulator or a negative allosteric modulator).

[00138] In other cases, a targeting moiety disclosed herein is not an antibody or antigen-binding fragment thereof.

[00139] A targeting moiety in a fusion polypeptide disclosed herein can be a polypeptide. Nonlimiting examples of polypeptides can be an agonist of IGF1R, IGF2R, TfRl, Nephrin, Nephi, Megalin, cKIT, GLUT4, GLUT1, Neuropilin, FOL1, FOL3, CCR5, CXCR1-4, EGFR, VEGFR1, VEGFR2, PDGFRA, or ADRB3.

[00140] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds a cell surface receptor. In certain embodiments, the cell surface receptor is membrane associated. Membrane proteins represent about a third of the proteins in living organisms and many membrane proteins are known in the field. Based on their structure, membrane proteins can be largely categorized into three main types: (1) integral membrane protein (IMP), which is permanently anchored or part of the membrane, (2) peripheral membrane protein, which is temporarily attached to the lipid bilayer or to other integral proteins, and (3) lipid-anchored proteins. The most common type of IMP is the transmembrane protein (TM), which spans the entire biological membrane. The cell surface receptor of the present disclosure includes single-pass and multi-pass membrane proteins. Single-pass membrane proteins cross the membrane only once, while multi-pass membrane proteins weave in and out, crossing several times. In some embodiments, the cell surface receptor can be a monomeric receptor. In some embodiments, the cell surface receptor can be a multimeric receptor. In some embodiments, the cell surface receptor can form a complex with other molecules (e.g., an integrin). The cell surface receptor can be a recycling receptor. For example, a recycling receptor as used herein refers to a cell surface receptor that specifically binds to a ligand (e.g., a targeting moiety) and leads to internalization of the cell surface receptor.

[00141] In some case, the cell surface receptor can be selected by expression levels in one cell type relative to other cell types, where the cell surface receptor is enrichment in the selected cell type and / or tissue type. In some embodiments, the cell surface receptor comprises a tissue-type specific protein. In some embodiments, the cell surface receptor comprises a cell-type specific protein. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein is not an antibody or an antigen binding domain of an antibody. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein is an antibody or an antigen binding domain of an antibody.

[00142] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to an extracellular domain of a receptor protein. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to an extracellular domain of a receptor selected from the group consisting of: IGF1R, IGF2R, TfRl, Nephrin, Nephi, Megalin, cKIT, GLUT4, GLUT1, Neuropilin, FOL1, FOL3, CCR5, CXCR1-4, EGFR, VEGFR1, VEGFR2, PDGFRA, ADRB3, NTRK2, GRIA1, GRIN1, GAB RAI, and GABBR1. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to an extracellular domain of transferrin receptor protein 1 (TfRl, CD71). In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to an extracellular domain of insulin-like growth factor 2 receptor (IGF2R). In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to an extracellular domain of insulinlike growth factor 1 receptor (IGF1R). In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises polypeptide sequence with less than 150 amino acids. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises polypeptide sequence with less than 100 amino acids.

[00143] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises polypeptide sequence with more than 50 amino acids. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises polypeptide sequence with more than 60 amino acids. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises polypeptide sequence with more than 70 amino acids. In some embodiments, targeting moiety in a fusion polypeptide disclosed herein comprises polypeptide sequence with 80 amino acids or more.

[00144] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polypeptide sequence selected from the sequences in Table 3. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost91%, atmost92%, atmost93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises 100% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence to any one of SEQ ID NOs: 31-151 or 350143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least20, atleast 21, atleast22, atleast23, atleast24, at least25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, atmost 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 31-151 or 350-143854

[00145] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to TfRl. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 31-131 or 350-5272. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, atleast 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast90%, atleast91%, atleast92%, atleast93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or atleast 99% sequence identity to any one of SEQ ID NOs: 31-131 or 350-5272. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to any one of SEQ ID NOs: 31-131 or 350-5272. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises 100% sequence identity to any one of SEQ ID NOs: 31-131 or 350-5272. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence of any one of SEQ ID NOs: 31-131 or 350-5272. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least5, atleast 10, atleast 11, atleast 12, atleast 13, atleast 14, atleast 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 31-131 or 350-5272. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has atmost 1, at most 2, atmost 3, at most 4, atmost 5, at most 10, atmost 11, at most 12, atmost 13, atmost 14, atmost 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 31-131 or 350-5272.

[00146] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to IGF1R. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 132-145 or 5273-71413. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprisesan amino acid sequence having at least 40%, at least 45%, atleast50%, atleast55%, atleast60%, atleast65%, atleast 70%, atleast 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 132- 145 or 5273-71413. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, at most91%, atmost92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 132-145 or 5273-71413. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises 100% sequence identity to any one of SEQ ID NOs: 132-145 or 5273-71413. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence of any one of SEQ ID NOs: 132-145 or 5273-71413. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at least 1, at least 2, at least 3, at least4, at least 5, at least 10, atleast 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 132-145 or 527371413. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 132-145 or 5273-71413.

[00147] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to IGF2R. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 146-151 or 71414-103440. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 40%, at least 45%, atleast50%, atleast55%, atleast60%, atleast65%, atleast 70%, atleast 75%, atleast 80%, at least85%, atleast90%, atleast91%, atleast92%, atleast93%, atleast94%, atleast95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 146151 or 71414-103440. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, at most 91%, at most 92%, at most 93%, atmost 94%, at most 95%, at most 96%, atmost 97%, at most 98%, or atmost 99% sequence identity to any one of SEQ ID NOs: 146-151 or 71414-103440. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises 100% sequence identity to any one of SEQ ID NOs: 146-151 or 71414-103440. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence of any one of SEQ ID NOs: 146-151 or 71414-103440. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at leasts, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relativeto any one of SEQ ID NOs: 146-151 or 71414103440. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, atmost 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, atmost20, atmost21, atmost22, atmost23, atmost24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 146-151 or 71414-103440.

[00148] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to Dnmt3a. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 103441-111640 In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 103441111640. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, atmost 91%, at most 92%, at most 93%, atmost 94%, at most 95%, at most 96%, at most 97%, at most 98%, or atmost 99% sequence identity to any one of SEQ ID NOs: 103441-111640. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises 100% sequence identity to any one of SEQ ID NOs: 103441-111640. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence of any one of SEQ ID NOs: 103441-111640 In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, atleast 11, atleast 12, atleast 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 103441-111640. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, atmost 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 103441-111640

[00149] In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein binds to Nephrin. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 111641-143854 In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, atleast 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 111641143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most60%, atmost65%, atmost70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost 91%, atmost92%, atmost93%, atmost94%, atmost95%, atmost96%, atmost97%, atmost98%, or at most 99% sequence identity to any one of SEQ ID NOs: 111641-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises 100% sequence identity to any one of SEQ ID NOs: 111641-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises an amino acid sequence of any one of SEQ ID NOs: 111641-143854 In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has atleast 1, atleast 2, at least 3, at least 4, at least 5, at least 10, atleast 11, atleast 12, atleast 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast20, atleast 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 111641-143854. In some embodiments, the targeting moiety in a fusion polypeptide disclosed herein comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, atmost 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 111641-143854

[00150] Provided herein, in some aspects, is a suitable composition for target cell uptake and internalization of a CRISPR-Cas effector fusion polypeptide in the form of a ribonucleoprotein (RNP), which helps in 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%, atleast 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 that not fused, or not fused the same configuration. Table 3. Exemplary targeting moiety amino acid sequences SEQ ID NO. Amino acid sequence Target protein 31 MWYSDDPAVALEIATKYGGKPWEEIEKAAKEAAKKGVAVWWGTDEEETIAYNTA LANDTGTGFEQAKKIADAIKAAVPNAKWLA TfRl 32 MAEEVYKLVEEMLEKGETDVKKIAKAVKEKFPEVNVIEVGNVAIFIVGGTEGLEERKAA AEKVYKEGIAAGWNTTELLTALLLATGADVWA TfRl 33 TADEVYKLVEEIGGSGPEAAERAAEAVEKKFPDVYWRLPDGTAVFWGHEGKEEKQA IALAVYEANKAAGITDPIERKINYLTAIPGAAWYG TfRl 34 AAAAAAARAAFEATLERLKADGTYDDPIAAGTALLLAVPGARVWYDKKYAVFITDEK ADEVYKLAEELLAKGATVEEISKAIKEAVPTVTVLLG TfRl 35 GAALEEARRLAREWERAKREGLDPLTTALRLLEAIPGASVWAAESYAVFITKEKAEE VYKLAEEFDPKSAEDIRELAELVREKFPEVEWLA TfRl 36 GGEEAYELAEKLLAKGATPEEIAEAVAKELAGRVIVVRVGDVVVLVEGDEAEAPRRQA LAEEVYERSRTLSPQERLEAFLTGVPGARVWG TfRl 37 AAARAAARAAFEGVLAQAKAEGITDPLEIALRLLEAIPDAAWYANPYTVFVSREHADE VYRLVEKAIEEGITDSREIAKRVREAVPDVEVLLG TfRl 38 EEERKELEEKVKKVLEEAKKEGITDPTEIAERILLETGARVWAGEKYAAFFAPPEYAEE VYKLAEEALKKVKDVEEVARIVREKVPEVFWLA TfRl 39 AAEAAAKEAAARAWEAGKKEGKSPLETAIALLEAVPGVRWYYRERFAAFFAPEEDA PKVYKLVEEASKKYENEEEVAAYVKEKVPTVEWIL TfRl 40 AADEVYKLVDELLEKGETNVEWAKAVREKFPEVTVITVGDVAVFIVGKEEEMEEREK KAKAVYEEAKKEGIDRETLLIRMLEATGADVWG TfRl 41 EEKWKEAEERFRKTLEKMKEEGVTDPAEIATALLLSIPDASVWATDKYAVFIAPKEDH DKVFELAEKLLEEGGTDVADIAKKVQEAIPSVKVILG TfRl 42 AADAVFAVAEELAGKASVEEIAE AIRER VEGNVLLIVKDDVLVAWGEIADPEAVRARF DEVYEASKDLDPQSRLTAILLATGADVWG TfRl 43 LADKVYEIAESLLEFDDVKAIAEAVEKAVPGVHVWIDDTAVFTVGGNKEEAEKKAKE VEEELKAKGITDKEERNLAFLEAIPNAKWIS TfRl 44 AAAREAAEAAVRAVLARAKAEGTSLEETNIALLEAVPDAAVWGYERYAVFITKKDAE GVYKLAEENLPSSEEGIRELAERVREAFPDVHVIVD TfRl 45 GMDKVYEIADKLIDEVENIEEIAKKIEEEVPGVRVIIIGNYAVAIEGGEENAEEYEAKAKK VIDEAKAKGITDPNEIGIALLLETGAGVWS TfRl 46 AADKVYAIAESSQGITDPREMAELVSAEVGGEVEVLVLDDFAVFWGGSEDREEIEERL KEWERGKKEGTPKEELAIELLLESGGAWFT TfRl 47 DAALEKAYKAASEALDKFGDNDMKGIAKYIADTVGDAVEVLYHKNWYVKGGVWS NNPAVALYLAENLAGAPKEEVFAAIDKLE TfRl 48 MGEEVYKIAESALGKMSPEEVADLVEEKIGGKVRAIRLPGGYWFVEGNDGFEEAEAR ARAAWEEIVAEGLTDDPTAVLTRLLLATGGSVAIA TfRl 49 MEKEIIEEVKELLKTKTKEETAVALVEKFPEIKWWTDKIVAWADDELGEKAYKAAE EALEKFPKDAAEEVAKYIKEKVGEGVKVI SI TfRl 50 MVWSEDPELALKLVEELANGTKEEVEKFVKEYSGGGVKWWLTNDPETAEKAYKA AEEALDKFGINNTEEIAKYIKEKVGEDAEWNI TfRl 51 DELYEEALKLGEKIVEEGKKNGWSFEEIGERLLTAVKGAALVYVTEEKWIVPEEVADE VYEAVSKMLEEGNFDEEEIIEKIKELAKGKAKWW TfRl 52 GEKIEEFKKKFKAAMEENKNNPTTTPAEIALEAYPEAGLVIAYGDLVLLVHKEVADEAY KIAEEILGDDEESWKAYEILKEKSGGKVTLVKI TfRl 53 MAEKIYEIAEKNLEGHSVAEIGELIEKEVPGARTIWGDVLAWVGGTEDDATLRARAE AWARGKAEGLSRLETLERILTEVPNAKVWG TfRl 54 EEAIEKLKEKI KEI VKEAKEKGLNTEETALRILTEVGAKLWLTDGYWIVLDPED ADKA YELAEELLKKYPNDSEIIVNKLKEAFPNVIWEV TfRl 55 ALDKVYEIAEKTLGKHNVKEVAEIVKKEVPEVEVIIVGDIAIFWGGDPAEKRAAAEAV LERAKAEGLNRLETLLAFLEAVPGAGVWG TfRl 56 EELRKRVEAAVEEVWKQKENMNKEELATALLLKSGASVWAAERYAVIVVPKEKAEE AYKIAEENLPENDEELEELAERIEEEVPGAWVLT TfRl 57 SAELAAKRAAFEAALAQSKSLNTTDALTLLLLATGASVGVWAGEYAVFLVPKEKEEEV YEIAEEMLKEGLKDVRELAERVEREVPGWVLLG TfRl 58 MEELEREERIVRELVEEGKREGIDKLTLAEKLLEAVPRARWWDEEYAVFIADPSIADK VYEIAEKALEEGVKDVREVAERVKKEVPGVHVIVA TfRl 59 MELDEKAYEAAREALGTGENRGKKMAEYIKEKVPEVKWLVGDDLVIVFRGDADVEA WATAEKLLKEVSKEQAALELVEKFPEIIWW TfRl 60 EEERERLRRLVEEVLERGKKEGVSPLELAEKLLLEIPGARVVAYDEKFAVFIADDETGEK VYKLAEEKLGKVSVEELAELVRKEFPSVEVLLA TfRl 61 EEEFEKARAIFDAIVEEAKAKGWDAVTTGIALLEGVPGARWAYTDRWAVFIADPKVA EEVYKLAEEAMEEVETVEEIAEWKEKVPEVKVLLS TfRl 62 DPLVEAALEAARIARERRAEDGGDDWWWIKDPEAAAEVAEAVGAEEVRQIGDYTVL FARGVSLEE AVAAAE AASRKYNSPYAVIGL TfRl 63 MSEEQKAYKVAEKELFGDSEENAKRIGEAISKETGLKVIVHGKVVVVLKGEVDEAAVK AAIDALEGKSDEDRATALLQQFPEIVLVW TfRl 64 VHVIVIEPDENTVKIAEKVKEEAGNDPEEIYKVAEKLVDEYPTTGFAVFITDPTGAAEKE ATARAIYAEAKAKGVDRLTTLERLLLGTGAAVWG TfRl 65 GMDEELATQAVEIAAQVGAKKAYAIVGDGFILVIIVGKGAAAKKAAEKILELAKKAGIK GEVYMVEGGDDEELIEELLKKWEEIKAKLGK TfRl 66 AAAQARADADVIVAHFGDLPERERTVLRLAAIAAAAGAEKVEAWEEGGRYHVRITGTD AAAKAGAAAARAWAAERGLDVEVTLG TfRl 67 MGDKVYEVAEKLLEGSSPEKIAEAVKKAVPGVQVFVEGDFVAFWGGEEDREEREARV REIVERAKREGWSTEETLLAILEGVPGATVWG TfRl 68 AYWVWSDGDLTPAAAAAAAEGATVRVEGNIVIAEVPDEATAMAVLTNAAAAVGDA RGFIIPAGEEGLEERIREEAERLRKREEEVRNGP TfRl 69 MEAAEKAYEVAEKYFGTGEENAKKIAEAIKKEAGDELDVIVVKDIVIVGKGFDKEEVK KRVEELLKEKSLEETALELVEEFPKIYWAV TfRl 70 DAEFEAAEAAAKKVIEEAKKKGLIGKDPITVMTDLLLAIDGASTVVYDKEYAVFIHKEK ADEVYKL ADEL VGKYNVKEIAEIVKKEIPGVR WML TfRl 71 MWVSDNPEAALAIAETNTGNMEEAKKMAKELTGGKGWIWAFADDEKAEKAYKVA EKAIEIGDNKENAKKIAEAIRKEVPGVEVIEL TfRl 72 MELEERVRKIVKEWEKGKKEGKSPEEIAIAILEAVPEASWYYSEDFWLFPHKVAEEA YKLAEKLLEELTDEEEIKKKLLEEFPEAILVEL TfRl 73 EKELEEKREKAKEIIEKGIKEGKSSVEIAEELLKEVGASWYINDGVAVFLAPPEVADKA YKIVESFGGKKSAEEVAKAWEETGGKVEVFLV TfRl 74 AAAQARADADVIVAHFGDLPERERTVLRLAAIAAAAGAEKVEAWEEGGRYHVRITGID EAAKEGAEAARAWAEERGLDVEVTLG TfRl 75 EEKRKEAIEKIKEVIEKYKKTGDLEALGIAVIEKSGASWVSLPNYWAVLDPKLADEVY KLAEELIEKTDDEEEIAKAIKEAFPEVEVIKL TfRl 76 EEERRAREARARELLAAARAAGIWESDPQALGTALLTGVPGARWWAERVAVFVTDE KADEVYELAEKNLPTSAEGVEELAELVRKELPDVEVILL TfRl 77 LEEKVYELAEEALKTLTDVKEIAEYVKEKVGDKVTVFIIGDIAVFIVGDLSEEELEEKKK KAEEIYKQPLSTTDKMIEILKATGASWVA TfRl 78 MELLEKVYEVARKAIGKGENEGRVIAEAIREETGADVLLIGDGLWVLTGNQDKEAIKE YYEKNKGDDEATAVEIAEKFGAGWW TfRl 79 DEAAEKAYEAAEEALEKYPGREDGKKRADYIKEAVGDELDVRLFHPEWWGRGFDEE AVRAYVEKNLGDLEKLALELIEKEPKIWWV TfRl 80 AARRVYEIVERAGGSGEEEAERVAEIVKKLVPDWIFVHEDGFWFWNPEGAEEQRRR LEEVKEEAKRRGITDPQEIAVAYLEAVPGAAWFR TfRl 81 SELEQGVYELAEKLYGEGKENMERIAKAIKEKFPEFTVILVEDIWVFRNADEEEIKEAV KRLLKEKSKEETALELVEKFPGWWVA TfRl 82 DEEEIQKAIEELLKEKSLEEAAIEIAQRFGAAWWTKTLWIKDDVILFGEGDREGKHIA EYIRRELEGFNGSKHERSTKAWEAAQKAF TfRl 83 AARRVYEIVERANGGGAEEARRVADIVRKLVPEVEVLIVDPEFWFWGREGAEEQRRR LEEVRERARREGLSRQELAVARLEAIPKAAWFG TfRl 84 MADEVYRLAEEALGLTDVREKAAAVLARVGGRVRAFVFDDIAVFVDGGEDRWAEIRA RLEAVWRAIKARGGALTTEEKLTAFLEAVPDAWAVA TfRl 85 AVWSENPEAALELVETVQDDPEKARALAKELTGGKKPIWAFATEPELGQKVYDVAK EAFPNYGGEEGAKAIADAIKDTLGDEVTVIAF TfRl 86 AAREVYEIVERALERGEEAERVAEIVKKLVGGEVTIFVHGDLWFWGREGEEEQRRRL EEVKEEAKRGLSPQEAAVLALEAVGAAWYV TfRl 87 MAAEEAAYKEAEKALEKGSGPELGRYAQEYLSKNVGGELEVHLGHPDIVWHRGEDP AEIDAAFEELAHLSLEDRGIEIARRFPGVWWA TfRl 88 VEVILIEPDEEIKKIAEEVKKEVPGGDPELIYKVAEKLTEKYPFKGTAVFVAGGLATWS GGDTELATKLTLAAQGNAEQREANVKKWEEYKK TfRl 89 MVWSEGDTETSIKLLLATQGDKESNEAAFKKVYAEAKAKGKGRFAVLVAPESVADEV YKVAEEYLNTEDLEEVAEKVKEAVPEVEVFLD TfRl 90 SLFEALEKAAKLLKERSGSDDDVLVVIKNPEAAEDAAKAINAEEVTQIGDWTVLFARGV SFDEAVAAAEAASQKYNTPFAVLKL TfRl 91 GVEEAYKIAEEALKGGNPKEIAKKIKEGVGDTIEVFDVGGEFAVWPKGAGDVEARKA AAEKAYKAAKAAGETGAKAAIAIAEAVGVPSVWA TfRl 92 EEEFKEKEKIAKEWEKGKKNGTPKEDILVDLLLATNARVWSDPYVAFLADEENGEKV YEIAEKLVGKKNGKEIAEVVKKEVPNVKVIVL TfRl 93 MEEIKKKEEKVEEVLERAKKEGITDPTEIALELLEATGASVWYDEKYAVFIDKENAEEV YKIAEELLGGKTVEEIAEEVKKKIPNVKVFLK TfRl 94 AAEKVYEIAERELGGGREDARRVAEIVRREVPDWWEVGDYVLFWGRHADAERLRA IAEEALARVKAEGLENDPEAAGLLFLEAVPDAAVWG TfRl 95 GGDEAYKIALEAGENASVEEVAERVRRELEGRVIVLRFGDIAVFVDAEAGDEETHRERL EAAWEEALKIEDPVERAELFLLAVGASWFR TfRl 96 DVVVADDEEAALKLLEAAQGDEETIRKNVAEVAKELEGKGVKFVVFVADPEVADQVY EIADETIKKVKDPEEIAEAVKAKIGGKVLVIVE TfRl 97 MVWSFGDPQAALELVEKYAGAPLEEVKKAIKELGSEAIVYVYAEDDEAIEKAYEVAE KYFGGSGLEEAKRVAKKIEEEVGGKVEWAE TfRl 98 AARRVYEIVERANLGPEEAKRVADIVKKLVPDVHFVHDGFWFWGREGEEEQRRRLE EVKEEAKRIGDPQEL AVLYLEAVPGAAWFR TfRl 99 MVEEIKKAAKELSSLPAEEAAIELTEKFGLAWWDRLVIVTANSDELSTKAYEIAREVY GGGEEDSRRIAEAIKEALGPDVEWIL TfRl 100 DEREEEARRRVEEVKERAKREGLSRQELAVALLEAVPDAAWWTEEAWFIVKDAAR RVYEIAERALPSSREELRRVADIVKKLVPDVIIIFH TfRl 101 AAEEVYKIAEEVGGDKSAKEVAKAVKEAIGGKVYIIEFDDDTWFFAGGGWYGNNLE VNLKLLESIGKNKEETEKNVKETYEKIKN TfRl 102 AAREVYEIVERANGGPAEEAERVAEIVKKLVPEVRIIRVAPDFWFWGDEGEEELRRRV EEVEERARREGWSLQERAVALLEAVPQAAWFG TfRl 103 MVVLTDNPEVGLELVEKLSQGTPEEVKAFIDEKLGGKGVVVYAVAETEEMQEKAYEV AEAAYRPLDAESAKEIAARIEEEVGEGVTVYSL TfRl 104 EEEFKEAEKKAKEVIKKAKEEGVGETELLIRLLEAIPNARVWANGYAVFLAEPEYADE VYKLAEKYGEKNNAEEVAEWEKELKGKVRVIKV TfRl 105 AAEKVYEIAEKEWEDEEGIKELAEKVKEEVEGEVLVFVKGNIAVFWGDFENKEEIEK KLEEAEKEIKEKGLSQTEALLTFLEKVNADWYG TfRl 106 MEMDERVYEVAEEALEASGGPEGAEAVARRVAEAVPGVKTIAVGTIVAIGRDFDEAAV RAAIEALAKTLSPEEAAEALIQAFPQITWW TfRl 107 MWFSVENVEFAEEWENFSNKPMEEIEKYLKEKGAKDTWAKATDPAKEEKAYEIAK KYFGSGPGEAEKIAEAIKEEVGEEDVLLV TfRl 108 AARRVYEIVERARGEDAEEAKRVADIVKKLVPEVHFVHDDTWFWGGNEEEQRRRLE EVKERAKREGWNRQDRAVLYLEAVPTAAWYT TfRl 109 MTEKVYELAEKEVGKDSIEEIAEAIKKAVPGVHVLLFDGKYAVFFSGGGVWTEGDTEL AIKLLEAIGSTVEETEKNVKKVIEAAKK TfRl 110 MVWSMSGTEAALQLVEEFPNGDFEEIKKKAKELAKGQPLVWRADDELGEKVYEAA REAFGGGDAEKAKEIGEYIKEKTGAEVIVL TfRl 111 DALEKKVWEVASKALDLGSGPEAMKRIAAEIAAKTGAEWAHGDIVYVSGGNVWAL GGEEAALQLVEEVAGKPLEEVLAKMRELA TfRl 112 MEEELKVYEVALKALEEAGENRGKQIAEYIKENAGDKFKVELISPDLVWTDGDVDLE GIKKLYEEKKGDLEAAGVAIAEKFPGWWW TfRl 113 MVWTDGDTEAALALLNAIGGTPEETRAAVEAALAEAKAKGVKAAVFHTGKNADEVY KLAEELLEVDDIEEIAEAVREKVPGVTVITL TfRl 114 MARRVYEIVERARGKDEAKRVADIVKKLVPEVQIFVFGDVWFWGDPATEEEQRRRL EEVMEEGKREGLDKQTLAVRCLEAVNAAVVWV TfRl 115 MVWAVEGKEAALKLAEKYPNGDPEEIKKEAKKLTGGKPVWAYGGTTNEEQLKIYK AAEEALEKNGDKEGAKEIAKYLKENGAKEVIVL TfRl 116 MKEEVKKLAEELKDISEEEIAEELLRKFPEIVWVANPLVWFADDETGEKAYKAAEKAI GKGENEGENIAKYIKEEAGDDVEVILL TfRl 117 MADEKVAVEAVEIAAQVGVKEAWLVKDGKVYILWGEGEKAKKALEEATKLAKELG FEVEARTIKPEELEEALEEGFRKL TfRl 118 SEKARQAYEAARKNFKGGGTVWLGGEVHAFGGGEEIKAQAEAIREESGGGGSVWSD NPELALALVEKYTGDPEAVAKAVEELG TfRl 119 MWLTDDGADAAVELVANYTGDPAAVKAALKELTGGGITWWFAPDPELAEKVYKV AEKEAGKDSAEGMKEAAKKIKEETGATDVIVL TfRl 120 HMEEIQKAIEELLKTISPEEAAIEIVQRFGAAWWTRDLWWCSDDHELSTRVWEAAQ KALETGGERQGKHIAEYIRREFEGEVEVILL TfRl 121 MWWGGDTEEGEKFAKIMEEELGGVTTPEERAEKAYEIAVKHYHNAMVLVGVKDPE AVKKAWEELKGDNAKAAEELVEKFGIDVAW TfRl 122 MEEAEKAYKAAKEALEKFGKNKGKKIADYIKKNGGDKFKVIQLEPEIVVVHKGDEKEI KERVEELLKTLSPEEVGLKLAEEFGAVWW TfRl 123 AADKVYEIAEKNLGSGEENAKRIAKLVEETLKGEVTVHMFGDVAVFWGDPEKAAEVR ARLEAVEAELKAKGITDPVERRLAFLEAVGAAWYG TfRl 124 MEEEIQKAIEELLKTKSKEEAAIEIVQRFGTAWWSSELWIHGGENHELSTRVWEAAQ KALETSGERDGKHIAEYIRRYTGGRVEVILI TfRl 125 MEEEMKVYEAAREALEKAGENRGRHIAAYIREKAGDKFDWLVGEDIWAGKGLDKE AIKKEAEELRKIGLEEAAIKLAEKFGAIWW TfRl 126 MVWSTDPEAALTLVEKAGGGDMAEVDKLVAELTKGEPWWSGDEVRVFHPTPEAV AVAEAIRKAVGGGNEWEKAEAAWKAAEEAF TfRl 127 MVWSDNPEAALKIVENYQDDPEAVKKAVEELGVKKLVAVLNGEVHVFNPTPESKKIA DYVKEKIGGGD AEEKAEQAYKAAEE AW TfRl 128 AADKVYELVESLLKEGVTDVNEIAEAVAKELPEVQVLIFGDVAVFWGKEEKAEEIRAA AEKVWKEALEKGVGREERLIRLLLATGGAVAVG TfRl 129 HETETRVWEAAQKALEETSGERQGKHISEYIRRYVPDVDVILHEGLVVISTGDVDEEEIK KAIEELLKKVSPEEAAIEIVQRFPEVSWW TfRl 130 EEERERRERLVREWERGKREGISKEELAIRVLEAIPDAAVWATERIVAFITREDADKV YEIAEKNLGNSEEEDRRLAELVRREVPGVEVIFV TfRl 131 MEAKEKAYEIAEEEYDKDSGPEAGKRIAEAIKKAIPGANVILIGERIVAVGVENEEEVRK KAEELEKEGLEKAAIELAEKFGIDWW TfRl 132 GREEEIKELSELLNVPPEVAELMLKAEELLKKTGDPKVKQLLHYAASAAVQGYPELAEK LLKEAL IGF1R 133 MDAIAEETAKRLGDPEAASKAVSVAYYLLQQGLAPEEWEQLREIAKEFNDEAFAWA EVLAELA IGF1R 134 GKEEEIKEMAEMLGVPEEVARKLVELEELAKKTGDPRLKQLLHWAASAALTGNPEAM NAYLDEGL IGF1R 135 SEKEEELIKFADYLESVYKGTGDPKYIEDLKELLEMAKELGAKKAVEYIEKKIKELGGSG GSGGS IGF1R 136 DDAEFVYNAVKADPSILKSAIYALISQSKKEGNIEEWKKLLELAKKAGDEAIAKAVKEL TKLVA IGF1R 137 GAVALLKELVPLYEARGEPELAELVALALALLEAGNPSAAYAVLSYAAKESGDPRIQEV LDLIGA IGF1R 138 GLREELEERLRETGDPEEWREFIERLRKEFNISEKTALSAAWGALQALGLPELLEALAKI MEEV IGF1R 139 DLLEEWKELLAKGVSPLEAAKLLIEKIAKEMNVSFKTALSALYGYASGEGNKELLEAIK ELMKEL IGF1R 140 AEELAEDAKFILDSGKSLKFAFYVLLQELKNKGIPEEEAIPLWEALAIASGDEELAKKV AEEVL IGF1R 141 MEELEKLLKEAKKAIEEGNEKKANELLIAAWQKATKLGDEEKLKELEELIDLFAEKFGG SGGSGG IGF1R 142 GAVKAAVDKALAGLNAELREDVTFILDHAVAEKNPGIALYFISRLAKETGDPALKALVA ELKKVA IGF1R 143 GLQKAAQELLELIESSGNIYAAIGSYYGYARRKGDPALLTVVDTAAELVETDEAAALAY VRDIAA IGF1R 144 AAERLARELRALGVPQDYVNVAVTLVATAARNPANTAAREALKVLIELLKEFNPEGAE VLARAAA IGF1R 145 DEELIEEAERIAI<EYQEI<YGLSEI<DFQVLFISAYQLLKI<GVPEEEVRI<IIEEMAEI<LGGSG GSGG IGF1R 146 DLEEKVEELVEEAEKTEDPEKRTALLNNAMLLAIRAKNEELIELVREARRELGGSGGSG GSGGSG IGF2R 147 GEEAEEKVESLVEAAREEKDPTTRFNLLADAYIIAYKAKNPELVELVREARKELGGSGG SGGSGG IGF2R 148 SDERIAEKLLRRARRLLEEGDEERAKLLLQLALMLAREAGTPLQEEIHRLARKLGGGSG GSGGSG IGF2R 149 SEERVAEKLLRKAKELLEKGDPHRALLLLTSAKLFAKEAGDPELLREIHELARRIAGGSG GSGGS IGF2R 150 DRSERSFERTRKRVEELEERGNPHEARLQLLSAFSVLRRLGDDELARKLQELLRRLIGGS GGSGG IGF2R 151 GAAAREKVNALIEAALKEKDPDKASNLFNNAYILAYDAGDEAAIKAVREARRKKFGGS GGSGGSG IGF2R pH responsive polypeptide domain

[00151] A fusion polypeptide of the present disclosure can include, in addition to the effector polypeptide, a pH responsive polypeptide domain. In some embodiments the fusion polypeptide comprises an effector polypeptide, and a pH responsive polypeptide domain (e.g., a CRISPR-Cas effector polypeptide covalently linked to a pH responsive polypeptide domain and optionally coupled to an EEP). In some embodiments the fusion polypeptide comprises an effector polypeptide, a targeting moiety, and a pH responsive polypeptide domain (e.g., a CRISPR-Cas effector polypeptide covalently linked to a targeting moiety and a pH responsive polypeptide domain and optionally coupled to an EEP, see for example FIG. 1). In some embodiments an N-terminus of the effector polypeptide is linked to a C-terminus of the targeting moiety and a C-terminus of the effector polypeptide is linked to an N-terminus of the pH responsive polypeptide domain. In some embodiments an N-terminus of the effector polypeptide is linked to a C-terminus of the pH responsive polypeptide domain and a C-terminus of the effector polypeptide is linked to an N-terminus of the targeting moiety. In some embodiments an N-terminus of the effector polypeptide is linked to a C-terminus of the targeting moiety and an N-terminus of the targeting moiety is linked to a C-terminus of the pH responsive polypeptide domain. In some embodiments a C-terminus of the effector polypeptide is linked to an N-terminus of the targeting moiety and a C-terminus of the targeting moiety is linked to an N-terminus of the pH responsive polypeptide domain. In some embodiments the fusion polypeptide comprises a CRISPR-Cas effector polypeptide and one or more heterologous polypeptides, where at least one of the one or more heterologous polypeptides is a pH responsive polypeptide domain.

[00152] In some embodiments, the pH responsive polypeptide domain is capable of functioning as an endosomal escape peptide as well, e.g., disrupting a membrane of a membrane-bound organelle (e.g., an endosome). In some of these embodiments, the pH responsive polypeptide domain in a fusion polypeptide or polynucleotide disclosed herein is capable of disrupting a membrane of a membrane-bound organelle (e.g., an endosome) and eliciting by itself endosomal escape of the fusion polypeptide or polynucleotide. In some embodiments, the pH responsive polypeptide domain is capable of binding to an endosomal escape peptide at a given pH level. The pH responsive polypeptide domain may bind to a EEP at neutral pH, but may be readily dissociated from it at acidic pH, for example, atpH 6.8, 6.7, 6.6, 6.5,6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0 or lower. For example, atapHof 6.8 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 6.6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, at a pH of 5.5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4. In some embodiments, ata pH of 5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

[00153] In some embodiments, at a pH of 6.8 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,200,500 or 1000 fold less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

[00154] In some embodiments, at a pH of 6.6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,200,500 or 1000 fold less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

[00155] In some embodiments, at a pH of 6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,200,500 or 1000 fold less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

[00156] In some embodiments, at a pH of 5.5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,200,500 or 1000 fold less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

[00157] In some embodiments, at a pH of 5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,200,500 or 1000 fold less than the affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

[00158] In some embodiments, the endosomal escape peptide is released from the protein complex when the protein complex is present within an endosome.

[00159] In some embodiments, the endosomal escape peptide is released from the protein complex when the protein complex is present within a lysosome.

[00160] In some embodiments, the pH responsive polypeptide domain binds to or more endosomal escape peptides at pH 7.4 In some embodiments, the protein complex comprises two or more copies of the endosomal escape peptide.

[00161] In some embodiments, the pH responsive polypeptide domain comprises an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polypeptide sequence selected from the sequences in Table 6. In some embodiments, the pH responsive polypeptide domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 336-349. In some embodiments, the pH responsive polypeptide domain comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, atleast60%, atleast65%, atleast70%, atleast75%, at least 80%, at least 85%, at least 90%, atleast91%, atleast92%, atleast93%, atleast94%, atleast95%, atleast96%, atleast97%, atleast 98%, or atleast 99% sequence identity to any one of SEQ ID NOs: 336-349. In some embodiments, the pH responsive polypeptide domain comprises an amino acid sequence having at most 40%, at most45%, atmost50%, atmost55%, atmost60%, atmost65%, atmost70%, atmost75%, atmost 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 336-349. In some embodiments, the pH responsive polypeptide domain comprises 100% sequence identity to any one of SEQ ID NOs: 336-349. In some embodiments, the pH responsive polypeptide domain comprises a sequence to any one of SEQ ID NOs: 336-349. In some embodiments, the pH responsive polypeptide domain comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 336-349. In some embodiments, the pH responsive polypeptide domain comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, atmost 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 336-349

[00162] In one aspect, provided herein is a composition comprising a fusion polypeptide comprising: (a) a CRISPR-Cas effector polypeptide; and (b) a targeting moiety that binds to an extracellular domain of a receptor expressed by a target cell, wherein the targeting moiety is not an antibody or an antigen binding domain of an antibody. In some embodiments, the fusion polypeptide further comprises a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain binds to an endosomal escape peptide at pH 7.4. In some embodiments, the fusion polypeptide comprising: (a) a CRISPR-Cas effector polypeptide; and (b) a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain binds to an endosomal escape peptide (often referred to as an EEP binding peptide) at pH 7.4. In some embodiments, the fusion polypeptide further comprises a targeting moiety that binds to an extracellular domain of a receptor expressed by a target cell. In some embodiments, the targeting moiety is not an antibody or an antigen binding domain of an antibody. In some cases, a 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) a heterologous polypeptide, e.g., a targeting moiety binds to an extracellular domain of a receptor e.g., a transferrin protein 1 (TfRl, CD71) and a pH responsive polypeptide domain that can bind to an EEP in a pH responsive manner, and one or more NLS. Suitable CRISPR-Cas effector polypeptides include fusion polypeptides that comprise: i) a CRISPR-Cas effector polypeptide; and ii) a heterologous polypeptide, e.g., a targeting moiety binds to an extracellular domain of transferrin receptor protein 1 (TfRl, CD71) and a pH responsive polypeptide domain that can bind to an EEP in a pH responsive manner. 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, such as any of the CRISPR-Cas effector polypeptides described herein. Linker

[00163] In some cases, a fusion polypeptide of the present disclosure comprises a linker. In some embodiments, the linker comprises one or more linker polypeptides. The linker polypeptide may have any of a variety of amino acid sequences. Polypeptides of the present disclosure 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.

[00164] Examples of linker polypeptides include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, and (GGGGS)n, 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, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, 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. A suitable spacer peptide includes, e.g., GIHGVPATT.

[00165] In some cases, a fusion polypeptide of the present disclosure comprises a linker between a targeting moiety and the polypeptide, such as a CRISPR-Cas effector polypeptide, present in the fusion polypeptide. In some cases, a fusion polynucleotide of the present disclosure comprises a linker between a targeting moiety and the polynucleotide, such as an siRNA present in the fusion polynucleotide. In some cases, the linker is a proteolytically cleavable linker. In some cases, a fusion polypeptide of the present disclosure comprises a first linker between a targeting moiety and a polypeptide disclosed herein, such as a CRISPR-Cas effector polypeptide, present in the fusion polypeptide, and a second linker between the polypeptide (or targeting moiety) and the compound disclosed herein. In some cases, a fusion polypeptide of the present disclosure comprises a first linker between a targeting moiety and a polynucleotide disclosed herein, such as an siRNA, present in the fusion polynucleotide, and a second linker between the polynucleotide (or targeting moiety) and the compound disclosed herein. In some cases, the firstand or second linker is a proteolytically cleavable linker.

[00166] The proteolytically cleavable linker can include a protease recognition sequence recognized by a protease selected from the group consisting of: alanine carb oxy peptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, IgA-specific serine endopeptidase, legumain, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E, picornain2A, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB, and Xaa-pro aminopeptidase.

[00167] 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-(Ser / Thr), e.g., Pro-Leu / Gln-Gly-Met-Thr-Ser or Pro-Leu / Gln-Gly-Met-Thr. 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.

[00168] 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, 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, 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. Additional suitable linkers comprising protease cleavage sites include linkers comprising one or more of the following amino acid sequences: LEVLFQGP, cleaved by PreScission protease (a fusion protein comprisinghumanrhinovirus 3C protease andglutathione-S-transferase; Walker etal. (1994) Biotechnol. 12:601); a thrombin cleavage site, e.g., CGLVPAGSGP; SLLKSRMVPNFN or SLLIARRMPNFN, cleaved by cathepsin B; SKLVQASASGVN or SSYLKASDAPDN, cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN cleaved by MMP-3 (stromelysin); SLRPLALWRSFN cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN cleaved by MMP-9; DVDERDVRGFASFL cleaved by a thermolysin-like MMP; SLPLGLWAPNFN cleaved by matrix metalloproteinase 2(MMP-2); SLLIFRSWANFN cleaved by cathespin L; SGVVIATVIVIT cleaved by cathepsin D; SLGPQGIWGQFN cleaved by matrix metalloproteinase l(MMP-l); KKSPGRVVGGSV cleaved by urokinase-type plasminogen activator; PQGLLGAPGILG cleaved by membrane type 1 matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin-1; GPQGLAGQRGIV cleaved by matrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE cleaved by tissue-type plasminogen activator(tPA); SLSALLSSDIFN cleaved by human prostate-specific antigen; SLPRFKIIGGFN cleaved by kallikrein (hK3); SLLGIAVPGNFN cleaved by neutrophil elastase; and FFKNIVTPRTPP cleaved by calpain (calcium activated neutral protease).

[00169] In some embodiments, the linker is conjugated to the polypeptide and the heterologous polypeptide to produce the fusion polynucleotide. In some embodiments, conjugating comprises chemically conjugating. Many chemical approaches have been developed to crosslink carbohydrate and protein, all of which can be used to conjugate a compound disclosed herein to a polypeptide described herein, polynucleotide described herein, or any combination thereof. For example, the Staudinger ligation employs a substituted phosphite to react with the azide-modified protein to form the carbohydrate-protein conjugation via the amide bond formation (C. Grandjean, A. Boutonnier, C. Guerreiro, J. M. Fournier, L. A. Mulard, J Org Chern 2005, 70, 7123-7132). For example, oxime conjugation introduces an aminooxy group on the protein to react with oligosaccharide containing aldehyde or keto group (J. Kubler-Kielb, V. Pozsgay, J Org Chern 2005, 70, 6987-6990). For example, Michael addition often uses thiol group addition to maleimide to form a stable thioester linkage (T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura, Y. C. Lee, Biomacromolecules 2005, 6, 880-884). For example, the method of copper (I)-catalyzed cycloaddition of azide to alkyens (click chemistry) provides efficient glycoconjugation (a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew Chern Int Ed Engl 2001, 40, 2004-2021; b) S. Hotha, S. Kashyap, J Org Chern 2006, 71, 364-367).

[00170] In some embodiments, chemically conjugating comprises a click chemistry reaction. Click chemistry comprises a group of biocompatible small molecule reactions commonly used in bioconjugation that are stereospecific, high yielding, wide in scope, and simple to perform. The primary click reaction utilizes copper-catalyzed coupling of a terminal alkyne with a terminal azide to exclusively form the 1,2,3-triazole unit. These reactions create only byproducts that can be removed without chromatography, and can be conducted in easily removable or benign solvents. Examples of the click chemistry reaction includes, but are not limited to, Huisgen azide-alkyne 1,3-dipolar cycloaddition, copper-catalyzed azide-alkyne cycloaddition, ruthenium-catalyzed azidealkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted alkyne-nitrone cycloaddition, and reactions of strained alkenes such as alkene and azide [3+2] cycloaddition, alkene and tetrazine inverse-demand Diels-Alder, and alkene and tetrazole photoclick reaction. In some embodiments, the click chemistry reaction comprises a copper-catalyzed azide-alkyne cycloaddition. The mechanism of the copper-catalyzed azide-alkyne cycloaddition is described in Himo, et al. J. Am. Chern. Soc. 2005. 127: 210-216. In some embodiments, the click chemistry reaction to form a fusion polypeptide as disclosed herein occurs at ambient temperatures. In some embodiments, the click chemistry reaction to form a fusion polypeptide as disclosed herein occurs in the presence of a metal catalyst, for example, a copper(I)-catalyzed azide-alkyne cycloaddition. In some embodiments, the click chemistry reaction is performed in the absence of copper.

[00171] In some embodiments, compositions disclosed herein comprise a polypeptide (such as a CRISPR-Cas effector polypeptide as disclosed herein) and a heterologous polypeptide (e.g., to form a fusion polypeptide). In some cases, a fusion polypeptide of the present disclosure comprises a linker between a heterologous polypeptide (such as a targeting moiety) and the polypeptide, such as a CRISPR-Cas effector polypeptide, present in the fusion polypeptide. In some cases, the heterologous polypeptide (such as a targeting moiety disclosed herein) is conjugated to an N-terminus or a C-terminus of the polypeptide. In some cases, the heterologous polypeptide comprises a targeting moiety (such as a targeting moiety as disclosed herein). In some cases, the composition further comprises a pH responsive domain. In some cases, the polypeptide is conjugated to the targeting moiety or the pH responsive domain. In some cases, the polypeptide is conjugated to an N-terminus or a C-terminus of the targeting moiety. In some cases, the polypeptide is conjugated to an N-terminus or a C-terminus of the pH responsive domain.

[00172] 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 a targeting moiety that binds to an extracellular domain of a receptor expressed by a target cell and facilitates cellular uptake. The present disclosure also provides fusion polypeptides comprising a CRISPR-Cas effector polypeptide and one or more heterologous polypeptides, wherein at least one of the one or more heterologous polypeptides is a pH responsive polypeptide domain that binds to an endosomal escape peptide that facilitates endosomal escape of a complex comprising the fusion polypeptide. The present disclosure also provides fusion polypeptides comprising a CRISPR-Cas effector polypeptide and two or more heterologous polypeptides, wherein at least one of the two or more heterologous polypeptides is a targeting moiety that binds to an extracellular domain of a receptor expressed by a target cell and facilitates cellular uptake, and at least one of the two or more heterologous polypeptides is a pH responsive polypeptide domain that binds to an endosomal escape peptide that facilitates endosomal escape of the CRISPR-Cas effector polypeptide.

[00173] The present disclosure provides compositions comprising a fusion polypeptide of the present disclosure and a guide nucleic acid. The present disclosure provides compositions and methods for delivery of RNPs in a cell. In one aspect, CRISPR-Cas-based RNPs are delivered using the compositions and methods described herein. In some embodiments, provided herein is an engineered RNP.

[00174] Provided herein, in some aspects, is a fused polypeptide, comprising a Cas endonuclease fused to a pH responsive binding peptide (EEP binder) that binds to an endosomal escape protein (EEP), such that the fused polypeptide is bound to an EEP at neutral pH, and such that when the fused polypeptide is in an endosomal compartment, the acidic pH of the endosomal compartment leads to the release of the EEP (and therefore the fused polypeptide) from the pH responsive binding peptide of the fused polypeptide. The released EEP can disrupt the endosome and therefore release the fused polypeptide from the endosome into the cytoplasm.

[00175] In some embodiments, the fusion polypeptide comprises a linker between the targeting moiety and the CRISPR-Cas effector polypeptide. In some embodiments, the linker between the targeting moiety and the EEP binder pH responsive polypeptide domain. In some embodiments, the linker between the targeting moiety and the CRISPR-Cas effector polypeptide is a proteolytically cleavable. In some embodiments, the fusion polypeptide comprises one or more nuclear localization sequences (NLSs). In some embodiments, the one ormoreNLSs comprise the amino acid sequence K(K / R)X(K / R), where X is any amino acid. In some embodiments, the one or more NLSs comprise the amino acid sequence PKKKRKV. In some embodiments, the one or more NLSs are at the N-terminus of the CRISPR-Cas effector polypeptide. Polynucleotide

[00176] Disclosed herein in some embodiments are polymers comprising polynucleotides. The terms “polynucleotide” and “nucleic acid,” can be used interchangeably herein, and can refer to a polymeric form of nucleotides of any length. In some embodiments a polymeric form of nucleotides can comprise ribonucleotides, deoxynucleotides, or combinations thereof. In some embodiments, a polynucleotide can comprise single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, or any combination thereof. In some embodiments a polynucleotide can comprise single-stranded polynucleotides. In some embodiments a single-stranded polynucleotide can comprise a sense or antisense strand. In some embodiments, a polynucleotide can comprise a double-stranded polynucleotide. Unless specifically limited, a polynucleotide can comprise nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as any sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chern. 260:2605-2608 (1985); andRossolini etal., Mol. Cell. Probes 8:91-98 (1994)). Nucleic acid modifications

[00177] In some embodiments, the polynucleotide can comprise any type of nucleic acid molecule (e.g., ribonucleic acid (RNA) molecule, deoxyribonucleic acid (DNA) molecule, xeno nucleic acid (XNA) molecule, etc.). In some embodiments, the polynucleotide comprises ribonucleic acid (RNA) molecules, deoxyribonucleic acid (DNA) molecules, xeno nucleic acid (XNA) molecules, or any combination thereof. In some embodiments, the polynucleotide is an engineered polynucleotide. An engineered polynucleotide can refer to a synthetically constructed polynucleotide. For example, the polynucleotide can be synthesized by chemical synthesis, enzymatic synthesis, or any combination thereof. In some embodiments, the polynucleotide is a synthetic polynucleotide. In some embodiments, the polynucleotide comprises at least one modification. In some embodiments, the polynucleotides disclosed herein can comprise nucleic acid molecules comprising a modification of a sugar, a phosphate backbone, or a nucleobase. For example, noncoding nucleic acid molecules can be modified through the addition of moieties onto the molecule. A modification can be a chemical modification, a synthetic modification, or a natural modification. For example, the polynucleotide disclosed herein can comprise a 2'-O-methoxyethoxy (2'-0Me) modification, a 2'-O-(2-methoxyethyl) (2'-O-moe) modification, a 2'-fluoro (2'-F) modification, a locked nucleic acid (LNA) modification, a pseudouridine (\p) modification, a phosphorothioate (PS) bond modification, a phosphorodiamidate morpholino oligomer (PMO) modification, a 2'-phosphorylation (2'-P) modification, or any combination thereof. In some embodiments, the polynucleotide comprises (i) a 5' end modification, (ii) a 3' end modification, or (iii) a 5' end modification and a 3' end modification. In some embodiments, the modification is in the middle of the polynucleotide (e.g., between the 3' and the 5' end).

[00178] Nucleic acid molecules of the present disclosure can be modified at the nucleobase. Nucleobase modifications include but are not limited to 2 ’-0-methylation (2’-O-Me), conversion of uridine to pseudouridine, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), 2’-fluoro (2’F), 2’-O-methoxyethyl (2’-M0E), ribose modification with bridged nucleic acids (e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), or constrained ethyl bridged nucleic acid (cEt) modifications), or nucleotides with alternative chemistries (e.g., phosphorodiamidate morpholino oligonucleotides (PMO), peptide nucleic acids (PNA), tricyclo DNA (tcDNA), unlocked nucleic acids (UNA) or glycol nucleic acids (GNA)).

[00179] Nucleic acid molecules of the present disclosure can be modified at the phosphate backbone. A phosphate backbone can be modified to comprise a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, a phosphorodithioate, a methylphosphontae, a 5’-phosphorothioate, a peptide nucleic acid, a 5’-(E)-vinylphosphonate, or a 5’-methyl phosphonate.

[00180] Additional moieties can be added or attached onto nucleic acid molecules of the present disclosure. Additional moieties can include, but are not limited to antibodies, lipophilic moieties, small molecules, and RNA aptamers (e.g., ribozymes, docosanoic acid, etc.). Additional moieties can be added to alter the pharmacological features (e.g., structural or chemical parameters) of the nucleic acid molecules of the present disclosure. Nucleic acid molecules of the present disclosure can be modified with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more moieties. An additional moiety can be added at the 5' end of a nucleic acid molecule. Alternatively, an additional moiety can be added at the 3' end of a nucleic acid molecule. Alternatively, an additional moiety can be added in the middle of a nucleic acid molecule (e.g., neither at the 3' end nor the 5' end). Effector nucleic acid molecules

[00181] In some embodiments, the polynucleotide is an effector nucleic acid molecule. "Effector nucleic acid molecules" can refer to nucleic acid sequences (e.g., microRNAs (miRNAs), small interfering RNAs (siRNAs), etc.) that directly interact with other molecules (e.g., RNA, polypeptides, etc.) to regulate gene expression (e.g., by inhibiting translation, causing degradation of a target mRNA, etc.), essentially acting as "effectors" to influence cellular processes. In some embodiments, the polynucleotide can be configured for polynucleotide-mediated gene expression alteration. Gene expression alteration can include, but is not limited to, changing the level of activity of a gene, such as turning it up (e.g., increasing expression) or down (e.g., decreasing expression) through various mechanisms. Gene expression alterations can also refer to changes in gene regulation mechanisms, such as translation, transcription, epigenetics, post-transcription, posttranslation, or any combination thereof. For example, in some cases, the polynucleotide can be configured for polynucleotide-mediated gene splicing, such as splice-switching oligonucleotides (SSOs) which can alter pre-mRNA splicing by binding to a target sequence and blocking the access of splicing factors to the pre-mRNA. Gene expression can be altered in a number of ways including, epigenetic alterations, DNA methylation, genome editing, site-directed mutagenesis, or any combination thereof. For example, in some cases, the polynucleotide can be configured for polynucleotide-mediated gene silencing, such as sequence-specific gene silencing. Sequence-specific gene silencing can refer to a decrease in gene expression relative to normal gene expression levels (e.g., in the absence of the polynucleotide disclosed herein). For example, polynucleotide-mediated gene silencing can refer to siRNA-mediated gene silencing (e.g., small interfering RNA, short interfering RNA, silencing RNA, etc.), where gene silencing can be elicited by binding of an siRNA, through complementarity, to a specific mRNA sequence, thereby targeting the specific mRNA sequence for cleavage and degradation.

[00182] In some embodiments, the polynucleotide can be complementary to a target nucleic acid sequence. In some embodiments, the polynucleotide can be complementary to a target ribonucleic acid sequence, such as a messenger ribonucleic acid sequence (mRNA), a pre-mRNA sequence, a coding ribonucleic acid sequence, a noncoding ribonucleic acid sequence, or any combination thereof. In some cases, the polynucleotide can selectively bind to a target amino acid sequence, such as an aptamer configured to bind a target protein. In some embodiments, the polynucleotide can comprise an siRNA, a microRNA (miRNA), an antisense oligonucleotide (ASO), a splice-switching oligonucleotide (SSO), an aptamer, a deoxy ribozyme, or any combination thereof. The creation of such polynucleotides is routine to those of skill in the art, including strategies for developing and designing polynucleotides that will be effective for use in the methods disclosed herein (such as inhibition of a target gene). For example, factors such as melting temperature, length, GC percentage, secondary structure, target sequence, splicing enhancer sites, binding affinity, and modificationscan be taken into account when designing polynucleotides of the present disclosure.

[00183] In some embodiments, the polynucleotide is an siRNA. Small interfering RNAs (siRNAs) generally comprise a noncoding double-stranded nucleic acid sequence that can be unwound into single stranded RNAs in order to bind to a target nucleic acid sequence, such as another RNA (e.g., mRNA) through Watson-Crick base pairing in a manner mediated by or independent of an argonaute-containing complex (e.g., RISC complex). An siRNA can function to specifically silence the expression of a target gene by binding to a complementary mRNA and causing degradation of the complementary mRNA, thus preventing translation into a protein. siRNAs can be classified into classes I, II, and III based on predicted silencing activities. Class I siRNAs are functional in mammalian cells, while Class III siRNAs are nonfunctional. In some embodiments, the siRNA is a class I siRNA. Strategies for developing and designing siRNAs of the present disclosure that will be effective (such as in silencing the expression of a target gene) can include, but are not limited to, factors such as distance of target region to transcription start site (e.g., avoiding 5' and 3'UTRs), nucleotide composition (e.g., %GC content, length, etc.), and absence of off-target effects and secondary structures in a target site.

[00184] In some embodiments, the polynucleotide is a short hairpin RNA (shRNA). A short hairpin RNA (shRNA) can refer to a small, artificial RNA molecule that can silence genes by degrading viral or messenger RNA. shRNA is processed in the cytoplasm by the dicer protein, which removes a loop structure to produce siRNA. In some embodiments, the shRNA is configured to produce an siRNA, such as an siRNA disclosed herein.

[00185] In some embodiments, the polynucleotide is an miRNA. In some embodiments, the miRNA is a primary miRNA (pri-miRNA). In some embodiments, the miRNA is a precursor miRNA. In some embodiments, the miRNA is a mature miRNA. MicroRNA (miRNA) can refer to a small, noncoding RNA molecule that regulates gene expression by binding to a target mRNA and preventing or delaying the translation of the target mRNA. Strategies for developing and designing miRNAs of the present disclosure that will be effective (such as in silencing the expression of a target gene) can include, but are not limited to, factors such as seed regions (e.g., including at least one seed match in the 3' UTR), secondary structure (e.g., including a terminal loop, a basal stem, and a flanking sequence on either side), and modifications.

[00186] In some embodiments, the polynucleotide can comprise an antisense oligonucleotide (ASO). Antisense oligonucleotides (ASOs) can refer to synthetic, single-stranded DNA or RNA molecules that bind to target RNA sequences and alter protein expression of the target RNA. For example, ASOs of the present disclosure can induce the degradation of mRNA and / or prevent or inhibit the progression of splicing or the translational machinery. ASOs can be modified to improve stability, uptake, and bioavailability. For example, modifications to the sugar ribose can increase affinity to the target RNA. Strategies for developing and designing ASOs of the present disclosure that will be effective (such as in inducing the degradation of a target gene) can include, but are not limited to, factors such as length, secondary structure, splicing enhancer sites, and modifications.

[00187] In some embodiments, the ASO is a splice-switching oligonucleotide (SSO). In some embodiments, the polynucleotide is an SSO. Splice-switching oligonucleotides (SSOs) are synthetic nucleic acids that can be used to disrupt the normal splicing process by blocking RNA-RNA base pairing and protein-RNA binding interactions. SSOs of the present disclosure can be designed to target different exons and have different lengths and modification contents. For example, AmNA (Amido-bridged nucleic acids) and GuNA (Guanidine-bridged nucleic acids) modified SSOs have been shown to have higher exon skipping activities than LNA (Locked nucleic acids) modified SSOs.

[00188] In some embodiments, the polynucleotide can comprise an aptamer. An aptamer can refer to a single-stranded nucleic acid molecule that binds to a target by folding into a three-dimensional structure. In some embodiments, the aptamer comprises DNA, RNA, XNA, or any combination thereof. In some embodiments, the aptamer binds to a polypeptide (e.g., protein), nucleic acid sequence, small molecule, or metal ion. In some embodiments, the aptamer is configured to alter gene expression. In some embodiments, the aptamer is an aptamer RNA switch. An RNA switch can refer to an RNA regulatory element that can change its structure (conformation) in response to the binding of a ligand (e.g., a polypeptide) in order to regulate gene expression of a target gene by either turning the target gene "on" or "off" depending on the ligand's presence. Strategies for developing and designing aptamers of the present disclosure that will be effective (such as in altering the expression of a target gene) can include, but are not limited to, factors such as secondary structure, and binding affinity.

[00189] In some embodiments, the polynucleotide is a deoxyribozyme (such as DNA enzymes, DNAzymes, catalytic DNA, etc.). Deoxyribozymes can refer to DNA sequences that can catalyze chemical reactions, including RNA and / or DNA cleavage, which can be used to control gene expression. In some embodiments, the deoxyribozyme catalyzes DNA cleavage. In some embodiments, the deoxyribozyme catalyzes RNA cleavage. In some embodiments, the deoxyribozyme is a ribonuclease. Nucleic acid molecule structure

[00190] In some embodiments, the polynucleotide can comprise a double-stranded nucleic acid molecule (e.g., a double-stranded polynucleotide). In some embodiments, the double-stranded nucleic acid molecule is any type of nucleic acid (e.g., ribonucleic acid (RNA), deoxyribonucleic acid (DNA), etc.) that contains two strands. In some embodiments, the double-stranded nucleic acid molecules are double-stranded oligonucleotides, which comprise short pieces of a nucleotide sequence (e.g., DNA, RNA). In some embodiments, the double-stranded nucleic acid molecule comprises an antisense strand configured to silence a target single-stranded nucleic acid sequence, such as a messenger RNA (mRNA). In some embodiments, the double-stranded nucleic acid molecule comprises an antisense strand configured to enhance expression of a target nucleic acid sequence (such as a target nucleic acid sequence as described herein). In some embodiments, the double-stranded nucleic acid molecule comprises an antisense strand configured to enhance expression of a single-stranded target nucleic acid sequence. The double-stranded nucleic acid molecule can be synthetic (e.g., engineered). Alternatively, or in addition to, the double-stranded nucleic acid molecule can be a chemically modified nucleic acid molecule, such as the modifications described herein. The double-stranded nucleic acid molecule can comprise two nucleic acid strands, a sense strand, and an antisense strand. The sense strand of the double-stranded nucleic acid molecule will have the same or substantially the same sequence as a target nucleic acid sequence. The antisense strand of the double-stranded nucleic acid molecule can be complementary or substantially complementary to a target nucleic acid sequence.

[00191] Double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least aboutll nucleotides, at least about 12 nucleotides, atleast about 13 nucleotides, atleast about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, atleastabout 18 nucleotides, atleastabout 19 nucleotides, atleastabout20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, atleastabout28 nucleotides, atleastabout29 nucleotides, atleastabout30 nucleotides, at least about 31 nucleotides, at least about 32 nucleotides, at least about 33 nucleotides, at least about34 nucleotides, at least about 35 nucleotides, at least about 36 nucleotides, at least about 37 nucleotides, atleastabout 38 nucleotides, atleastabout 39 nucleotides, atleastabout40 nucleotides, at least about 41 nucleotides, at least about 42 nucleotides, at least about 43 nucleotides, at least about 44 nucleotides, at least about 45 nucleotides, at least about 46 nucleotides, at least about 47 nucleotides, at least about 48 nucleotides, at least about 49 nucleotides, at least about 50 nucleotides, at least about 51 nucleotides, at least about 52 nucleotides, at least about 53 nucleotides, at least about 54 nucleotides, at least about 55 nucleotides, at least about 56 nucleotides, at least about 57 nucleotides, atleastabout 58 nucleotides, atleastabout 59 nucleotides, atleastabout60 nucleotides, or more nucleotides in length.

[00192] Double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be at most about 60 nucleotides, at most about 59 nucleotides, at most about 58 nucleotides, at most about 57 nucleotides, at most about 56 nucleotides, at most about 55 nucleotides, at most about 54 nucleotides, at most about 53 nucleotides, at most about 52 nucleotides, at most about 51 nucleotides, at most about 50 nucleotides, at most about 49 nucleotides, at most about 48 nucleotides, at most about 47 nucleotides, at most about 46 nucleotides, at most about 45 nucleotides, at most about 44 nucleotides, at most about 43 nucleotides, at most about 42 nucleotides, at most about 41 nucleotides, at most about 40 nucleotides, at most about 39 nucleotides, at most about 38 nucleotides, at most about 37 nucleotides, at most about 36 nucleotides, at most about 35 nucleotides, at most about 34 nucleotides, at most about 33 nucleotides, at most about 32 nucleotides, at most about 31 nucleotides, at most about 30 nucleotides, at most about 29 nucleotides, at most about 28 nucleotides, at most about 27 nucleotides, at most about 26 nucleotides, at most about 25 nucleotides, at most about 24 nucleotides, at most about 23 nucleotides, at most about 22 nucleotides, at most about 21 nucleotides, at most about 20 nucleotides, at most about 19 nucleotides, at most about 18 nucleotides, at most about 17 nucleotides, at most about 16 nucleotides, at most about 15 nucleotides, at most about 14 nucleotides, at most about 13 nucleotides, at most about 12 nucleotides, at most about 11 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, or fewer nucleotides in length.

[00193] In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 55 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 50 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 45 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 40 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 35 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 30 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 25 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 20 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 15 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 5 to about 10 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 10 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 15 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 20 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 25 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 30 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 35 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 40 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 45 to about 60 nucleotides in length. In some embodiments, the double-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, etc.) can be between about 50 to about 60 nucleotides in length.

[00194] The antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding with an entire sense strand of the double-stranded noncoding nucleic acid molecule. Alternatively, the antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding with a part of a sense strand of a double-stranded noncoding nucleic acid molecule. The antisense strand of a double-stranded noncoding nucleic acid molecule can also form complementary binding with an entire target strand. Alternatively, the antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding with a part of a target strand. The antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding to one or more portions of a target strand (e.g., an mRNA strand) including a 5’ UTR, a 3 ’UTR, a regulatory region, and / or a coding sequence. A regulatory region of a target strand (e.g., a mRNA) can comprise a promoter region, an enhancer region, an operator region, or a repressor region.

[00195] The antisense strand of the double-stranded noncoding nucleic acid molecule can form complementary binding to part or all of a portion of an mRNA target strand. The antisense strand of the double-stranded noncoding nucleic acid molecule can be completely complementary (e.g., 100% complementary) to their target strand counterparts. Alternatively, the antisense strand of the doublestranded noncoding nucleic acid molecule and can have imperfect complementarity to their target strand counterparts. An antisense strand of the double-stranded noncoding nucleic acid molecule can have at least about 50% complementarity, at least about 55% complementarity, at least about 60% complementarity, at least about 65% complementarity, at least about 70% complementarity, at least about 75% complementarity, at least about 80% complementarity, at least about 85% complementarity, at least about 90% complementarity, at least about 91% complementarity, at least about 92% complementarity, at least about 93% complementarity, at least about 94% complementarity, at least about 95% complementarity, at least about 96% complementarity, at least about 97% complementarity, at least about 98% complementarity, at least about 99% complementarity, or more to its corresponding target strand. An The antisense strand of the doublestranded noncoding nucleic acid molecule can have at most about 99% complementarity, at most about 98% complementarity, at most about 97% complementarity, at most about 96% complementarity, at most about 95% complementarity, at most about 94% complementarity, at most about 93% complementarity, at most about 92% complementarity, at most about 91% complementarity, at most about 90% complementarity, at most about 85% complementarity, at most about 80% complementarity, at most about 75% complementarity, at most about 70% complementarity, at most about 65% complementarity, at most about 60% complementarity, at most about55% complementarity, at most about 50% complementarity, orless to its corresponding target strand.

[00196] In some embodiments, the polynucleotide can comprise a single-stranded nucleic acid molecule (e.g., a single-stranded polynucleotide). In some embodiments, the single-stranded nucleic acid molecule is any type of nucleic acid (e.g., ribonucleic acid (RNA), deoxyribonucleic acid (DNA), etc.) that contains one strand. In some embodiments, the single-stranded nucleic acid molecules are single-stranded oligonucleotides, which comprise short pieces of a nucleotide sequence (e.g., DNA, RNA). In some embodiments, the single-stranded nucleic acid molecule is configured to silence a target single-stranded nucleic acid sequence, such as a messenger RNA (mRNA). In some embodiments, the single-stranded nucleic acid molecule is configured to enhance expression of a target nucleic acid sequence (such as a target nucleic acid sequence as described herein). In some embodiments, the single-stranded nucleic acid molecule is configured to enhance expression of a single-stranded target nucleic acid sequence. The single-stranded nucleic acid molecule can be synthetic (e.g., engineered). Alternatively, or in addition to, the single-stranded nucleic acid molecule can be a chemically modified nucleic acid molecule, such as the modifications described herein.

[00197] Single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 31 nucleotides, at least about 32 nucleotides, at least about33 nucleotides, at least about 34 nucleotides, at least about 35 nucleotides, at least about 36 nucleotides, at least about 3 7 nucleotides, at least about 3 8 nucleotides, at least about 3 9 nucleotides, at least about 40 nucleotides, at least about 41 nucleotides, at least about 42 nucleotides, at least about 43 nucleotides, at least about 44 nucleotides, at least about 45 nucleotides, at least about 46 nucleotides, at least about 47 nucleotides, at least about 48 nucleotides, at least about 49 nucleotides, at least about 50 nucleotides, at least about 51 nucleotides, at least about 52 nucleotides, at least about 53 nucleotides, at least about 54 nucleotides, at least about 55 nucleotides, at least about 56 nucleotides, at least about 57 nucleotides, at least about 58 nucleotides, at least about 59 nucleotides, at least about 60 nucleotides, or more nucleotides in length.

[00198] Single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be at most about 60 nucleotides, at most about 59 nucleotides, at most about 58 nucleotides, at most about 57 nucleotides, at most about 56 nucleotides, at most about 55 nucleotides, at most about 54 nucleotides, at most about 53 nucleotides, at most about 52 nucleotides, at most about 51 nucleotides, at most about 50 nucleotides, at most about 49 nucleotides, at most about 48 nucleotides, at most about 47 nucleotides, at most about 46 nucleotides, at most about 45 nucleotides, at most about 44 nucleotides, at most about 43 nucleotides, at most about 42 nucleotides, at most about 41 nucleotides, at most about 40 nucleotides, at most about 39 nucleotides, at most about 38 nucleotides, at most about 37 nucleotides, at most about 36 nucleotides, at most about 35 nucleotides, at most about 34 nucleotides, at most about 33 nucleotides, at most about 32 nucleotides, at most about 31 nucleotides, at most about 30 nucleotides, at most about 29 nucleotides, at most about 28 nucleotides, at most about 27 nucleotides, at most about 26 nucleotides, at most about 25 nucleotides, at most about 24 nucleotides, at most about 23 nucleotides, at most about 22 nucleotides, at most about 21 nucleotides, at most about 20 nucleotides, at most about 19 nucleotides, at most about 18 nucleotides, at most about 17 nucleotides, at most about 16 nucleotides, at most about 15 nucleotides, at most about 14 nucleotides, at most about 13 nucleotides, at most about 12 nucleotides, at most about 11 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, or fewer nucleotides in length.

[00199] In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 55 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 50 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 45 nucleotides in length. In some embodiments, the singlestranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 40 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 35 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 30 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 25 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 20 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 15 nucleotides in length. In some embodiments, the singlestranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 5 to about 10 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 10 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 15 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 20 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 25 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 30 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 35 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 40 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 45 to about 60 nucleotides in length. In some embodiments, the single-stranded nucleic acids of the present disclosure (e.g., siRNAs, miRNAs, ASOs, SSOs, aptamers, deoxyribozymes, etc.) can be between about 50 to about 60 nucleotides in length. Fusion polynucleotides

[00200] In some aspects, the present disclosure relates to a fusion polynucleotide. Fusion polynucleotides can refer to polynucleotides that are fused with a protein, such as by fusing an endosomal escape peptide or a targeting moiety of the present disclosure to a polynucleotide of interest. In some embodiments, the fusion polynucleotide comprises: i) a polynucleotide (e.g., any polynucleotide disclosed herein can be used in a fusion polynucleotide of the present disclosure, such as an siRNA); and ii) one or more heterologous polypeptide, e.g., a targeting moiety that binds to an extracellular domain of a receptor such as a transferrin protein 1 (TfRl, CD71) ); and optionally also includes one or more nuclear localization signals (NLSs), such as an NLS as disclosed herein (see for example NLS sequences in Table 2).

[00201] In some cases, nucleic acid modifications (such as nucleic acid modifications disclosed herein), and / or amino acid modifications (such as amino acid modifications disclosed herein) can be used to conjugate a polynucleotide (such as a polynucleotide disclosed herein) to a polypeptide (such as a targeting moiety disclosed herein). For example, the polynucleotide can be chemically modified with a reactive group at either the 5' or 3' end, then a bifunctional crosslinker can be used to attach the polynucleotide to a reactive site on the polypeptide, such as a primary amine group on a lysine residue; common methods can include using click chemistry (e.g., azide-alkyne cycloaddition) or NHS ester coupling, where the polynucleotide is modified with an azide or amine group respectively, and the polypeptide is activated with a complementary reactive group like a DBCO (dibenzocyclooctyne) or NHS ester. Targeting moiety

[00202] A fusion polynucleotide of the present disclosure can include, in addition to the polynucleotide, a targeting moiety. In some embodiments the fusion polynucleotide comprises a polynucleotide and a targeting moiety (e.g., an siRNA linked to a targeting moiety, see for example FIG. 1). In some embodiments a 5' end of the polynucleotide is linked to a C-terminus of the targeting moiety. In some embodiments a 3' end of the polynucleotide is linked to an N-terminus of the targeting moiety. In some embodiments the fusion polynucleotide comprises a polynucleotide, a targeting moiety, and an EEP (e.g., an siRNA linked to a targeting moiety and an EEP, see for example FIG. 1). In some embodiments a 5' end of the polynucleotide is linked to a C-terminus of the targeting moiety and a 3' end of the polynucleotide is linked to an N-terminus of the EEP. In some embodiments a 5' end of the polynucleotide is linked to a C-terminus of the EEP and a 3' end of the polynucleotide is linked to an N-terminus of the targeting moiety. In some embodiments a 5' end of the polynucleotide is linked to a C-terminus of the targeting moiety and an N-terminus of the targeting moiety is linked to a C-terminus of the EEP. In some embodiments a 3' end of the polynucleotide is linked to an N-terminus of the targeting moiety and a C-terminus of the targeting moiety is linked to an N-terminus of the EEP. In some embodiments the fusion polynucleotide comprises a polynucleotide, a targeting moiety, and a pH responsive polypeptide domain (e.g., an siRNA linked to a targeting moiety and a pH responsive polypeptide domain and coupled to an EEP, see for example FIG. 1). In some embodiments a 5' end of the polynucleotide is linked to a C-terminus of the targeting moiety and a 3' end of the polynucleotide is linked to an N-terminus of the pH responsive polypeptide domain. In some embodiments a 5' end of the polynucleotide is linked to a C-terminus of the pH responsive polypeptide domain and a 3' end of the polynucleotide is linked to an N-terminus of the targeting moiety. In some embodiments a 5' end of the polynucleotide is linked to a C-terminus of the targeting moiety and an N-terminus of the targeting moiety is linked to a C-terminus of the pH responsive polypeptide domain. In some embodiments a 3' end of the polynucleotide is linked to an N-terminus of the targeting moiety and a C-terminus of the targeting moiety is linked to an N-terminus of the pH responsive polypeptide domain.

[00203] In some embodiments the fusion polypeptide comprises a polynucleotide and one or more heterologous polypeptides, where at least one of the one or more heterologous polypeptides is a targeting moiety. A targeting moiety can be used to direct a polynucleotide to a target cell or tissue. A targeting moiety in a fusion polynucleotide disclosed herein can be but is not limited to a lipophilic moiety, a small molecule, a peptide (e.g., a polypeptide), an RNA molecule, a nanoparticle, an antibody, a single-domain antibody, a miniprotein, or an antigen binding fragment thereof. A targeting moiety in a fusion polynucleotide disclosed herein can be specific to an antigen or receptor on the target cell or tissue (e.g., Asialoglycoprotein receptor (ASGPR)). In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein specifically binds to a target protein. In some embodiments, the target protein is a receptor expressed by a target cell. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein binds to an extracellular domain of a receptor expressed by a target cell and facilitates cellular uptake.

[00204] A targeting moiety in a fusion polynucleotide disclosed herein can be a lipophilic moiety. A lipophilic moiety can comprise one or more fatty acid groups or salts thereof. A lipophilic moiety can comprise one or more lipids. Lipids are fatty acids and their derivatives which are insoluble in water but soluble in organic solvents. In some embodiments, a lipophilic moiety can be unsaturated. Alternatively, or in addition to, a lipophilic moiety can be monosaturated. Alternatively, or in addition to, a lipophilic moiety can be poly saturated. In some embodiments, a double bond of an unsaturated lipophilic moiety can be in a cis conformation. Alternatively, or in addition to, a double bond of an unsaturated lipophilic moiety can be in a trans conformation. Non-limiting examples of lipophilic moieties can be a triglyceride, a phospholipid, a sterol, an oil, a wax, a hormone, a vitamin, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis- O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03- (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[00205] A targeting moiety in a fusion polynucleotide disclosed herein can be a small molecule. A small molecule can be a sugar, an amino acid, a phenolic compound, an alkaloid, a sterol, a lipid, a fatty acid, or other small chemical compound. A chemical compound can be a molecule that has a molecular weight of less than 1000 Daltons. Alternatively, or in addition to, a small molecule is a molecule with a size on the order of 1 nm.

[00206] A targeting moiety in a fusion polynucleotide disclosed herein can be a sugar or sugar moiety. A sugar can be a monosaccharide. Alternatively, a sugar can be a disaccharide. Alternatively, a sugar can be a polysaccharide. Non-limiting examples of sugars include glucose, dextrose, fructose, galactose, a sugar alcohol, a pentose, xylose, ribose, sucrose, cellulose, starch, lactose, maltose, trehalose, lactulose, cellobiose, chitobiose, glycogen, or chitin. A small molecule can be an amino sugar such as but not limited to N-acetyl Galactosamine (GalNAc), N-acetylglucosamine or sialic acid.

[00207] A targeting moiety in a fusion polynucleotide disclosed herein can be an RNA molecule. An RNA molecule can comprise an aptamer, a ribozyme, or a hairpin RNA.

[00208] A targeting moiety in a fusion polynucleotide disclosed hereincan be an antibody or an antigen-binding fragment thereof. An antibody, also known as an immunoglobulin, is a blood protein produced to counteract a specific antigen. Antibodies can be Y-shaped proteins which comprise variable binding sites that are specific to particular epitopes. An antibody can be a monoclonal antibody. Alternatively, an antibody can be a polyclonal antibody. An antibody can be a singledomain antibody. An antibody can be an antibody fragment. An antibody can be an agonist. Alternatively, an antibody can be an antagonist. Alternatively, an antibody can be an allosteric modulator (e.g., a positive allosteric modulator or a negative allosteric modulator).

[00209] A targeting moiety in a fusion polynucleotide disclosed herein can be a polypeptide. Nonlimiting examples of polypeptides can be an agonist of IGF1R, IGF2R, TfRl, Nephrin, Nephi, Megalin, cKIT, GLUT4, GLUT1, Neuropilin, FOL1, FOL3, CCR5, CXCR1-4, EGFR, VEGFR1, VEGFR2, PDGFRA, or ADRB3.

[00210] In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein binds a cell surface receptor. In certain embodiments, the cell surface receptor is membrane associated. Membrane associated proteins represent about a third of the proteins in living organisms and many membrane proteins are known in the field. Based on their structure, membrane proteins can be largely categorized into three main types: (1) integral membrane protein (IMP), which is permanently anchored or part of the membrane, (2) peripheral membrane protein, which is temporarily attached to the lipid bilayer or to other integral proteins, and (3) lipid-anchored proteins. The most common type of IMP is the transmembrane protein (TM), which spans the entire biological membrane. The cell surface receptor of the present disclosure includes single-pass and multi-pass membrane proteins. Single-pass membrane proteins cross the membrane only once, while multipass membrane proteins weave in and out, crossing several times. In some embodiments, the cell surface receptor can be a monomeric receptor. In some embodiments, the cell surface receptor can be a multimeric receptor. In some embodiments, the cell surface receptor can form a complex with other molecules (e.g., an integrin). The cell surface receptor can be a recycling receptor. For example, a recycling receptor as used herein refers to a cell surface receptor that specifically binds to a ligand (e.g., a targeting moiety) and leads to internalization of the cell surface receptor.

[00211] In some case, the cell surface receptor can be selected by expression levels in one cell type relative to other cell types, where the cell surface receptor is enrichment in the selected cell type and / or tissue type. In some embodiments, the cell surface receptor comprises a tissue-type specific protein. In some embodiments, the cell surface receptor comprises a cell-type specific protein. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein is not an antibody or an antigen binding domain of an antibody. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein is an antibody or an antigen binding domain of an antibody.

[00212] In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein binds to an extracellular domain of a receptor protein. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein binds to an extracellular domain of a receptor selected from the group consisting of: IGF1R, IGF2R, TfRl, Nephrin, Nephi, Megalin, cKIT, GLUT4, GLUT1, Neuropilin, FOL1, FOL3, CCR5, CXCR1-4, EGFR, VEGFR1, VEGFR2, PDGFRA, ADRB3, NTRK2, GRIA1, GRIN1, GABRA1, and GABBR1. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein binds to an extracellular domain of transferrin receptor protein 1 (TfRl, CD71). In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein binds to an extracellular domain of insulin-like growth factor 2 receptor (IGF2R). In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein binds to an extracellular domain of insulin-like growth factor 1 receptor (IGF1R).

[00213] In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises polypeptide sequence with less than 150 amino acids. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises polypeptide sequence with less than 100 amino acids. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises polypeptide sequence with more than 50 amino acids. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises polypeptide sequence with more than 60 amino acids. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises polypeptide sequence with more than 70 amino acids. In some embodiments, targeting moiety in a fusion polynucleotide disclosed herein comprises polypeptide sequence with 80 amino acids or more.

[00214] In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polypeptide sequence selected from the sequences in Table 3. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any oneofSEQID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises an amino acid sequence having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, atmost75%, atmost80%, atmost85%, atmost90%, atmost91%, atmost92%, atmost93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises 100% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises a sequence to any one of SEQ ID NOs: 31-151 or 350143854. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least20, atleast 21, atleast22, atleast23, atleast24, at least25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 31-151 or 350-143854. In some embodiments, the targeting moiety in a fusion polynucleotide disclosed herein comprises a sequence that has at most 1, at most 2, at most 3, at most 4, atmost 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, atmost 16, atmost 17, atmost 18, atmost 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 31-151 or 350-143854 Linker

[00215] In some cases, a fusion polynucleotide of the present disclosure comprises a linker. In some embodiments, the linker is configured to link a polynucleotide of the present disclosure and a heterologous polypeptide of the present disclosure. In some cases, a fusion polynucleotide of the present disclosure comprises a linker between a heterologous polypeptide (such as a targeting moiety) and the polynucleotide, such as an siRNA, present in the fusion polynucleotide. In some cases, the linker is a proteolytically cleavable linker. In some cases, a polymer conjugate of the present disclosure comprises a linker between a heterologous polypeptide (such as a targeting moiety) and a polynucleotide disclosed herein, such as an siRNA, present in the fusion polynucleotide, and a linking group (as disclosed herein) between the polynucleotide (or heterologous polypeptide) and the compound disclosed herein. In some cases, the heterologous polypeptide (such as a targeting moiety disclosed herein) is conjugated to a 5' end or a 3' end of the polynucleotide.

[00216] In some cases, the linker is configured to covalently link a polynucleotide of the present disclosure and a polypeptide of the present disclosure. In some cases, the polynucleotide, polypeptide, or a combination thereof comprise at least one reactive moiety. In some cases, the polynucleotide and the polypeptide are covalently linked by an enzymatic reaction. For example, SNAP-tag, a modified form of DNA repair enzyme named human O6alkylguanine-DNA-alkyltransferase, can be used to selectively form a covalent bond with a polynucleotide comprising a benzylguanine (e.g., RNA-TAG can be used to label the polynucleotide with a modified substrate analog, such as benzylguanine), thus allowing the conjugation of a polynucleotide of the present disclosure and a polypeptide of the present disclosure. As an additional example, RNAylation, mediated by the T4 phage ADP-ribosyltransferase (ART) ModB, can be used to covalently link the polynucleotide to the polypeptide through an N-glycosidic bond. In general, ARTs, a class of enzymes ubiquitous across all kingdoms of life, attach an ADP-ribose moiety from the redox cofactor nicotinamide adenine dinucleotide (NAD+, is referred as NAD in the following) to a target protein, nucleic acid, or small molecule in a covalent manner. ARTs generally exhibit high substrate specificity for NAD, such as a NAD-capped-RNA substrate. In some embodiments, a polynucleotide if the present disclosure comprises a NAD-cap (e.g., a NAD at a 5' end of the polynucleotide).

[00217] In some cases, the linker is configured to noncovalently link a polynucleotide of the present disclosure and a polypeptide of the present disclosure. For example, a polypeptide of the present disclosure can comprise a targeting moiety and a nucleic acid (DNA, RNA, etc.) binding domain, wherein the nucleic acid binding domain is configured to bind a polynucleotide of the present disclosure (e.g., an siRNA, ASO, etc.). Thus, forming a noncovalent linkage between the polynucleotide and the polypeptide.

[00218] In some cases, a fusion polynucleotide of the present disclosure comprises a linker. In some embodiments, the linker is conjugated to the polynucleotide and the heterologous polypeptide to produce the fusion polynucleotide. In some embodiments, conjugating comprises chemically conjugating. Many chemical approaches have been developed to crosslink nucleotide and protein, all of which can be used to conjugate a polypeptide described herein, and a polynucleotide described herein. For example, oxime conjugation introduces an aminooxy group on the protein to react with oligosaccharide containing aldehyde or keto group (J. Kubler-Kielb, V. Pozsgay, J Org Chern 2005, 70, 6987-6990). For example, Michael addition often uses thiol group addition to maleimide to form a stable thioester linkage (T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura, Y. C. Lee, Biomacromolecules 2005, 6, 880-884). For example, the method of copper (I)-catalyzed cycloaddition of azide to alkyens (click chemistry) provides efficient glycoconjugation (a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew Chem IntEd Engl 2001, 40, 2004-2021; b) S. Hotha, S. Kashyap, J Org Chem 2006, 71, 364-367).

[00219] In some embodiments, chemically conjugating comprises a click chemistry reaction. Click chemistry comprises a group of biocompatible small molecule reactions commonly used in bioconjugation that are stereospecific, high yielding, wide in scope, and simple to perform. The primary click reaction utilizes copper-catalyzed coupling of a terminal alkyne with a terminal azide to exclusively form the 1,2,3-triazole unit. These reactions create only byproducts that can be removed without chromatography, and can be conducted in easily removable or benign solvents. Examples of the click chemistry reaction includes, but are not limited to, Huisgen azide-alkyne 1,3-dipolar cycloaddition, copper-catalyzed azide-alkyne cycloaddition, ruthenium-catalyzed azidealkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted alkyne-nitrone cycloaddition, and reactions of strained alkenes such as alkene and azide [3+2] cycloaddition, alkene and tetrazine inverse-demand Diels-Alder, and alkene and tetrazole photoclick reaction. In some embodiments, the click chemistry reaction comprises a copper-catalyzed azide-alkyne cycloaddition. The mechanism of the copper-catalyzed azide-alkyne cycloaddition is described in Himo, et al. J. Am. Chem. Soc. 2005. 127: 210-216. In some embodiments, the click chemistry reaction to form a fusion polynucleotide as disclosed herein occurs at ambient temperatures. In some embodiments, the click chemistry reaction to form a fusion polynucleotide as disclosed herein occurs in the presence of a metal catalyst, for example, a copper(I)-catalyzed azide-alkyne cycloaddition. In some embodiments, the click chemistry reaction is performed in the absence of copper.

[00220] In some embodiments, compositions disclosed herein comprise a polynucleotide (such as a polynucleotide as disclosed herein) and a heterologous polypeptide (e.g., to form a fusion polynucleotide). In some cases, a fusion polynucleotide of the present disclosure comprises a linker between a heterologous polypeptide (such as a targeting moiety) and the polynucleotide, such as an siRNA, present in the fusion polynucleotide. In some cases, the heterologous polypeptide (such as a targeting moiety disclosed herein) is conjugated to a 5' end or a 3' end of the polynucleotide. In some cases, the heterologous polypeptide comprises a targeting moiety (such as a targeting moiety as disclosed herein). In some cases, the composition further comprises a pH responsive domain. In some cases, the polynucleotide is conjugated to the targeting moiety or the pH responsive domain. In some cases, the polynucleotide is conjugated to an N-terminus or a C-terminus of the targeting moiety. In some cases, the polynucleotide is conjugated to an N-terminus or a C-terminus of the pH responsive domain. Nucleic acids, expression vectors, and modified host cells

[00221] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure. The present disclosure provides a nucleic acid comprising a polynucleotide 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 present disclosure provides a recombinant expression vector comprising a polynucleotide of the present disclosure. The nucleic acids and recombinant expression vectors are useful for producing a fusion polypeptide of the present disclosure, a fusion polynucleotide of the present disclosure, or any combination thereof. In some cases, the nucleotide sequence encoding the fusion polypeptide, fusion polynucleotide, or any combination thereof 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. “Operably linked” may refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence 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.

[00222] 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. The terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” may be used interchangeably herein, and can refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate expression of a coding sequence and / or production of an encoded polypeptide in a host cell.

[00223] 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.

[00224] 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.

[00225] 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 carried out using standard methods of protein purification.

[00226] 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.

[00227] 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. Methods of Modifying

[00228] The present disclosure provides methods of modifying a target nucleic acid and / or modifying a polypeptide associated with a target nucleic acid.

[00229] In some cases, the methods provided herein comprise editing a locus within a target cell, the method comprising introducing into the target cell one or more of the compositions disclosed herein. In some embodiments, the locus comprises two or more target nucleic acids (e.g., two or more target sequences, two or more target genes, two or more target mRNAs, etc.). In some embodiments, the target cell is a eukaryotic cell. 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. In some embodiments, the target cell is a tissue cell, such as cells from a kidney, brain, heart, or muscle.

[00230] In some embodiments, the target cell expresses a cell surface receptor. In some embodiments, the target cell expresses a cell-type specific receptor. In some embodiments, the target cell expresses a tissue-type specific receptor. In some embodiments, the targeting moiety disclosed herein binds the cell surface receptor. In some embodiments, the targeting moiety binds to an extracellular domain of the cell surface receptor. In some embodiments, the cell surface receptor is selected from the group consisting of: IGF1R, IGF2R, TfRl, Nephrin, Nephi, Megalin, cKIT, GLUT4, GLUT1, Neuropilin, FOL1, FOL3, CCR5, CXCR1-4, EGFR, VEGFR1, VEGFR2, PDGFRA, ADRB3, NTRK2, GRIA1, GRIN1, GABRA1, and GABBR1.

[00231] Provided herein, in some aspects, are methods of modifying a target nucleic acid (e.g., a target sequence, a target gene, etc.), the method comprising contacting the target nucleic acid with the one or more of the compositions disclosed herein. Also provided herein, in some aspects, are methods of modifying a target nucleic acid in a target cell, the method comprising introducing into the target cell one or more of the compositions disclosed herein. In some cases, a method of the present disclosure comprises contacting a eukaryotic cell comprising a target nucleic acid with a fusion polynucleotide of the present disclosure. 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.

[00232] For example, a CRISPR-Cas effector 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.).

[00233] 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.

[00234] A polypeptide of the present disclosure, a polynucleotide of the present disclosure, a fusion polynucleotide of the present disclosure, or a fusion polypeptide of the present disclosure (e.g., a CRISPR-Cas effector fusion polypeptide 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).

[00235] 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. A target nucleic acid sequence can be a coding RNA (e.g., an mRNA). Alternatively, a target nucleic acid sequence can be a non-coding RNA (e.g., a tRNA, an rRNA, an miRNA, a noncoding RNA, a small noncoding RNA, a long noncoding RNA, an siRNA, or a piwi-interacting RNA). Target nucleic acid sequences can be synthetic nucleic acid sequences. Alternatively, target nucleic acid sequence can be native to a cell or organism. In some embodiments, the target nucleic acid sequence is associated with a disease or a condition, such as a disease or a condition disclosed herein. For example, modulation of transcription of the target nucleic acid sequence (e.g., in the case of a DNA target) may be therapeutically effective to treat the disease or the condition. In another example, modulation of expression of the target nucleic acid sequence (e.g., in the case of an RNA target) may be therapeutically effective to treat the disease or the condition. In some embodiments, the modulation of the expression of the target nucleic acid sequence comprises post-transcriptional modifications, such as capping, splicing and polyadenylation of the RNA target.

[00236] In some embodiments, the target nucleic acid sequence comprises at least a portion of a target gene. In some embodiments, the target gene encodes a DNA repair protein (e.g., to inhibit unwanted microhomology-mediated end joining and / or nonhomologous end-joining and prevent proteins from functioning, or to recruit DNA repair enzymes to increase homology-directed repair), DNA methylation protein, histone methylation protein, histone demethylation protein, histone acetyltransferase (HAT) protein, histone deacetylation (HDAC) protein, histone crotonylation protein (e.g., an activating protein), histone phosphorylation protein, histone ubiquitination protein, or a receptor protein. In some embodiments, the target gene is selected from the group consisting of: 53BPl,LigIV, DNA-PK, CYREN, POLQ, Ku70, Ku80, XRCC4,RAD51, MRE11, RAD50, NBS1, CtIP (RBBP8), EXO1, DNA2, Dnmtl, UHRF1, UHFR2, Dnmt3a, Dnmt3b, Dnmt3L, MMSET, EZH2, SMYD3, SETDB1, NSD1, NSD3, SUV39H1 / 2, G9a / EHMT2, GLP, DOT1L, PRMT1-8, PRMT5, JHDM1 / KDM2, LSD1 / KDM1 A, LSD2, KDM5B, HBO1, BRPF and JADE (e.g, subunits that determine which histone tails HBO1 acetylates), p300, CBP, GCN5, PCAF, SIRT1-7, HDAC1-11, Tafl4, ATM Kinase, HASPIN, Rad6, Brel, PRC1 / BMH, IGF1R, IGF2R, TfRl, Nephrin, Nephi, Megalin, cKIT, GLUT4, GLUT1, Neuropilin, FOL1, FOL3, CCR5, CXCR1-4, EGFR, VEGFR1, VEGFR2, PDGFRA, ADRB3, NTRK2, GRIA1, GRIN1, GABRA1, and GABBR1.

[00237] Modulation of a target nucleic acid can result in increased expression of a target gene, e.g., expression of a protein encoded by the tar...

Claims

1. A composition comprising a fusion polypeptide comprising:(a) an effector polypeptide; and(b) a targeting moiety that specifically binds to an extracellular domain of a receptor expressed by a target cell, wherein the targeting moiety is not an antibody or an antigen binding domain of an antibody.

2. The composition of claim 1, wherein the fusion polypeptide further comprises a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain is capable of binding to an endosomal escape peptide at pH 7.4, and wherein when the fusion polypeptide is present at a pH lower than 7.0, the pH responsive polypeptide domain contains one or more amino acids that are protonated.

3. The composition of claim 1 or 2, wherein the fusion polypeptide further comprises an endosomal escape peptide, wherein the endosomal escape peptide is capable of disrupting a membrane of a membrane-bound organelle.

4. A composition comprising a fusion polypeptide comprising:(a) an effector polypeptide; and(b) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 31-151 or 350-143854.

5. The composition of claim 4, wherein the fusion polypeptide further comprises a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain is capable of binding to an endosomal escape peptide at pH 7.4, and wherein when the fusion polypeptide is present at a pH lower than 7.0, the pH responsive polypeptide domain contains one or more amino acids that are protonated.

6. The composition of claim 4 or 5, wherein the fusion polypeptide further comprises an endosomal escape peptide, wherein the endosomal escape peptide is capable of disrupting a membrane of a membrane-bound organelle.

7. A composition comprising a fusion polypeptide comprising:(a) an effector polypeptide; and(b) a pH responsive polypeptide domain, wherein the pH responsive polypeptide domain is capable of binding to an endosomal escape peptide at pH 7.4, and wherein when the fusionpolypeptide is present at a pH lower than 7.0, the pH responsive polypeptide domain contains one or more amino acids that are protonated.

8. A composition comprising a fusion polypeptide comprising:(a) an effector polypeptide; and(b) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 336-349.

9. The composition of claim 7 or 8, wherein the fusion polypeptide further comprises a targeting moiety that binds specifically to an extracellular domain of a receptor expressed by a target cell.

10. The composition of claim 9, wherein the targeting moiety is not an antibody or an antigen binding domain of an antibody.

11. A composition comprising a fusion polypeptide comprising:(a) an effector polypeptide; and(b) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 152-335.

12. A composition comprising a protein complex, the protein complex comprising:(a) the fusion polypeptide of the composition of claim 7 or 8, and(b) an endosomal escape peptide, wherein the endosomal escape peptide is bound to the fusion polypeptide, and wherein the endosomal escape peptide is capable of disrupting a membrane of a membrane-bound organelle.

13. The composition of claim 11 or 12, wherein the fusion polypeptide further comprises a targeting moiety that binds specifically to an extracellular domain of a receptor expressed by a target cell.

14. The composition of claim 13, wherein the endosomal escape peptide is bound to a pH responsive polypeptide domain.

15. The composition of any one of claims 1-3, 9-10 and 13-14, wherein the targeting moiety binds to an extracellular domain of transferrin receptor protein 1 (TfRl), IGF1R, IGF2R, Dnmt3a, orNephrin.

16. The composition of any one of claims 1-3, 9-10 and 13-15, wherein the targeting moiety comprises a polypeptide sequence with less than 150 amino acids.

17. The composition of any one of claims 1-3, 9-10 and 13-16, wherein the targeting moiety comprises a polypeptide sequence with less than 100 amino acids.

18. The composition of claim 16 or 17, wherein the targeting moiety comprises a polypeptide sequence with more than 50 amino acids.

19. The composition of claim 18, wherein the targeting moiety comprises a polypeptide sequence with more than 60 amino acids.

20. The composition of claim 18, wherein the targeting moiety comprises a polypeptide sequence with more than 70 amino acids.

21. The composition of claim 18, wherein the targeting moiety comprises a polypeptide sequence with 80 amino acids or more.

22. The composition of any one of claims 1-3, 9-10 and 13-21, wherein the targeting moiety comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence any one of SEQ ID NOs: 31-151 or 350-143854.

23. The composition of any one of claims 2, 3, 5-7, and 14-22, wherein the pH responsive polypeptide domain contains one or more amino acids that are protonated at a pH of 6.8 or less, and wherein the one or more amino acids that are protonated at a pH of 6.8 or less are not protonated at a pH of 7.4.

24. The composition of any one of claims 2, 3, 5-7, and 14-22, wherein the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 6.6 or less, and wherein the one or more amino acids that are protonated at a pH of 6.6 or less are not protonated at a pH of 7.4.

25. The composition of any one of claims 2, 3, 5-7, and 14-22, wherein the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 6 or less, and wherein the one or more amino acids that are protonated at a pH of 6 or less are not protonated at a pH of 7.4.

26. The composition of any one of claims 2, 3, 5-7, and 14-22, wherein the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 5.5 or less, and wherein the one or more amino acids that are protonated at a pH of 5.5 or less are not protonated at a pH of 7.4.

27. The composition of any one of claims 2, 3, 5-7, and 14-22, wherein the pH responsive polypeptide domain contains one or more amino acids are protonated at a pH of 5 or less, and wherein the one or more amino acids that are protonated at a pH of 5 or less are not protonated at a pH of 7.4.

28. The composition of any one of claims 2, 3, 5-7, and 14-27, wherein the pH responsive polypeptide domain contains one or more amino acids that are protonated when the fusion polypeptide is present within an endosome.

29. The composition of any one of claims 2, 3, 5-7, and 14-28, wherein the pH responsive polypeptide domain contains one or more amino acids that are protonated when the fusion polypeptide is present within a lysosome.

30. The composition of any one of claims 2, 3, 5-7, and 14-29, wherein the pH responsive polypeptide domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 336-349.

31. The composition of any one of claims 2, 3,5-7, and 14-30, wherein ata pH of 6.8 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

32. The composition of any one of claims2, 3, 5-7, and 14-30, wherein ata pH of 6.6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

33. The composition of any one of claims 2, 3, 5-7, and 14-30, wherein at a pH of 6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

34. The composition of any one of claims 2, 3,5-7, and 14-30, wherein ata pH of 5.5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

35. The composition of any one of claims 2, 3, 5-7, and 14-30, wherein at a pH of 5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

36. The composition of any one of claims2, 3, 5-7, and 14-30, wherein ata pH of 6.8 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least2,3,4, 5,6, 7,8,9, 10, 15,20,25,30,40,50,60,70,80,90, 100, 200, 500 or 1000 foldless than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

37. The composition of any oneof claims2, 3, 5-7, and 14-30, wherein ata pH of 6.6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least2,3,4, 5,6, 7,8,9, 10, 15,20,25,30, 40,50, 60,70,80,90, 100, 200, 500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

38. The composition of any oneof claims 2, 3, 5-7, and 14-30, wherein at a pH of 6 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least2,3,4, 5,6,7,8,9, 10, 15,20,25,30, 40,50, 60,70,80,90, 100, 200, 500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

39. The composition of any oneof claims 2, 3,5-7, and 14-30, wherein ata pH of 5.5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least2,3,4, 5,6,7,8,9, 10, 15,20,25,30, 40,50, 60,70,80,90, 100, 200, 500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

40. The composition of any oneof claims 2, 3, 5-7, and 14-30, wherein at a pH of 5 or less the endosomal escape peptide has an affinity to the pH responsive polypeptide domain that is at least2,3,4, 5,6, 7,8,9, 10, 15,20,25,30, 40,50, 60,70,80,90, 100, 200, 500 or 1000 fold less than an affinity of the endosomal escape peptide to the pH responsive polypeptide domain at a pH of 7.4.

41. The composition of any oneof claims 3, 6, and 12-40, wherein the endosomal escape peptide is released from the protein complex when the protein complex is present within an endosome.

42. The composition of any oneof claims 3, 6, and 12-41, wherein the endosomal escape peptide is released from the protein complex when the protein complexis present within a lysosome.

43. The composition of any oneof claims 3, 6, and 12-42, wherein the endosomal escape peptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 152-335.

44. The composition of any one of claims 2, 3, 5-7, and 14-43, wherein the pH responsive polypeptide domain is capable of binding to two or more endosomal escape peptides at pH 7.4.

45. The composition of any one of claims 12-44, wherein the protein complex comprises two or more copies of the endosomal escape peptide.

46. The composition of any one of claims 1-45, wherein the fusion polypeptide further comprises a linker linking the effector polypeptide and one or more other peptide sequences of the fusion polypeptide.

47. The composition of claim 46, wherein the linker is a proteolytically cleavable linker.

48. The composition of any one of claims 1-47, wherein the fusion polypeptide further comprises one or more nuclear localization sequences (NLSs).

49. The composition of claim 48, wherein the one or more NLSs comprise the amino acid sequence K(K / R)X(K / R), and where X is any amino acid.

50. The composition of claim 48, wherein the one or more NLSs comprise the amino acid sequence PKKKRKV.

51. The composition of any one of claims 1-50, wherein the one or more NLSs are at the N-terminus of the effector polypeptide.

52. The composition of any one of claims 1-51, wherein the effector polypeptide is a CRISPR-Cas effector polypeptide.

53. The composition of any one of claims 1-51, wherein the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide, a Type V CRISPR-Cas effector polypeptide, or a Type VI CRISPR-Cas effector polypeptide.

54. The composition of any one of claims 1-51, wherein the CRISPR-Cas effector polypeptide is a SpyCas9 or a variant thereof.5 5. The composition of any one of claims 1-51, wherein the CRISPR-Cas effector polypeptide is GeoCas9 or a variant thereof.

56. The composition of any one of claims 1-55, wherein the CRISPR-Cas effector polypeptide is catalytically active.

57. The composition of any one of claims 1-56, wherein the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide.

58. The composition of any one of claims 1-55, wherein the CRISPR-Cas effector polypeptide is catalytically inactive.

59. The composition of any one of claims 1-58, wherein the fusion polypeptide further comprises at least one additional heterologous polypeptide.

60. The composition of claim 59, wherein the at least one additional heterologous polypeptide is a deaminase, a base editor, a reverse transcriptase, a transcription modulator, or an epigenetic modulator.

61. A composition comprising:(a) a fusion polypeptide of the composition of any one of claims 1-60, or a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide and / or endosomal escape polypeptide; and(b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.

62. The composition of claim 61, wherein the guide nucleic acid is a single-molecule guide nucleic acid.

63. The composition of claim 61, further comprising a donor nucleic acid.

64. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of the composition of any one of claims 1-60.

65. The nucleic acid of claim 64, wherein the nucleotide sequence is operably linked to a transcriptional control element, optionally wherein the transcriptional control element is a promoter.

66. A recombinant expression vector comprising the nucleic acid of claim 65.

67. A cell comprising the composition of any one of claims 1-63 or the nucleic acid of claim 64 or 65, or the recombinant expression vector of claim 66.

68. The cell of claim 67, wherein the cell is a eukaryotic cell.

69. The cell of claim 67 or 68, wherein the cell is in vitro.

70. The cell of claim 67 or 68, wherein the cell is in vivo.

71. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with:(a) the composition of any one of claims 1-63; and(b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.

72. A method of modifying a target nucleic acid in a eukaryotic cell, the method comprising introducing into the eukaryotic cell:(a) the composition of any one of claims 1-63; and(b) a guide nucleic acid, or a nucleic acid comprising a nucleotide sequence encoding the guide nucleic acid.

73. The method of claim 72, further comprising introducing into the cell a donor nucleic acid.

74. The method of claim 72 or 73, wherein the cell is in vitro.

75. The method of claim 72 or 73, wherein the cell is in vivo.

76. The method of any one of claims 72-75, 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.

77. The method of any one of claims 72-76, wherein the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.

78. The method of any one of claims 72-77, wherein the modifying comprises genome editing.

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