WO2024124238A1 - Endonucléases de modification génique - Google Patents

Endonucléases de modification génique Download PDF

Info

Publication number
WO2024124238A1
WO2024124238A1 PCT/US2023/083377 US2023083377W WO2024124238A1 WO 2024124238 A1 WO2024124238 A1 WO 2024124238A1 US 2023083377 W US2023083377 W US 2023083377W WO 2024124238 A1 WO2024124238 A1 WO 2024124238A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
nucleic acid
endonuclease
seq
cell
Prior art date
Application number
PCT/US2023/083377
Other languages
English (en)
Inventor
Basem AL-SHAYEB
Jacob BORRAJO
Mohammad Kamyab JAVANMARDI
Kushagra Sharma
Original Assignee
Amber Bio Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amber Bio Inc. filed Critical Amber Bio Inc.
Publication of WO2024124238A1 publication Critical patent/WO2024124238A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/22—Endodeoxyribonucleases producing 3'-phosphomonoesters (3.1.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)

Definitions

  • the disclosure relates to compositions, systems, and methods that modify target RNA, as well as methods of detecting a nucleic acid.
  • the instant application contains a sequence listing, which has been submitted in XML format via EFS-Web.
  • Bacterial adaptive immune systems employ CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage.
  • CRISPRs clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated proteins
  • the CRISPR-Cas systems thereby confer adaptive immunity in bacteria and archaea via RNA-guided nucleic acid interference.
  • processed CRISPR array transcripts assemble with Cas protein-containing surveillance complexes that recognize nucleic acids bearing sequence complementarity to the virus derived segment of the crRNAs, known as the spacer.
  • CRISPR-Cas tools have been widely used for gene editing, gene activation, gene inactivation, protein imaging, and beyond.
  • the RNA-guided endonucleases of the CRISPR-Cas9 system including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), can be used as a gene-editing tool in certain organisms.
  • SpCas9 polypeptides are capable of high-efficiency gene modifications, limitations remain due to off-target activities, such as the undesirable production of modifications within the genome at sites other than the desired target.
  • current endonuclease may be restricted in use due to protospacer adjacent motif (PAM) specificities and packaging constraints for delivery of system components.
  • PAM protospacer adjacent motif
  • composition comprising an endonuclease comprising a sequence, optionally comprising a higher eukaryotes and prokaryotes nucleotide- binding domain (HEPN) domain, or a fragment or variant thereof, having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity to SEQ ID NO: 1, or having about 1 to about 20 amino acid modifications e.g.
  • the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • compositions comprising an endonuclease comprising a sequence, optionally comprising one or more higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) domains, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity to SEQ ID NO: 1 , or having about 1 to about 20 amino acid modifications (e.g.
  • the sequence comprises a fragment or variant of a HEPN domain. In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the present disclosure provides a composition comprising a nucleic acid encoding an endonuclease comprising a sequence, optionally comprising one or more HEPN domains, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%%, or at least about 97%, or at least about 98%, or at least about 99%) identity to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g.
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof. In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the present disclosure provides a composition comprising a nuclease system, comprising (a) an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g.
  • RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule.
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof.
  • the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the composition further comprises one or more donor polynucleotides and/or is suitable for introducing one or more donor polynucleotides into a target nucleic acid molecule.
  • the endonuclease is suitable for introducing one or more excisions into a target nucleic acid molecule.
  • the present disclosure provides a composition comprising a chimeric protein comprising: an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to o SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g.
  • nucleic acid-modulating domain or a nucleic acid-modifying domain, or nucleic acid- interacting/binding domain comprising a sequence comprising a catalytic domain, or a fragment or variant thereof, wherein (a) and (b) do not naturally occur together in a same reading frame.
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof.
  • the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the nucleic acid-modulating domain or a nucleic acid-modifying domain is a nucleic acid-interacting domain, e.g. selected from MCP, lambdaN, PP7, QBeta, SLBP, and TBP/TAR.
  • the endonuclease reduces or enhances collateral activity for nucleic acid detection.
  • the present disclosure provides a composition comprising a complex comprising chimeric protein and an RNA molecule, wherein the chimeric protein comprises an endonuclease comprising a sequence, optionally comprising one or more HEPN domains, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g.
  • nucleic acid-modulating domain or a nucleic acid-modifying domain comprising a sequence comprising a catalytic domain, or a fragment or variant thereof, wherein (a) and (b) do not naturally occur together in a same reading frame and the RNA molecule comprises a sequence complementary to one strand of a target nucleic acid molecule.
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof. In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the nucleic acid-modulating domain or the nucleic acidmodifying domain has one or more of 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, debranching activity, transesterification activity, photolyase activity and glycosylase activity.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain is selected from a deaminase, reverse transcriptase, transposase, integrase, and recombinase.
  • the deaminase is a cytidine or cytosine deaminase, or a fragment or variant thereof.
  • the cytidine or cytosine deaminase is selected from activation-induced cytidine deaminase (AID), cytidine deaminase 1 (CDA1), and apolipoprotein B mRNA-editing complex (APOBEC), or a fragment or variant thereof.
  • AID activation-induced cytidine deaminase
  • CDA1 cytidine deaminase 1
  • APOBEC apolipoprotein B mRNA-editing complex
  • the APOBEC is selected from A3 A, AB3, APOBEC1, APOBEC3C, AP0BEC3D, APOBEC3F, APOBEC3G, and APOBEC3H, or a fragment or variant thereof.
  • the APOBEC has an amino acid sequence of one of SEQ ID NO: 39 [A3A], SEQ ID NO: 40 [AB3], SEQ ID NO: 41 [APOBEC1], SEQ ID NO: 42 [APOBEC3C], SEQ ID NO: 43 [APOBEC3D], SEQ ID NO: 44 [APOBEC3F], SEQ ID NO: 45 [APOBEC3G], and SEQ ID NO: 46 [APOBEC3H], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the deaminase is a DNA-specific adenine or adenosine deaminase, or a fragment or variant thereof.
  • the DNA-specific adenine or adenosine deaminase is selected from tRNA-specific adenosine deaminase 7.10 (TadA 7.10), tRNA-specific adenosine deaminase 6.3 (TadA 6.3), tRNA-specific adenosine deaminase 7.8 (TadA 7.8), tRNA-specific adenosine deaminase 7.9 (TadA 7.9), and tRNA-specific adenosine deaminase 8e (TadA8e (TadA-8e V106W)) or a fragment or variant thereof.
  • the TadA has an amino acid sequence of one of SEQ ID NO: 48 [TadA 7.10], SEQ ID NO: 49 [TadA 6.3], SEQ ID NO: 50 [TadA 7.8], SEQ ID NO: 51 [TadA 7.9], and SEQ ID NO: 52 [TadA 8e], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the deaminase is a RNA-specific adenine or adenosine deaminase, or a fragment or variant thereof.
  • the RNA-specific adenine or adenosine deaminase is an adenosine deaminases acting on RNA (ADAR) enzyme, or a fragment or variant thereof.
  • the ADAR is selected from AD ARI, ADAR2, and ADAR3, or a fragment or variant thereof.
  • the ADAR has an amino acid sequence of one of SEQ ID NO: 53 [ADARl] and SEQ ID NO: 54 [ADAR2] or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the catalytic deaminase domain of ADARl comprises amino acids 833-1226 of SEQ ID NO: 53.
  • the catalytic deaminase domain of ADAR2 comprises amino acids 299-701 of SEQ ID NO: 54.
  • the deaminase further comprises a nuclear localization signal.
  • the endonuclease further comprises a uracil glycosylase inhibitor (UGI), or a fragment or variant thereof.
  • the RNA molecule is a guide RNA (gRNA).
  • the gRNA comprises a sequence that interacts with the endonuclease.
  • the endonuclease forms a complex with the gRNA.
  • the composition is suitable for base editing.
  • the composition is suitable for DNA base editing.
  • the composition is suitable for RNA base editing.
  • the composition is suitable for catalyzing OT nucleotide conversions or A>G nucleotide conversions in a target nucleic acid.
  • the composition comprises both an adenosine deaminase and a cytidine deaminase.
  • the composition is suitable for dual base editing.
  • the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase (M-MLV RT) or M-MLV RT(D200N/L603W/T330P/T306K/W313F), or a fragment or variant thereof.
  • M-MLV RT Moloney murine leukemia virus reverse transcriptase
  • M-MLV RT M-MLV RT(D200N/L603W/T330P/T306K/W313F)
  • the M-MLV RT has an amino acid sequence of SEQ ID NO: 55 [M- MLV RT] or SEQ ID NO 56 [M-MLV RT(D200N/L603W/T330P/T306K/W313F)] or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the composition further comprises a dominant negative human MutL homolog (MLH1). In embodiments, the composition is suitable for use with a dominant negative MEHL
  • the RNA molecule is or comprises a prime editing guide RNA (pegRNA).
  • the endonuclease forms a complex with the pegRNA.
  • the pegRNA serves as a template for transcription of a new DNA sequence.
  • the pegRNA binds to a DNA strand opposite from a typical gRNA binding site.
  • the pegRNA comprises a gRNA containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence.
  • the RNA molecule is or comprises a gRNA.
  • the gRNA comprises a sequence that interacts with the endonuclease.
  • the endonuclease forms a complex with the gRNA.
  • the composition comprises both a gRNA and a pegRNA.
  • the composition is suitable for prime editing.
  • the transposase is selected from Tnl, Tn2, Tn3, Tn5, Tn7, Tn9, TnlO, Tn552, Tn903, TnlOOO/Gamma-delta, Tn/O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping beauty, piggyBac, Tol2, Most, Himarl, Hermes, Tol2, Minos, Tel, P-element, MuA, Tyl , Chapaev, transib, Tcl/mariner, and Tc3 donor DNA system.
  • the transposase is a transposon 7-like (Tn7-like) transposon system, or a fragment or variant thereof.
  • the transposase is one or more of transposon 7 protein A (TnsA), transposon 7 protein B (Tns B), transposon 7 protein C (Tns C), and transposition of integron protein Q (TniQ), or a fragment or variant thereof.
  • the Tn7-like transposon system is derived from Vibrio cholerae Tn6677.
  • the transposase has an amino acid sequence of one or more of SEQ ID NO: 57 [TnsA], SEQ ID NO: 58 [TnsB], SEQ ID NO: 59 [TnsC], and SEQ ID NO: 60 [TniQ], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the integrase is a serine-recombinase, or a fragment or variant thereof.
  • the serine-recombinase is Bxbl, or a fragment or variant thereof.
  • the recombinase is a Gin invertase or Tn3 resolvase, or a fragment or variant thereof.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain comprises one or more modifications, (e.g., without limitation, mutations) to reduce activity relative to an unmutated form.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain comprises one or more modifications, (e.g., without limitation, mutations) to increase activity relative to an unmutated form.
  • sequence of (a) is disposed at the N-terminus of the chimeric protein and the sequence of (b) is disposed at the C-terminus of the chimeric protein.
  • sequence of (a) is disposed at the C-terminus of the chimeric protein and the sequence of (b) is disposed at the N-terminus of the chimeric protein.
  • the composition further comprises a linker that joins the sequence of (a) and the sequence of (b).
  • the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids.
  • the linker is substantially comprised of glycine and serine residues.
  • the linker is (GGS)n, wherein nis 1, or 2, or 3, or 4, or 5.
  • the endonuclease is suitable for creating a double stranded break in a nucleic acid. In embodiments, the endonuclease is suitable for creating a nick in a nucleic acid. In embodiments, the endonuclease is suitable for nucleic acid modification by homology-directed repair (HDR). In embodiments, the endonuclease is suitable for nucleic acid modification by non- homologous end joining (NHEJ). In embodiments, the endonuclease recognizes a PAM. In embodiments, the endonuclease recognizes a plurality of PAMs.
  • HDR homology-directed repair
  • NHEJ non- homologous end joining
  • the endonuclease comprises one or more modifications, (e.g., without limitation, mutations) to reduce catalytic activity relative to an unmutated form.
  • the endonuclease comprises one or more modifications, (c.g., without limitation, mutations) to render the endonuclease substantially catalytically inactive relative to an unmutated form.
  • the endonuclease comprises one or more modifications, (e.g., without limitation, modifications, (e.g., without limitation, mutations) to increase catalytic activity relative to an unmutated form.
  • the endonuclease comprises one or more modifications, (e.g., without limitation, mutations) to render the endonuclease substantially catalytically hyperactive relative to an unmutated form.
  • the endonuclease has nickase activity.
  • the endonuclease comprises one or more modifications, (e.g., without limitation, mutations) to produce nickase activity.
  • the endonuclease has collateral cleavage activity.
  • the endonuclease comprises one or more modifications, (e.g., without limitation, mutations) to produce collateral cleavage activity.
  • the endonuclease has at least about 75% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 80% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 85% identity to SEQ ID NO: 1
  • the endonuclease has at least about 90% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 95% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 97% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 99% identity to SEQ ID NO: 1.
  • the endonuclease has about 1 to about 15 amino acid modifications. In embodiments, the endonuclease has about 1 to about 10 amino acid modifications. In embodiments, the endonuclease has about 1 to about 5 amino acid modifications. In embodiments, the endonuclease has about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20 amino acid modifications. In embodiments, the amino acid modifications are selected from substitutions and deletions.
  • the endonuclease (or chimeric protein) comprises a domain from a different endonuclease.
  • the different endonuclease is a Cas endonuclease.
  • the domain is a PAM-interacting domain.
  • the target nucleic acid is or comprises single-stranded RNA (ssRNA).
  • the target nucleic acid is or comprises doublestranded RNA (dsRNA).
  • dsRNA doublestranded RNA
  • the target nucleic acid is or comprises single- stranded DNA (ssDNA).
  • the target nucleic acid is or comprises double-stranded DNA (dsDNA).
  • the target nucleic acid is about 2 to about 6 nucleotides upstream of a PAM sequence.
  • the RNA molecule is or comprises a guide ribonucleic structure configured to form a complex with the endonuclease.
  • the guide ribonucleic structure comprises (a) a CRISPR RNA (crRNA) suitable for hybridizing to a target nucleic acid molecule and/or (b) a transactivating CRISPR RNA (tracrRNA) suitable for interacting with the endonuclease or (ii) lacks a (a) a crRNA suitable for hybridizing to a target nucleic acid molecule and/or (b) a tracrRNA suitable for interacting with the endonuclease.
  • the RNA molecule is or comprises a gRNA.
  • the gRNA comprises a sequence that interacts with the endonuclease.
  • the endonuclease forms a complex with the gRNA.
  • the RNA molecule is or comprises the nucleic acid sequence of SEQ ID NO: SEQ ID NO: 28, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the RNA molecule has perfect sequence complementarity to one strand of a target nucleic acid molecule. In embodiments, the RNA molecule has partial sequence complementarity to one strand of a target nucleic acid molecule.
  • the composition further comprises a viral vector.
  • the viral vector is or comprises an AAV.
  • the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2/1, AAV2/5, AAV2/8, AAV2/9, AAV3/1, AAV3/5, AAV3/8, and AAV3/9.
  • the composition further comprises a non-viral vector.
  • the composition further comprises a lipid nanoparticle (LNP) liposomes, lipoplexes or polymeric nanoparticle.
  • LNP lipid nanoparticle
  • the LNP comprises one or more of ionizable lipids, amino lipids, anionic lipids, neutral lipids, amphipathic lipids, helper lipids, structural lipids, PEG lipids, and lipoids.
  • the composition further comprises a virusdike particle (VLP).
  • VLP virusdike particle
  • the present disclosure provides a nucleic acid encoding the chimeric protein of any one of the embodiments and/or aspects disclosed herein.
  • the nucleic acid is or comprises a DNA molecule or an RNA molecule.
  • the RNA is or comprises mRNA or modified mRNA (mmRNA).
  • the DNA is or comprises a vector or plasmid.
  • the nucleic acid comprises a codon optimized sequence.
  • the nucleic acid comprises one or more modifications. In embodiments, the modifications are one or more of base modifications and backbone modifications.
  • the present disclosure provides a viral vector comprising the nucleic acid of any one of the embodiments and/or aspects disclosed herein.
  • the viral vector is or comprises an AAV.
  • the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2/1, AAV2/5, AAV2/8, AAV2/9, AAV3/1, AAV3/5, AAV3/8, and AAV3/9.
  • the present disclosure provides a viral vector comprising the nucleic acid of any one of the embodiments and/or aspects disclosed herein.
  • the viral vector is or comprises a VLP.
  • the endonuclease mediates a trans-splicing event.
  • the endonuclease mediates an exon skipping or exon inclusion event.
  • the present disclosure provides a lipid nanoparticle comprising the nucleic acid of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a cell comprising a nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, or the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein.
  • the cell is a prokaryotic cell. In embodiments, the cell is a eukaryotic cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is a human cell. In embodiments, the cell is an immortalized cell. In embodiments, the cell is harvested from a subject.
  • the present disclosure provides a pharmaceutical composition comprising the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, or the cell of any one of the embodiments and/or aspects disclosed herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a composition comprising an RNA molecule comprising a nucleic acid sequence of SEQ ID NO: 28, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the RNA molecule interacts with an endonuclease comprising a sequence comprising, optionally a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97% to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g.
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof. In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the RNA molecule comprises one or more modifications.
  • the modifications are one or more of base modifications and backbone modifications.
  • the RNA molecule comprises a sequence complementary to one strand of a target nucleic acid molecule.
  • the RNA molecule has perfect sequence complementarity to one strand of a target nucleic acid molecule.
  • the RNA molecule has partial sequence complementarity to one strand of a target nucleic acid molecule.
  • the present disclosure provides a composition comprising a nucleic acid encoding an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, in conjunction with an RNA containing a repeat having at least about 70% identity to SEQ ID NO: 28.
  • the composition has least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%%, or at least about 97%, or at least about 98%, or at least about 99%) identity to SEQ ID NO: 28, or has about 1 to about 20 nucleotide modifications e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof.
  • the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the present disclosure provides a kit comprising a container comprising the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein and with instructions for use in modulating and/or modifying a nucleic acid.
  • the present disclosure provides a method of modulating and/or modifying a nucleic acid in a cell, comprising contacting the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a method of modulating and/or modifying a nucleic acid in a subject in need thereof, comprising administering an effective amount of the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein to the subject.
  • the modulating and/or modifying is selected from one or more of cleaving, nicking, methylating, labeling, and mutating the nucleic acid. In embodiments, the modulating and/or modifying is selected from one or more of cleaving the nucleic acid; inserting a nucleic acid, editing the nucleic acid; modulating transcription from the nucleic acid; isolating the nucleic acid, binding the nucleic acid, and imaging the nucleic acid.
  • the present disclosure provides a method of disrupting, correcting, and/or replacing a gene in a cell, comprising contacting the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a method of disrupting, correcting, and/or replacing a gene in a subject in need thereof, comprising administering an effective amount of the composition of any one of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein to the subject.
  • the present disclosure provides a method of treating, ameliorating or preventing a disease or disorder in a subject, comprising (a) contacting a cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein, and (b) administering an effective amount of the cell to the subject.
  • the present disclosure provides a method of treating, ameliorating or preventing a disease or disorder in a subject, comprising administering an effective amount of the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein to the subj ect.
  • the present disclosure provides use of the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein in the manufacture of a medicament for the treating, ameliorating or preventing of a disease or disorder.
  • the present disclosure provides a method of detecting and/or quantifying a nucleic acid in a sample, comprising contacting the sample with a composition of any one of the embodiments and/or aspects disclosed herein.
  • the nucleic acid is a target and/or reporter nucleic acid.
  • the method comprises detection of a reporter signal, the reporter signal being generated upon endonuclease cleavage.
  • the reporter signal is a fluorescent signal.
  • the endonuclease has collateral cleavage activity.
  • the composition disclosed herein, or a system for targeting a nucleic acid for trans-splicing system disclosed herein comprises a repair RNA (repRNA) sequence, comprising: (a) one or more exons and/or introns; (b) a splice donor and/or splice acceptor, wherein the repRNA is suitable for trans-splicing.
  • the trans-splicing system comprises a splice donor, a splice acceptor, and replaces an internal exon.
  • the repRNA is operably linked to the RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule or the gRNA.
  • the present disclosure provides a system for targeting a nucleic acid for trans- splicing, the system comprising: (a) an endonuclease of any one of the embodiments disclosed herein, and optionally an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; (b) an RNA-binding polypeptide that associates with the endonuclease; and (c) a repair RNA (repRNA) sequence, comprising: (i) one or more exons and/or introns; (ii) a splice donor and/or splice acceptor.
  • repRNA repair RNA
  • the RNA molecule is a gRNA.
  • the endonuclease is not linked, associated, and/or fused with an RNA binding protein.
  • the repRNA is not operably linked to one or more gRNAs. In embodiments, the repRNA is provided in trans to one or more gRNAs.
  • the repRNA further comprises a ribozyme site.
  • the ribozyme site is a hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof.
  • the ribozyme site is a HDV ribozyme site.
  • the ribozyme site is upstream of the one or more exons and/or introns of the repRNA.
  • the present disclosure provides a system for targeting a nucleic acid for trans-splicing, the system comprising: (a) an endonuclease of any one of claims 1-108 and an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; and (b) a repair RNA (repRNA) sequence, comprising: (i) one or more exons and/or introns; (ii) a splice donor and/or splice acceptor.
  • repRNA repair RNA
  • the RNA molecule is a gRNA.
  • the endonuclease is not linked, associated, and/or fused with an RNA binding protein.
  • the repRNA is operably linked to one or more gRNAs.
  • the composition comprises a gRNA, repRNA, and a Cas endonuclease operably linked to a single promoter or a bidirectional promoter.
  • the gRNA and repRNA are located on a first side of the bidirectional promoter, and the Cas endonuclease is located on a second side of the bidirectional promoter.
  • FIG. 1 is an image showing the amino acid sequence of the Casl3K2G system for SEQ ID NO: 1 (Casl3K2Gl).
  • the red or black arrows in FIG. 1 indicate the placement of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain in the Casl3K2Gl (SEQ ID NO: 1) protein.
  • HEPN prokaryotes nucleotide-binding
  • FIG. 2 is an image showing the design of guide RNAs (gRNAs) to target multiple sites across the coding sequence of eGFP in HEK293T cells.
  • gRNAs guide RNAs
  • FIG. 3 is an image showing the gRNA structure for the Casl3K2G system (SEQ ID NO: 2).
  • FIG. 4 is an image showing a maximum likelihood phylogenetic tree of for SEQ ID NO: 1 (Casl3_K2Gl), SEQ ID NO: 3 (13d_WP_005358205), SEQ ID NO: 4 (13d_WP_009985792), SEQ ID NO: 5 (13d_WP_041337480), SEQ ID NO: 6 (13d_WP_074833651), SEQ ID NO: 7 (13d_WP_075424065), SEQ ID NO: 8 (Casl3btl), SEQ ID NO: 9 (Casl3bt2), SEQ ID NO: 10 (Casl3bt8), SEQ ID NO: 11 (Casl3X.2), SEQ ID NO: 12 (Casl3bt9), SEQ ID NO: 13
  • the present disclosure provides, inter alia, compositions and methods related to new families of CRISPR-Cas effector proteins, including nucleic acids encoding CRISPR-Cas effector proteins, and RNA components to induce DNA targeting, and methods of use thereof.
  • the present disclosure is based, in part, on the discovery of compositions and methods related to type VI CRISPR-Cas effector proteins, optionally, complexed with a guide nucleic acid, that are capable of modifying a target nucleic acid.
  • the present disclosure also provides methods of modifying a target nucleic acid using an endonuclease or chimeric protein of the present disclosure and, optionally, a guide RNA.
  • Casl3 enzymes were identified as RNA-guided RNA-targeting proteins. While Cas9 cleaves DNA to interrupt DNA replication, Casl3 digests RNA to abate transcription.
  • the CRISPR-Cas 13 system can be divided into six subtypes (a, bl, b2, c, d, X, Y). Each subtype carries Casl3, which is a single effector protein. All Casl3 proteins exhibit two distinct RNase activities. One is RNA-targeting degradation, the other is pre-crRNA processing. About six total Casl3 variants have been identified to date.
  • the present endonucleases (e.g., SEQ ID NO: 1), or fragments or variants thereof), without wishing to be bound by theory, belong to a Casl3K2G system.
  • compositions comprising an endonuclease comprising a sequence, or a fragment or variant thereof, having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to SEQ ID NO: 1, or having about 1 to about 20 amino acid modifications (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).
  • amino acid modifications e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or
  • compositions comprising an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to SEQ ID NO: 1, or having about 1 to about 20 amino acid modifications (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).
  • amino acid modifications e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof. In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the sequence comprises one or more truncated HEPN domains.
  • one or more HEPN domains are located according to the positions of the arrows in FIG. 1 and/or by reference to Table 1 or Table 2
  • a polypeptide of the present disclosure e.g., endonuclease or chimeric protein
  • a nucleic acid e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, and the like
  • the endonuclease or chimeric protein of the present disclosure is provided directly as a protein (e.g., without an associated guide RNA or with an associate guide RNA, i.e., as a ribonucleoprotein complex).
  • an endonuclease or chimeric protein or nucleic acid of the present disclosure can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art.
  • the endonuclease is suitable for creating a double stranded break in a nucleic acid.
  • the endonuclease is suitable for creating a nick in a nucleic acid.
  • the endonuclease is suitable for nucleic acid modification by HDR.
  • the endonuclease is suitable for nucleic acid modification by NHEJ.
  • the endonuclease recognizes a PAM. In embodiments, the endonuclease recognizes a plurality of PAMs (e.g., about 2, or about 3, or about 4, or about 5, or about 6, or about 8, or about 10 PAMs). In embodiments, the PAM sequence is about 1 to about 20, or about 2 to about 12, or about 2 to about 6, or about 2, or about 3, or about 4, or about 5, or about 6, or about 8, or about 10 nucleotides in length.
  • the endonuclease (or chimeric protein) comprises one or more mutations to reduce catalytic activity relative to an unmutated form.
  • the one or more mutations to reduce catalytic activity relative to an unmutated form are in one or more HEPN domains of the present endonucleases.
  • One of skill in the art may select the one or more mutations to reduce catalytic activity relative to an unmutated form by reference, e.g., to FIG. 1 and/or by reference to Table 1 or Table 2 and/or by reference to structural information about other endonucleases known in the art, e.g.
  • the endonuclease (or chimeric protein) comprises one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form.
  • the one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form are in one or more HEPN domains of the present endonucleases.
  • One of skill in the art may select the one or more mutations to render the endonuclease substantially catalytically inactive relative to an unmutated form by reference, e.g., to FIG. 1 and/or by reference to Table 1 or Table 2 and/or by reference to structural information about other endonucleases known in the art, e.g.
  • the endonuclease (or chimeric protein) comprises one or more mutations to increase catalytic activity relative to an unmutated form.
  • the one or more mutations to increase catalytic activity relative to an unmutated form are in one or more HEPN domains of the present endonucleases.
  • One of skill in the art may select the one or more mutations to increase catalytic activity relative to an unmutated form by reference, e.g., to FIG. 1 and/or by reference to Table 1 or Table 2 and/or by reference to structural information about other endonucleases known in the art, e.g.
  • the endonuclease (or chimeric protein) comprises one or more mutations to render the endonuclease substantially catalytically hyperactive relative to an unmutated form.
  • the one or more mutations to render the endonuclease substantially catalytically hyperactive relative to an unmutated form are in one or more HEPN domains of the present endonucleases.
  • One of skill in the art may select the one or more mutations to render the endonuclease substantially catalytically hyperactive relative to an unmutated form by reference, e.g., to FIG. 1 and/or by reference to Table 1 or Table 2 and/or by reference to structural information about other endonucleases known in the art, e.g.
  • the endonuclease has nickase activity.
  • the endonuclease (or chimeric protein) comprises one or more mutations to produce nickase activity.
  • the one or more mutations to produce nickase activity relative to an unmutated form are in one or more HEPN domains of the present endonucleases.
  • One of skill in the art may select the one or more mutations to produce nickase activity relative to an unmutated form by reference, e.g., to FIG. 1 and/or by reference to Table 1 or Table 2 and/or by reference to structural information about other endonucleases known in the art, e.g.
  • the endonuclease has collateral cleavage activity.
  • the endonuclease (or chimeric protein) comprises one or more mutations to produce, increase, remove, or decrease collateral cleavage activity.
  • the one or more mutations to produce, increase, remove, or decrease collateral cleavage activity relative to an unmutated form are in one or more HEPN domains of the present endonucleases.
  • One of skill in the art may select the one or more mutations to produce, increase, remove, or decrease collateral cleavage activity relative to an unmutated form by reference, e.g., to FIG.
  • one of skill in the art may select residues to alter in light of a desired percent sequence identity and/or select amino acid modifications by reference to the domains of e.g., to FIG. 1 and/or by reference to Table 1 or Table 2 and/or reference to the phylogenetic information of FIG. 4 and/or by reference to structural information about other endonucleases known in the art, e.g.
  • the amino acid modifications are amino acid mutations or amino acid substitutions. In embodiments, the amino acid substitutions are conservative and/or nonconservative substitutions. In embodiments, the amino acid modifications are amino acid truncations of two or more amino acids (e.g. about to about 100, or about 2 to about 90, or about 2 to about 80, or about 2 to about 70, or about 2 to about 60, or about 2 to about 50, or about 2 to about 40, or about 2 to about 30, or about 2 to about 20, or about 2 to about 10, or about 20 to about 100, or about 50 to about 100, or about 70 to about 100 amino acids).
  • two or more amino acids e.g. about to about 100, or about 2 to about 90, or about 2 to about 80, or about 2 to about 70, or about 2 to about 60, or about 2 to about 50, or about 2 to about 40, or about 2 to about 30, or about 2 to about 20, or about 2 to about 10, or about 20 to about 100, or about 50 to about 100, or about 70 to about 100 amino acids).
  • “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • non-conservative substitutions are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
  • the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine P-alanine, GABA and 8-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohe
  • the percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows.
  • a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using, e.g., the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14 or the like.
  • This stand-alone version of BLASTZ can be obtained online at or at ncbi.nlm.nih.gov. Instructions explaining how to use the B12seq program can be found in the readme fde accompanying BLASTZ.
  • B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options may be set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C: ⁇ seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared e.g., C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C: ⁇ output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting.
  • the following command can be used to generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql.txt -j c: ⁇ seq2.txt -p blastn -o c: ⁇ output.txt -q -1 -r 2.
  • B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C: ⁇ seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C: ⁇ seq2.txt); -p is set to blastp; - o is set to any desired file name (e.g., C: ⁇ output.txt); and all other options are left at their default setting.
  • -i is set to a file containing the first amino acid sequence to be compared (e.g., C: ⁇ seql.txt)
  • -j is set to a file containing the second amino acid sequence to be compared (e.g., C: ⁇ seq2.txt)
  • -p is set to blastp
  • - o is set to any desired file name (e.g., C: ⁇ output.txt); and all other options
  • the following command can be used to generate an output file containing a comparison between two amino acid sequences: C: ⁇ B12seq -i c: ⁇ seql.txt -j c: ⁇ seq2.txt -p blastp -o c: ⁇ output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
  • the percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e.g., of SEQ ID NO: 1), or by an articulated length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It also is noted that the length value will always be an integer.
  • the endonuclease has at least about 75% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 80% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 85% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 90% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 95% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 97% identity to SEQ ID NO: 1. In embodiments, the endonuclease has at least about 99% identity to SEQ ID NO: 1.
  • the endonuclease has about 1 to about 15 amino acid modifications. In embodiments, the endonuclease has about 1 to about 10 amino acid modifications. In embodiments, the endonuclease has about 1 to about 5 amino acid modifications. In embodiments, the endonuclease has about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20 amino acid modifications. In embodiments, the amino acid modifications are selected from substitutions and deletions.
  • the endonuclease is selected from Table 1 below.
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof. In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1.
  • the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1.
  • the endonuclease comprises about 1 to about 20 amino acid modifications (e.g.
  • the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications) to SEQ ID NO: 1.
  • the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications to SEQ ID NO: 1.
  • the present disclosure provides a composition comprising a nucleic acid encoding an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% identity to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).
  • the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the HEPN domain is located according to the positions of the arrows in FIG. 1.
  • the endonuclease is selected from Table 1 below.
  • the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1.
  • the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1.
  • the endonuclease comprises about 1 to about 20 amino acid modifications (e.g.
  • the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications) to SEQ ID NO: 1.
  • the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications to SEQ ID NO: 1.
  • the present disclosure provides a composition comprising a nuclease system, comprising (a) an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% identity to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications); and (b) an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule.
  • a nuclease system comprising (a) an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% identity to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g. about
  • the HEPN domain is located according to the positions of the arrows in FIG. 1.
  • the sequence comprises at least two HEPN domains, or fragments or variants thereof.
  • the endonuclease is selected from Table 1 below.
  • the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1.
  • the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1.
  • the endonuclease comprises about 1 to about 20 amino acid modifications (e.g.
  • the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications) to SEQ ID NO: 1.
  • the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications to SEQ ID NO: 1.
  • the present endonuclease, or a fragment or variant thereof, having at least about 70% identity to SEQ ID NO: 1 has the N terminal M residue removed.
  • the present endonuclease, or a fragment or variant thereof, having at least about 70% identity to SEQ ID NO: 1 has the N terminal M residue removed and a SV40 NLS SV40 sequence is added to the N-terminus (MSPKKKRKVEAS (SEQ ID NO: 78)).
  • the present endonuclease, or a fragment or variant thereof, having at least about 70% identity to SEQ ID NO: 1 has the N terminal M residue removed and a SV40 NLS SV40 sequence is added to the N-terminus (MSPKKKRKVEAS (SEQ ID NO: 78)) and a HA tag is added to the C-terminus (GSGPKKKRKVAAAYPYDVPDYA (SEQ ID NO: 77)).
  • the catalytic domain is selected from Table 2.
  • a mutation of any of the present endonucleases is of one or more of the residues of Table 2.
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification one or more positions that have an R or H residue in the wild type sequence.
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification at one or more positions in a HEPN domain.
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification at one or more positions in a region of the endonuclease at about 1 to about 300 amino acids, or about 1 to about 200 amino acids, or about 1 to about 150 amino acids, or about 1 to about 100 amino acids, or about 20 to about 160 amino acids, or about 40 to about 160 amino acids, or about 50 to about 150 amino acids, or about 140 to about 160, or about 150 amino acids from the N-terminus of the endonuclease.
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification at about 1 to about 300 amino acids, or about 1 to about 250 amino acids, or about 1 to about 200 amino acids, or about 1 to about 100 amino acids, or about 1 to about 80 amino acids, or about 1 to about 50 amino acids, or about 30 to about 100 amino acids, or about 30 to about 70 amino acids, or about 40 to about 60, or about 50 amino acids from the C-terminus of the endonuclease.
  • the amino acid modification is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification is amino acid modification is a hydrophilic amino acid. In embodiments, the amino acid modification is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification is arginine (R) or lysine (K). In embodiments, the amino acid modification is a polar and neutral charged hydrophilic amino acid. In embodiments, the amino acid modification is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the amino acid modification is a polar and negatively charged hydrophilic amino acid.
  • the amino acid modification is selected from aspartate (D) or glutamate (E).
  • the amino acid modification is an aromatic, polar and positively charged hydrophilic amino acid.
  • the amino acid modification is histidine (H).
  • the amino acid modification is a hydrophobic amino acid.
  • the amino acid modification is a hydrophobic, aliphatic amino acid.
  • the amino acid modification is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V).
  • the amino acid modification is a hydrophobic, aromatic amino acid.
  • the amino acid modification is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification at position R150.
  • the amino acid modifications are selected from substitutions and deletions.
  • the amino acid modification to SEQ ID NO: 1 at R150 is an essential or non- essential amino acid.
  • the amino acid modification at R150 is a hydrophilic or hydrophobic amino acid.
  • the amino acid modification at R150 is amino acid modification is a hydrophilic amino acid. In embodiments, the amino acid modification at R150 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification atR150 is lysine (K). In embodiments, the amino acid modification at R150 is a polar and neutral charged hydrophilic amino acid. In embodiments, the amino acid modification at R150 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the amino acid modification at R150 is a polar and negatively charged hydrophilic amino acid.
  • the amino acid modification at R150 is selected from aspartate (D) or glutamate (E). In embodiments, the amino acid modification at R150 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R150 is histidine (H). In embodiments, the amino acid modification at R150 is a hydrophobic amino acid. In embodiments, the amino acid modification at R150 is a hydrophobic, aliphatic amino acid. In embodiments, the amino acid modification at R150 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). In embodiments, the amino acid modification at R150 is a hydrophobic, aromatic amino acid. In embodiments, the amino acid modification at R150 is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification at position H155.
  • the amino acid modifications are selected from substitutions and deletions.
  • the amino acid modification to SEQ ID NO: 1 at Hl 55 is an essential or non- essential amino acid.
  • the amino acid modification at Hl 55 is a hydrophilic or hydrophobic amino acid.
  • the amino acid modification at Hl 55 is amino acid modification is a hydrophilic amino acid. In embodiments, the amino acid modification at Hl 55 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H155 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at Hl 55 is a polar and neutral charged hydrophilic amino acid. In embodiments, the amino acid modification at Hl 55 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the amino acid modification at Hl 55 is a polar and negatively charged hydrophilic amino acid.
  • the amino acid modification at Hl 55 is selected from aspartate (D) or glutamate (E). In embodiments, the amino acid modification at Hl 55 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at Hl 55 is a hydrophobic amino acid. In embodiments, the amino acid modification at Hl 55 is a hydrophobic, aliphatic amino acid. In embodiments, the amino acid modification at Hl 55 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). In embodiments, the amino acid modification at Hl 55 is a hydrophobic, aromatic amino acid. In embodiments, the amino acid modification at Hl 55 is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification at position R840.
  • the amino acid modifications are selected from substitutions and deletions.
  • the amino acid modification to SEQ ID NO: 1 at R840 is an essential or non- essential amino acid.
  • the amino acid modification at R840 is a hydrophilic or hydrophobic amino acid.
  • the amino acid modification at R840 is amino acid modification is a hydrophilic amino acid. In embodiments, the amino acid modification at R840 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R840 is lysine (K). In embodiments, the amino acid modification at R840 is a polar and neutral charged hydrophilic amino acid. In embodiments, the amino acid modification at R840 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the amino acid modification at R840 is a polar and negatively charged hydrophilic amino acid.
  • the amino acid modification at R840 is selected from aspartate (D) or glutamate (E). In embodiments, the amino acid modification at R840 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R840 is histidine (H). In embodiments, the amino acid modification at R840 is a hydrophobic amino acid. In embodiments, the amino acid modification at R840 is a hydrophobic, aliphatic amino acid. In embodiments, the amino acid modification at R840 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). In embodiments, the amino acid modification at R840 is a hydrophobic, aromatic amino acid. In embodiments, the amino acid modification at R840 is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).
  • the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 and an amino acid modification at position H845.
  • the amino acid modifications are selected from substitutions and deletions.
  • the amino acid modification to SEQ ID NO: 1 at H845 is an essential or non- essential amino acid.
  • the amino acid modification at H845 is a hydrophilic or hydrophobic amino acid.
  • the amino acid modification at H845 is amino acid modification is a hydrophilic amino acid. In embodiments, the amino acid modification at H845 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H845 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H845 is a polar and neutral charged hydrophilic amino acid. In embodiments, the amino acid modification at H845 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the amino acid modification at H845 is a polar and negatively charged hydrophilic amino acid.
  • the amino acid modification at H845 is selected from aspartate (D) or glutamate (E). In embodiments, the amino acid modification at H845 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H845 is a hydrophobic amino acid. In embodiments, the amino acid modification at H845 is a hydrophobic, aliphatic amino acid. In embodiments, the amino acid modification at H845 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). In embodiments, the amino acid modification at H845 is a hydrophobic, aromatic amino acid. In embodiments, the amino acid modification at H845 is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).
  • the endonuclease (or chimeric protein) comprises a domain from a different endonuclease.
  • the different endonuclease is a Cas endonuclease.
  • the domain is one or more of a PAM-interacting domain.
  • the domain is derived from one or more of Cas9, Cas 12a (Cpfl), Casl2e (CasX), Cas 12d (CasY), Cas 12b (C2cl), Casl3a (C2c2), Casl3b, Casl3c, Casl3d, Casl3X/ Casl3bt, Casl3Y, Casl2c (C2c3), GeoCas9, CjCas9, NmeCas9, Casl2J (CasPhi), Casl2L (CasLambda), Casl2f (Cas 14), Cas 12g, Casl2h, Casl2i, Casl2k, NmeCas9, Nme2Cas9, CjCas9, GeoCas9, BlatCas9, PpCas9, and Casl4.
  • the domain is derived from a Cas from one or more of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitis, Streptococcus thermophilis, or Treponema denticola.
  • the composition further comprises one or more donor polynucleotides.
  • the endonuclease is suitable for introducing one or more donor polynucleotides into a target nucleic acid molecule.
  • the donor polynucleotide comprises a transgene.
  • the donor polynucleotide comprises a sequence which corrects a mutation.
  • a donor polynucleotide is of any length, for example between about 2 and about 10000 nucleotides in length (or any integer value therebetween or thereabove), for instance, between about 100 and about 1000 nucleotides in length (or any integer therebetween), or between about 200 and about 500 nucleotides in length.
  • the endonuclease comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the NLS comprises the consensus sequence K(K/R)X(K/R) (SEQ ID NO: 32).
  • the NLS comprises the consensus sequence (K/R)(KZR)Xio-i2(K/R)3/s (SEQ ID NO: 33), where (K/R)3/s represents at least three of the five amino acids is either lysine or arginine.
  • the NLS comprises the c-myc NLS.
  • the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 34).
  • the NLS is the nucleoplasmin NLS.
  • the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 35).
  • the NLS comprises the SV40 Large T-antigen NLS.
  • the SV40 Large T-antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 36).
  • the NLS comprises three SV40 Large T-antigen NLSs (e.g, DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 37)).
  • the NLS is or comprises SEQ ID NO: 72.
  • the NLS comprises mutations/variations in the above sequences such that they contain 1 or more substitutions, additions, or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10 substitutions, additions, or deletions).
  • the endonuclease (or chimeric protein) comprises a polypeptide permeant domain to promote uptake by a cell.
  • the permeant domain is a peptide, peptidomimetic, or non-peptide carrier.
  • the permeant peptide is derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 38).
  • the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally occurring tat protein.
  • the permeant peptide is a poly-arginine motif, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like.
  • a poly-arginine motif for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like.
  • the endonuclease (or chimeric protein) comprises a polypeptide that promotes or is suitable for VLP delivery, including, without limitation, a retroviral gag polyprotein comprising a matrix polypeptide, a capsid polypeptide, and a nucleocapsid polypeptide (optionally with one or more heterologous protease cleavage sites (e.g.
  • TEV cleavage site a PreScission (fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease) cleavage site, a human rhinovirus 3C protease cleavage site, an enterokinase cleavage site, an Epstein-Barr virus protease cleavage site, a cathepsin D cleavage site, and/or a thrombin cleavage site) between one or both of: the matrix polypeptide and the capsid polypeptide; and the capsid polypeptide and the nucleocapsid polypeptide, e.g., a lentiviral gag polyprotein, e.g., a bovine immunodeficiency virus gag polyprotein, a murine leukemia virus (MLV) a gag protein, a simian immunodeficiency virus gag polyprotein, a feline immunodeficiency virus gag poly
  • the polypeptide that promotes or is suitable for VLP delivery is co-delivered with a protease to promote cleavage of the chimeric protein.
  • the cleavage of the chimeric protein occurs between the endonuclease and the polypeptide that promotes or is suitable for VLP delivery.
  • the protease is fused to a polypeptide that promotes or is suitable for VLP delivery.
  • the endonuclease (or chimeric protein) or a delivery vehicle e.g. one or more lipids, associated therewith comprises a polypeptide or other moiety that interacts with an targeting moiety, e.g. an antibody or antibody-like molecule, a ligand to bind a receptor or a receptor (or fragment thereof) to bind a ligand, aptamer, and the like, for targeted delivery.
  • an targeting moiety e.g. an antibody or antibody-like molecule
  • the endonuclease (or chimeric protein) or a delivery vehicle e.g.
  • one or more lipids, associated therewith comprises a targeting moiety, e.g., an antibody or antibody-like molecule, a ligand to bind a receptor or a receptor (or fragment thereof) to bind a ligand, aptamer, and the like, for targeted delivery.
  • a targeting moiety e.g., an antibody or antibody-like molecule, a ligand to bind a receptor or a receptor (or fragment thereof) to bind a ligand, aptamer, and the like, for targeted delivery.
  • the endonuclease is suitable for introducing one or more excisions into a target nucleic acid molecule.
  • the excision is a double-strand DNA break in two strands of the target nucleic acid molecule.
  • the excision is a nick in one or more strands of the target nucleic acid molecule.
  • the present disclosure provides a composition comprising a chimeric protein comprising: an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) to SEQ ID NO: 1 or having about 1 to about 20 amino acid modifications (e.g.
  • nucleic acid-modulating domain or a nucleic acid-modifying domain comprising a sequence comprising a catalytic domain, or a fragment or variant thereof, wherein (a) and (b) do not naturally occur together in a same reading frame.
  • the endonuclease reduces or enhances non-specific degradation of transcripts. In embodiments, the endonuclease reduces or enhances collateral activity, e.g., for use in nucleic acid detection. In embodiments, the endonuclease reduces or enhances collateral activity, e.g. for use in nucleic acid detection using an electrochemical method.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has one or more of 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, debranching activity, transesterification activity, photolyase activity and glycosylase activity.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain is a METTL3 methyltransferase domain, a METTL3: METTL1 fusion domain, or a fragment or variant thereof.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain is a nucleic acid interacting/binding domain.
  • nucleic acid interacting/binding domains are MCP, lambdaN, PP7, QBeta, SLBP, and TBP/TAR.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain is a splice-modulating domain.
  • the splice-modulating domain is the RS-rich domain of SRSF1, the Gly-rich domain of hnRNP Al, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1.
  • the endonuclease (or chimeric protein) induces exon skipping. In embodiments, the endonuclease (or chimeric protein) induces exon inclusion.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain is a degradation domain.
  • the degradation domain is a E2 ubiquitin or ubiquitin-like domain.
  • the degradation domain comprises a ubiquitin core catalytic (UBC) domain.
  • the degradation domain is SUMO, NEDD8, ATG8, ATG12, ISG15, UFM1, FAT10, URM1, or FUBI, or a fragment or variant thereof.
  • the degradation domain is UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcHlO), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F (NCE2), UBE2G1 (UBE2G), UBE2G2 (UBC 7), UBE2H (UBCH), UBE2I (Ubc9), UBE2J1 (NCUBE1), UBE2I2 (NCUBE2), UBE2K (HIP2), UBE2L3 (UbcH7), UBE2L6 (UbcH8), UBE2M (Ubcl2), UBE2N (Ubcl3), UBE2NL, UBE20 (E2-
  • the degradation domain is a protease, e.g. a protease that is conditionally modulated by another molecule, e.g. a protease inhibitor, e.g. a matrix metalloprotease (MMP) and TIMP-1, TIMP-2, TIMP-3, or TIMP-4.
  • a protease inhibitor e.g. a matrix metalloprotease (MMP) and TIMP-1, TIMP-2, TIMP-3, or TIMP-4.
  • MMP matrix metalloprotease
  • the degradation domain is modulated by a small molecule.
  • the degradation domain is active in the presence of a small molecule.
  • the degradation domain is inactive in the presence of a small molecule.
  • the small molecule is an antiviral drug.
  • the small molecule is one or more of abscisic acid (ABA), rapamycin (or rapalog), FK506, Cyclosporine A, FK1012, Gibberellin3-AM, FKCsA, AP1903/AP20187, and auxin.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has nuclease activity, such as that provided by a restriction enzyme e.g., FokI nuclease).
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c- m ethyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 e.g. plants), ZMET2, CMT1, CMT2 (e.g. plants), and the like).
  • a methyltransferase e.g., Hhal DNA m5c- m ethyltransferase (M.Hhal), DNA methyltransferas
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has 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).
  • a demethylase e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1).
  • a deaminase e.g., a cytosine deaminase enzyme such as rat APOBEC1.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has integrase and/or resolvase activity (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).
  • integrase and/or resolvase activity 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.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase).
  • a recombinase e.g., catalytic domain of Gin recombinase
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain is selected from a deaminase, reverse transcriptase, transposase, integrase, and recombinase.
  • the deaminase is a cytidine or cytosine deaminase, or a fragment or variant thereof.
  • the cytidine or cytosine deaminase is selected from AID, CDA1, and APOB EC, or a fragment or variant thereof.
  • the APOBEC is selected from A3A, AB3, APOBEC1, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H, or a fragment or variant thereof.
  • the APOBEC has an amino acid sequence of one of SEQ ID NO: 39 [A3A], SEQ ID NO: 40 [AB3], SEQ ID NO: 41 [APOBEC1], SEQ ID NO: 42 [APOBEC3C], SEQ ID NO: 43 [APOBEC3D], SEQ ID NO: 44 [APOBEC3F], SEQ ID NO: 45 [APOBEC3G], and SEQ ID NO: 46 [APOBEC3H], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the APOBEC has an amino acid sequence selected from one of SEQ ID NOs: 32-39
  • the deaminase is a DNA-specific adenine or adenosine deaminase or adenosine base editor (ABE), or a fragment or variant thereof.
  • the ABE is generated by replacing APOB EC 1 component of BE3 with natural or engineered E. coli TadA, human ADAR2, mouse ADA, or human ADAT2.
  • ABE comprises an evolved TadA variant.
  • the ABE is ABE 1 .2 (TadA*-XTEN-nCas9- NLS).
  • TadA* comprises A106V and D108N mutations.
  • the ABE is a second-generation ABE.
  • the ABE is ABE2.1, which comprises additional mutations D147Y and E155V in TadA* (TadA*2.1).
  • the ABE is ABE2.2, ABE2.1 fused to catalytically inactivated version of human alkyl adenine DNA glycosylase (AAG with E125Q mutation).
  • the ABE is ABE2.3, ABE2.1 fused to catalytically inactivated version of . coli Endo V (inactivated with D35A mutation).
  • the ABE is ABE2.6 which has a linker twice as long (32 amino acids, (SGGS) 2 - XTEN-(SGGS) 2 (“(SGGS) 2 ” disclosed as SEQ ID NO: 47)) as the linker in ABE2.1.
  • the ABE is ABE2.7, which is ABE2.1 tethered with an additional wild-type TadA monomer.
  • the ABE is ABE2.8, which is ABE2.1 tethered with an additional TadA*2.1 monomer.
  • the ABE is ABE2.9, which is a direct fusion of evolved TadA (TadA*2.1) to the N-terminus of ABE2.1.
  • the ABE is ABE2.10, which is a direct fusion of wild type TadA to the N-terminus of ABE2.1.
  • the ABE is ABE2.11, which is ABE2.9 with an inactivating E59A mutation at the N-terminus of TadA* monomer.
  • the ABE is ABE2.12, which is ABE2.9 with an inactivating E59A mutation in the internal TadA* monomer.
  • the ABE is a third generation ABE.
  • the ABE is ABE3.1, which is ABE2.3 with three additional TadA mutations (L84F, H123Y, and I156F).
  • the ABE is a fourth generation ABE.
  • the ABE is ABE4.3, which is ABE3.1 with an additional TadA mutation A142N (TadA*4.3).
  • the ABE is a fifth generation ABE.
  • the ABE is ABE5.1, which is generated by importing a consensus set of mutations from surviving clones (H36L, R51L, S146C, and K157N) into ABE3.1.
  • the ABE is ABE5.3, which has a heterodimeric construct containing wild-type E. coli TadA fused to an internal evolved TadA*.
  • the ABE is ABE5.2, ABE5.4, ABE5.5, ABE5.6, ABE5.7, ABE5.8, ABE5.9, ABE5.10, ABE5.11, ABE5.12, ABE5.13, or ABE5.14, as shown in Table 6 of US Pat. No. 11,142,760 (herein incorporated herein by reference).
  • the ABE is a sixth generation ABE.
  • the ABE is ABE6.1, ABE6.2, ABE6.3, ABE6.4, ABE6.5, or ABE6.6, as shown in below Table 6 of US Pat. No. 11,142,760 (herein incorporated herein by reference).
  • the ABE is a seventh generation ABE.
  • the ABE is ABE7.1, ABE7.2, ABE7.3, ABE7.4, ABE7.5, ABE7.6, ABE7.7, ABE7.8, ABE 7.9, or ABE7.10, as shown in Table 6 of US Pat. No. 1 1,142,760 (herein incorporated herein by reference).
  • the base editor is an eighth generation ABE (ABE8).
  • the ABE8 contains a TadA*8 variant. In embodiments, the ABE8 has a monomeric construct containing a TadA*8 variant (ABE8.x-m).
  • the DNA-specific adenine or adenosine deaminase is selected from tRNA- specific adenosine deaminase 7.10 (TadA 7.10), tRNA-specific adenosine deaminase 6.3 (TadA 6.3), tRNA-specific adenosine deaminase 7.8 (TadA 7.8), tRNA-specific adenosine deaminase 7.9 (TadA 7.9), and tRNA-specific adenosine deaminase 8e (TadA8e (TadA-8e V106W)) or a fragment or variant thereof.
  • the TadA has an amino acid sequence of one of SEQ ID NO: 48 [TadA 7.10], SEQ ID NO: 49 [TadA 6.3], SEQ ID NO: 50 [TadA 7.8], SEQ ID NO: 51 [TadA 7.9], and SEQ ID NO: 52 [TadA 8e], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the TadA has an amino acid sequence selected from one of SEQ ID NOs: 48-52 as shown in Table 6 below.
  • the deaminase is a RNA-specific adenine or adenosine deaminase, or a fragment or variant thereof.
  • the RNA-specific adenine or adenosine deaminase is an adenosine deaminases acting on RNA (ADAR) enzyme, or a fragment or variant thereof.
  • the ADAR is selected from AD ARI, ADAR2, and ADAR3, or a fragment or variant thereof.
  • the ADAR has an amino acid sequence of one of SEQ ID NO: 53 [ADAR1] and SEQ ID NO: 54 [ADAR2], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the ADAR has an amino acid sequence of any one of SEQ ID NOs 53-54 as shown in Table 7.
  • the catalytic deaminase domain of AD ARI comprises amino acids 833-1226 of SEQ ID NO: 53.
  • the catalytic deaminase domain of ADAR2 comprises amino acids 299-701 of SEQ ID NO: 54.
  • Table 7 ADAR amino acid sequences
  • the deaminase further comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the NLS comprises the consensus sequence K(K/R)X(K/R) (SEQ ID NO: 32).
  • the NLS comprises the consensus sequence (K/R)(K/R)Xio-i2(K/R)35 (SEQ ID NO: 33), where (K/R)3/s represents at least three of the five amino acids is either lysine or arginine.
  • the NLS comprises the c-myc NLS.
  • the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 34).
  • the NLS is the nucleoplasmin NLS.
  • the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 35).
  • the NLS comprises the SV40 Large T-antigen NLS.
  • the SV40 Large T-antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 36).
  • the NLS comprises three SV40 Large T-antigen NLSs e.g., DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 37)).
  • the NLS may comprise mutations/variations in the above sequences such that they contain 1 or more substitutions, additions or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10 substitutions, additions, or deletions).
  • the endonuclease further comprises a UGI, or a fragment or variant thereof.
  • the endonuclease (or chimeric protein) comprises 1, or 2, or 3, or UGIs.
  • the endonuclease (or chimeric protein) comprises a UGI at the N- and/or C- terminus.
  • Illustrative UGI proteins for use in the present disclosure include, for example, those published in Wang et al., J. Biol. Chem. 264:1163-1171(1989); Lundquist et al., J. Biol. Chem. 272:21408-21419(1997); Ravishankar etal., Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al., J. Mol. Biol. 287:331-346(1999), each of which is incorporated herein by reference in their entirety.
  • the RNA molecule is a gRNA.
  • the gRNA comprises a sequence that interacts with the endonuclease.
  • the endonuclease forms a complex with the gRNA.
  • the composition is suitable for base editing, see, e.g., Rees, H. A., and Liu, D. R. (2016). Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770-788. doi: 10.1038/s41576-018-0059-l, hereby incorporated by reference in its entirety.
  • the composition is suitable for DNA base editing.
  • the composition is suitable for RNA base editing.
  • the composition is suitable for catalyzing OT nucleotide conversions or A>G nucleotide conversions in a target nucleic acid.
  • the composition comprises both an adenosine deaminase and a cytidine deaminase.
  • the composition is suitable for dual base editing, see, e.g., Nature Biotechnology volume 38, 856-860 (2020), hereby incorporated by reference in its entirety.
  • the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase (M-MLV RT) or M-MLV RT(D200N/L603W/T330P/T306K/W313F), or a fragment or variant thereof.
  • M-MLV RT Moloney murine leukemia virus reverse transcriptase
  • M-MLV RT M-MLV RT(D200N/L603W/T330P/T306K/W313F)
  • the M-MLV RT has an amino acid sequence of SEQ ID NO: 55 [M- MLV RT] or SEQ ID NO 56 [M-MLV RT(D200N/L603W/T330P/T306K/W313F)] or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the Moloney murine leukemia virus reverse transcriptase (M-MLV RT) or M-MLV RT(D200N/L603W/T330P/T306K/W313F) is selected from SEQ ID NO: 55 or SEQ ID NO: 56, as shown in Table 8 below.
  • Table 8 Moloney murine leukemia virus reverse transcriptase sequences
  • the composition further comprises a dominant negative human MutL homolog (MLH1). In embodiments, the composition is suitable for use with a dominant negative MLH1.
  • MH1 dominant negative human MutL homolog
  • the RNA molecule is or comprises a prime editing guide RNA (pegRNA).
  • the endonuclease forms a complex with the pegRNA.
  • the pegRNA serves as a template for transcription of a new DNA sequence.
  • the pegRNA binds to a DNA strand opposite from a typical gRNA binding site.
  • the pegRNA comprises a gRNA containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence.
  • the RNA molecule is or comprises a gRNA.
  • the gRNA comprises a sequence that interacts with the endonuclease.
  • the endonuclease forms a complex with the gRNA.
  • the composition comprises both a gRNA and a pegRNA.
  • the composition is suitable for prime editing see, e.g., Nature 576, 149-157 (2019), hereby incorporated by reference in its entirety.
  • the transposase is selected from Tnl, Tn2, Tn3, Tn5, Tn7, Tn9, TnlO, Tn552, Tn903, TnlOOO/Gamma-delta, Tn/O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping beauty, piggyBac, Tol2, Most, Himarl, Hermes, Tol2, Minos, Tel, P-element, MuA, Tyl , Chapaev, transib, Tcl/mariner, and Tc3 donor DNA system.
  • the transposase is a transposon 7-like (Tn7-like) transposon system, or a fragment or variant thereof.
  • the transposase is one or more of transposon 7 protein A (TnsA), transposon 7 protein B (Tns B), transposon 7 protein C (Tns C), and transposition of integron protein Q (TniQ), or a fragment or variant thereof.
  • the Tn7-like transposon system is derived from Vibrio cholerae Tn6677.
  • the transposase has an amino acid sequence of one or more of SEQ ID NO: 57 [TnsA], SEQ ID NO: 58 [TnsB], SEQ ID NO: 59 [TnsC], and SEQ ID NO: 60 [TniQ], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the transposase has an amino acid sequence of one or more of SEQ ID NOs 57-60, as shown in Table 9 below.
  • the integrase is a serine-recombinase, or a fragment or variant thereof.
  • the serine-recombinase is Bxbl, or a fragment or variant thereof.
  • the recombinase is a Gin invertase or Tn3 resolvase, or a fragment or variant thereof.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain can increase transcription, e.g. transcriptional activators such as VP 16, VP64, VP48, VP 160, p65 subdomain (e.g., from NFkB), and the activation domain of EDLL and/or TAL activation domain e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, Pl 60, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TE), VP
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain can decrease transcription, e.g. transcriptional repressors such as the Kriippel associated box (KRAB or SKD); KOX1 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, JARID 1 C/SMCX, JARID1D/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, H
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has enzymatic activity that modifies a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA).
  • a target nucleic acid e.g., ssRNA, dsRNA, ssDNA, dsDNA.
  • enzymatic activity examples include but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METT, DRM3 (e.g. plants), ZMET2, CMT1, CMT2 (e.g.
  • 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 AP0BEC1), 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 (EM); Tn3 resolvase; and the like), transposase activity
  • TET Ten
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has debranching activity, including, for example, lariat debranching enzyme or DBR1.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has transesterification activity, including, for example, a relaxase or Spoi l.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain has activity that modifies a protein associated with a target nucleic acid (e.g. , ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like).
  • a target nucleic acid e.g. , ssRNA, dsRNA, ssDNA, dsDNA
  • a histone e.g., an RNA binding protein, a DNA binding protein, and the like.
  • enzymatic activity that modifies a protein associated with a target nucleic acid
  • enzymatic activity that modifies a protein associated with a target nucleic acid
  • methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1 A), Vietnamese histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, D0T1L, 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
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain is one or more effector domains, including proteins and/or enzymes, such that those can cleave RNA (e.g., a PIN endonuclease domain, an NYN domain, an SMR domain from SOT1, or an RNase domain from Staphylococcal nuclease), those that can affect RNA stability e.g., tristetraprolin (TTP) or domains from UPF1, EXOSC5, and STAU1), those that can edit a nucleotide or ribonucleotide (e.g., a cytidine deaminase, PPR protein, adenosine deaminase, ADAR family protein, or APOB EC family protein), those that can activate translation (e.g., eIF4E and other translation initiation factors, a domain of the yeast poly(A)-binding protein or GLD2), those that can re
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain comprises one or more mutations to reduce activity relative to an unmutated form.
  • the nucleic acid-modulating domain or the nucleic acid-modifying domain comprises one or more mutations to increase activity relative to an unmutated form.
  • sequence of (a) is disposed at the N-terminus of the chimeric protein and the sequence of (b) is disposed at the C-terminus of the chimeric protein.
  • sequence of (a) is disposed at the C-terminus of the chimeric protein and the sequence of (b) is disposed at the N-terminus of the chimeric protein.
  • the composition further comprises a linker that joins the sequence of (a) and the sequence of (b).
  • the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids.
  • the linker is substantially comprised of glycine and serine residues.
  • the linker is (GGS)n, wherein n is 1, or 2, or 3, or 4, or 5.
  • the target nucleic acid is referred to as a target sequence.
  • the target nucleic acid is or comprises a sequence of contiguous nucleotides present in a target RNA or target DNA.
  • a stretch of contiguous nucleotides refers to a string of nucleotides that are covalently linked and immediately adjacent to one another.
  • the target nucleic acid is or comprises at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the target nucleic acid is or comprises less than about 300, 250, 200, 100, 150, or 50 nucleotides in length.
  • the target nucleic acid is or comprises about 5-10, about 5-15, about 5-20, about 10-20, about 10-30, about 10-40, about 10- 50, about 10-60, about 10-70, about 10-80, about 10-90, about 10-100, about 50-100, about 50- 150, about 50-200, about 50-250, about 50-300, about 100-200, about 100-300, or about 200-300 nucleotides in length.
  • the target nucleic acid is 10-50 nucleotides in length, e.g., 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37
  • the target nucleic acid is or comprises ssRNA. In embodiments, the target nucleic acid is or comprises dsRNA. In embodiments, the target nucleic acid is or comprises ssDNA. In embodiments, the target nucleic acid is or comprises dsDNA. In embodiments, the target nucleic acid is about 2 to about 6 nucleotides upstream of a PAM sequence.
  • the target nucleic acid is proximal to an exon. In embodiments, the target nucleic acid is upstream of an exon. In embodiments, the target nucleic acid is downstream of an exon. In embodiments, the target nucleic acid overlaps with an exon.
  • the target nucleic acid is proximal to an intron. In embodiments, the target nucleic acid is upstream of an intron. In embodiments, the target nucleic acid is downstream of an intron. In embodiments, the target nucleic acid overlaps with an intron.
  • the composition further comprises a viral vector.
  • the viral vector is or comprises an AAV.
  • the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2/1, AAV2/5, AAV2/8, AAV2/9, AAV3/1, AAV3/5, AAV3/8, and AAV3/9.
  • the composition further comprises a non-viral vector.
  • the present compositions take the form of, or delivery of the present compositions is effected using, a nanoparticle, e.g., any particle having a diameter of less than about 1000 nm.
  • nanoparticles suitable for use in delivering the present compositions to a target cell have a diameter of about 500 nm or less, e.g., from about 25 nm to about 35 nm, from about 35 nm to about 50 nm, from about 50 nm to about 75 nm, from about 75 nm to about 100 nm, from about 100 nm to about 150 nm, from about 150 nm to about 200 nm, from about 200 nm to about 300 nm, from about 300 nm to about 400 nm, or from about 400 nm to about 500 nm.
  • nanoparticles suitable for use in delivering the present compositions to a target cell have a diameter of from about 25 nm to about 200 nm. In embodiments, nanoparticles suitable for use in delivery have a diameter of about 100 nm or less. In embodiments, nanoparticles suitable for use in delivery have a diameter of from about 35 nm to about 60 nm.
  • the composition further comprises a lipid nanoparticle (LNP) liposomes, lipoplexes or polymeric nanoparticle.
  • LNP lipid nanoparticle
  • the LNP comprises one or more of ionizable lipids, amino lipids, anionic lipids, neutral lipids, amphipathic lipids, helper lipids, structural lipids, PEG lipids, and lipoids.
  • DOTAP 1,2- ditetradecanoyl-sn-glycero-3-phosphocholine
  • a liposome is used to deliver a composition of the present disclosure to a target cell.
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid fdm is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus.
  • a liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2- distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.
  • DSPC 1,2- distearoryl-sn-glycero-3-phosphatidyl choline
  • sphingomyelin egg phosphatidylcholines and monosialoganglioside.
  • the composition is in the form of a lipoplex.
  • Lipoplexes that utilize cationic lipids have proven utility for gene transfer. Cationic lipids, due to their positive charge, naturally complex with the negatively charged DNA. Also, as a result of their charge, they interact with the cell membrane. Endocytosis of the lipoplex then occurs, and the DNA is released into the cytoplasm. The cationic lipids also protect against degradation of the DNA by the cell.
  • the composition is in the form of a polyplex.
  • Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions.
  • One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis) such as inactivated adenovirus must occur.
  • endosome-lytic agents to lyse the endosome that is made during endocytosis
  • endosome-lytic agents to lyse the endosome that is made during endocytosis
  • polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.
  • the composition is in the form of a dendrimer
  • a highly branched macromolecule with a spherical shape may be also be used to genetically modify stem cells.
  • the surface of the dendrimer particle may be functionalized to alter its properties.
  • a cationic dendrimer e. ., one with a positive surface charge.
  • charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer.
  • the dendrimer-nucleic acid complex can be taken up into a cell by endocytosis.
  • the present disclosure provides a nucleic acid encoding the chimeric protein of any one of the embodiments and/or aspects disclosed herein.
  • the nucleic acid is or comprises a DNA molecule or an RNA molecule.
  • the RNA is or comprises mRNA or modified mRNA (mmRNA).
  • the DNA is or comprises a vector or plasmid.
  • the nucleic acid comprises a codon optimized sequence.
  • the nucleic acid comprises one or more modifications.
  • the modifications are one or more of base modifications and backbone modifications.
  • sugar-based particles may be used, for example GalNAc, can be used to deliver a composition of the present disclosure to a target cell.
  • the present disclosure provides a viral vector comprising the nucleic acid of any one of the embodiments and/or aspects disclosed herein. In aspects, the present disclosure provides an expression vector comprising the nucleic acid of any one of the embodiments and/or aspects disclosed herein.
  • the expression vector is selected from viral expression vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis
  • a retroviral vector e.g., Murine Leuk
  • a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector.
  • a recombinant expression vector of the present disclosure is a recombinant lentivirus vector.
  • a recombinant expression vector of the present disclosure is a recombinant retroviral vector.
  • the viral vector is or comprises an AAV.
  • the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2/1, AAV2/5, AAV2/8, AAV2/9, AAV3/1, AAV3/5, AAV3/8, and AAV3/9.
  • the composition further comprises a VLP, e.g. a structure that in at least one attribute resembles a virus, but which has not been demonstrated to be infectious.
  • the VLP is a nonreplicating, noninfectious viral shell that contains a viral capsid but lacks all or part of the viral genome, in particular, the replicative components of the viral genome.
  • the VLP is composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface, and structural proteins e.g., VP1, VP2).
  • the VLP resembles the structure of a bacteriophage, being non-repl icative and noninfectious, and lacking at least the gene or genes coding for the replication machinery of the bacteriophage, and also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.
  • the VLP comprises a polypeptide that promotes or is suitable for VLP delivery, including, without limitation, a retroviral gag polyprotein comprising a matrix polypeptide, a capsid polypeptide, and a nucleocapsid polypeptide (optionally with one or more heterologous protease cleavage sites (e.g.
  • TEV cleavage site a PreScission (fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease) cleavage site, a human rhinovirus 3C protease cleavage site, an enterokinase cleavage site, an Epstein-Barr virus protease cleavage site, a cathepsin D cleavage site, and/or a thrombin cleavage site) between one or both of: the matrix polypeptide and the capsid polypeptide; and the capsid polypeptide and the nucleocapsid polypeptide, e.g., a lentiviral gag polyprotein, e.g., a bovine immunodeficiency virus gag polyprotein, a murine leukemia virus (MLV) a gag protein, a simian immunodeficiency virus gag polyprotein, a feline immunodeficiency virus gag poly
  • the polypeptide that promotes or is suitable for VLP delivery is co-delivered with a protease to promote cleavage of the chimeric protein.
  • the cleavage of the chimeric protein occurs between the endonuclease and the polypeptide that promotes or is suitable for VLP delivery.
  • the protease is fused to a polypeptide that promotes or is suitable for VLP delivery.
  • any of a number of transcription and/or translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like may be used in the expression vector.
  • a nucleotide sequence encoding a present RNA is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • a nucleotide sequence encoding a present protein, or a present fusion polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • the transcriptional control element is a promoter.
  • the promoter is a constitutively active promoter. In embodiments, the promoter is a regulatable promoter. In embodiments, the promoter is an inducible promoter. In embodiments, the promoter is a tissue-specific promoter. In embodiments, the promoter is a cell type-specific promoter. In embodiments, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in embodiments, the transcriptional control element is functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, and the like).
  • mPB mobilized peripheral blood
  • BM bone marrow
  • eukaryotic promoters include EFla, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • LTRs long terminal repeats
  • a nucleotide sequence encoding a present RNA and/or a present fusion polypeptide is operably linked to an inducible promoter. In embodiments, a nucleotide sequence encoding a present RNA and/or a present chimeric protein is operably linked to a constitutive promoter.
  • the promoter is derived from viruses and can therefore be referred to as viral promoters, or they are derived from any organism, including prokaryotic or eukaryotic organisms.
  • the promoter is used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III).
  • RNA polymerase e.g., pol I, pol II, pol III.
  • Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter (e.g.
  • SEQ ID NO: 76 is or comprising SEQ ID NO: 76, or a variant thereof) such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1 ;31(17)), a human Hl promoter (Hl), and the like.
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • U6 small nuclear promoter U6 small nuclear promoter
  • Hl human Hl promoter
  • a nucleotide sequence encoding a present RNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an Hl promoter, and the like).
  • a promoter operable in a eukaryotic cell e.g., a U6 promoter, an enhanced U6 promoter, an Hl promoter, and the like.
  • a promoter operable in a eukaryotic cell e.g., a U6 promoter, an enhanced U6 promoter, an Hl promoter, and the like.
  • a promoter operable in a eukaryotic cell e.g., a U6 promoter, an enhanced U6 promoter, an Hl promoter, and the like.
  • the RNA may need to be mutated if there are several Ts in a row (coding for Us in the RNA).
  • a nucleotide sequence encoding a present protein is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EFla promoter, an estrogen receptor- regulated promoter, and the like).
  • the inducible promoter includes, but are not limited to, one of T7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline- regulated promoter, Steroid-regulated promoter, Metal -regulated promoter, estrogen receptor- regulated promoter, and the like.
  • Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; estrogen and/or an estrogen analog; IPTG; and the like.
  • inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol -regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast,
  • the promoter is a spatially restricted promoter (e.g., cell type specific promoter, tissue specific promoter, and the like) such that in a multi-cellular organism, the promoter is active (e.g., “ON”) in a subset of specific cells.
  • Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell; prokaryotic cell).
  • the promoter is a reversible promoter.
  • Suitable reversible promoters including reversible inducible promoters are known in the art.
  • Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art.
  • Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), and the like), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, and the like), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, and the like), metal regulated promoters (e.g., metallothionein promoter systems, and the like), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene
  • the vector contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector includes appropriate sequences for amplifying expression.
  • the vector includes nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag (e.g., GSGPKKKRKVAAAYPYDVPDYA (SEQ ID NO: 77)), fluorescent protein, and the like) that are fused to the present protein, e.g., at the N- or C- terminus or between the N- or C- terminus, thus resulting in a fusion present polypeptide.
  • protein tags e.g., 6xHis tag, hemagglutinin tag (e.g., GSGPKKKRKVAAAYPYDVPDYA (SEQ ID NO: 77)
  • fluorescent protein e.g., fluorescent protein, and the like
  • nucleic acid e.g., a nucleic acid comprising a donor polynucleotide sequence, one or more nucleic acids encoding a present protein and/or a present RNA, and the like
  • any convenient method is used to introduce a nucleic acid (e.g., an expression construct) into a cell.
  • Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome- mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.
  • PEI polyethyleneimine
  • introducing the recombinant expression vector into cells can occur in any culture media and under any culture conditions that promote the survival of the cells. In embodiments, introducing the recombinant expression vector into a target cell is carried out in vivo or ex vivo. In embodiments, introducing the recombinant expression vector into a target cell is carried out in vitro.
  • the present protein (e.g., endonuclease, chimeric protein) is provided as a nucleic acid.
  • the present protein (e.g., endonuclease, chimeric protein) is provided as RNA.
  • the present protein (e.g., endonuclease, chimeric protein) is provided as DNA.
  • the RNA is generated by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the present protein). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, and the like).
  • a nucleic acid into a host cell e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like.
  • Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like.
  • a target cell e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like.
  • Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection,
  • nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g., Angel and Yanik (2010) PLoS ONE 5(7): el 1756, and the commercially available TRANSMESSENGER reagents from Qiagen, STEMFECT RNA Transfection Kit from Stemgent, and TRANSIT-mRNA Transfection Kit from Mirus Bio LLC. See also Beumer et al. (2008) PNAS 105(50): 19821-19826.
  • the present disclosure provides a lipid nanoparticle comprising the nucleic acid of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a cell comprising a nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, or the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein.
  • the cell is a prokaryotic cell. In embodiments, the cell is a eukaryotic cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is a human cell. In embodiments, the cell is an immortalized cell. In embodiments, the cell is harvested from a subject.
  • the cell derived from the subject is derived from a biological sample.
  • the biological sample comprises a biopsy, tissue or bodily fluid.
  • the biological sample comprises one or more of tumor cells, cultured cells, stem cells, and differentiated cells.
  • biological sample refers to a sample obtained or derived from a source of interest (e.g., a cell), as described herein.
  • a source of interest comprises an organism, such as an animal or human.
  • a biological sample is a biological tissue or fluid.
  • Non-limiting examples of biological samples include bone marrow, blood, blood cells, ascites, (tissue or fine needle) biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, swabs (e.g., skin swabs, vaginal swabs, oral swabs, and nasal swabs), washings or lavages such as a ductal lavages or broncheoalveolar lavages, aspirates, scrapings, specimens (e.g., bone marrow specimens, tissue biopsy specimens, and surgical specimens), feces, other body fluids, secretions, and/or excretions, and cells therefrom, and the like.
  • swabs e.g., skin swabs, vaginal swabs, oral swabs, and nasal sw
  • the present disclosure provides a modified cell comprising a composition of the present disclosure.
  • the present disclosure provides a modified cell comprising a composition of the present disclosure, where the modified cell is a cell that does not normally comprise a composition of the present disclosure.
  • the present disclosure provides a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding a composition of the present disclosure.
  • a genetically modified cell that is genetically modified with an mRNA comprising a nucleotide sequence encoding a composition of the present disclosure.
  • a genetically modified cell that is genetically modified with a recombinant expression vector comprising a composition of the present disclosure comprising a composition of the present disclosure.
  • the cells are primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; and the like.
  • a cell that serves as a recipient for a composition of the present disclosure is referred to as a “host cell” or a “target cell.”
  • the host cell or a target cell can be a recipient of a composition or system of the present disclosure.
  • a host cell or a target cell can be a recipient of a RNP of the present disclosure.
  • a host cell or a target cell can be a recipient of a single component of a k system of the present disclosure.
  • Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, homworts, liverworts, mosses, dicotyledons, monocotyledons, and the like), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochlor opsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • seaweeds e.g. kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, and the like)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); and the like), and the like.
  • the cell is a cell that does not originate from a natural organism (e.g
  • the cell is an in vitro cell (e.g., established cultured cell line).
  • a cell can be an ex vivo cell (cultured cell from an individual).
  • the cell is in vivo cell (e.g., a cell in an individual).
  • the cell is an isolated cell.
  • the cell is a cell inside of an organism.
  • the cell is an organism.
  • the cell is a cell in a cell culture (e.g., in vitro cell culture).
  • the cell is cell in a collection of cells.
  • the cell is a prokaryotic cell or derived from a prokaryotic cell, cell culture (e. , in vitro cell culture).
  • the cell is a mammalian cell or derived from a mammalian cell. In embodiments, the cell is a rodent cell or derived from a rodent cell. In embodiments, the cell is a human cell or derived from a human cell. In embodiments, the cell is a microbe cell or derived from a microbe cell. In embodiments, the cell is a fungal cell or derived from a fungal cell. In embodiments, the cell is an insect cell. In embodiments, the cell is an arthropod cell. In embodiments, the cell is a protozoan cell.
  • the suitable cells include a stem cell (e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, and the like); a somatic cell, e.g., a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, and the like.
  • a stem cell e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell
  • a germ cell e.g., an oocyte, a sperm, an oogonia, a spermatogonia, and the like
  • a somatic cell e.g., a fibroblast, an oli
  • the suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and
  • the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.
  • the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage.
  • the immune cell is a cytotoxic T cell.
  • the immune cell is a helper T cell.
  • the immune cell is a regulatory T cell (Treg).
  • the cell is a stem cell.
  • Stem cells include adult stem cells.
  • Adult stem cells are also referred to as somatic stem cells.
  • Adult stem cells are resident in differentiated tissue but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found.
  • somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.
  • Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc.
  • the stem cell is a human stem cell.
  • the stem cell is a rodent (e.g., a mouse; a rat) stem cell.
  • the stem cell is a non-human primate stem cell.
  • the stem cell is a hematopoietic stem cell (HSC).
  • HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34 + and CD3". HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.
  • the stem cell is a neural stem cell (NSC).
  • NSCs neural stem cells
  • a neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively.
  • Methods of obtaining NSCs are known in the art.
  • the stem cell is a mesenchymal stem cell (MSC).
  • MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.
  • the cell is a plant cell.
  • the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes, Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like.
  • a major agricultural plant e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice
  • the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, com stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns
  • the cell is an arthropod cell.
  • the cell can be a cell of a sub-order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopter
  • the cell is an insect cell.
  • the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.
  • the present disclosure provides a pharmaceutical composition comprising the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, or the cell of any one of the embodiments and/or aspects disclosed herein, and a pharmaceutically acceptable carrier.
  • compositions described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art.
  • Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.
  • salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenyl acetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxy benzoate, methyl
  • Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH- lower alkylamines), such as mono-; bis, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert- butylamine, or tris-(hydroxymethyl)
  • compositions described herein are in the form of a pharmaceutically acceptable salt.
  • the present invention pertains to pharmaceutical compositions comprising the compositions described herein and a pharmaceutically acceptable carrier or excipient.
  • Any pharmaceutical compositions described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle.
  • Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
  • pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutically acceptable excipients are sterile when administered to a subject.
  • Water is a useful excipient when any agent described herein is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions.
  • Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents. Other examples of suitable pharmaceutical excipients are described in Remington ’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
  • the present invention includes the described pharmaceutical compositions (and/or additional therapeutic agents) in various formulations.
  • Any inventive pharmaceutical composition (and/or additional therapeutic agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powder, frozen suspension, desiccated powder, or any other form suitable for use.
  • the composition is in the form of a capsule.
  • the composition is in the form of a tablet.
  • the pharmaceutical composition is formulated in the form of a soft-gel capsule.
  • the pharmaceutical composition is formulated in the form of a gelatin capsule.
  • the pharmaceutical composition is formulated as a liquid.
  • inventive pharmaceutical compositions can also include a solubilizing agent.
  • the agents can be delivered with a suitable vehicle or delivery device as known in the art.
  • Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.
  • the formulations comprising the inventive pharmaceutical compositions (and/or additional agents) of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).
  • any pharmaceutical compositions (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
  • Routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically.
  • Administration can be local or systemic. In embodiments, the administration is by parenteral injection.
  • the mode of administration can be left to the discretion of the practitioner and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.
  • compositions described herein is formulated in accordance with routine procedures as a composition adapted for oral administration.
  • Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.
  • Selectively permeable membranes surrounding an osmotically active driving any compositions described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
  • These delivery platforms can provide an essentially zero order delivery profde as opposed to the spiked profiles of immediate release formulations.
  • a timedelay material such as glycerol monostearate or glycerol stearate can also be useful.
  • Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.
  • the excipients are of pharmaceutical grade.
  • Suspensions in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.
  • Dosage forms suitable for parenteral administration include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
  • Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl paraben
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as EDTA
  • buffers such as acetates, citrates or phosphates
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the carrier should be stable under the conditions of manufacture and storage and should be preserved against microorganisms.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
  • compositions provided herein can be made into aerosol formulations (e.g., “nebulized”) to be administered via inhalation.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Any inventive pharmaceutical compositions (and/or additional agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.
  • Such dosage forms can be useful for providing controlled-or sustained-release of one or more active ingredients using, for example, hydropropyl cellulose, hydropropylmethyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein.
  • the invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.
  • Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
  • a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release , supra ⁇ vol. 2, pp. 115-138 (1984)).
  • Other controlled-release systems discussed in the review by Langer, 1990, Science 249: 1527-1533 may be used.
  • compositions preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
  • trans-splicing system comprising the compositions or systems of the present disclosure.
  • the trans-splicing system comprises a repair template comprising a splice donor and/or a splice acceptor.
  • the trans- splicing system further comprises a repair template lacking a splice donor and/or a splice acceptor.
  • the trans-splicing system comprises a splice donor and a splice acceptor. In embodiments, the trans-splicing system comprises a splice donor, a splice acceptor, and the trans- splicing system is suitable for replacing an internal exon in a gene.
  • the trans-splicing system comprises a repair template comprising a splice donor and/or a splice acceptor. In embodiments, the trans-splicing system comprises a repair template comprising a splice donor and a splice acceptor. In embodiments, the trans-splicing system comprises a repair template comprising a splice donor and a splice acceptor, and the trans-splicing system and/or repair template is suitable for replacing an internal exon in a gene. In embodiments, there is provided a method of trans-splicing an exon in a target nucleic acid, e.g.
  • the method comprising contacting the cell with the trans-splicing system disclosed herein, wherein the trans-splicing system comprises a repair template comprising a splice donor and a splice acceptor.
  • the present disclosure provides a method of trans-splicing an exon in a target nucleic acid, e.g. a pre-mRNA in a cell in a subject in need thereof, comprising administering an effective amount of the trans-splicing system disclosed herein, wherein the trans- splicing system comprises a repair template comprising a splice donor and a splice acceptor.
  • the trans-splicing system further comprises a repair template lacking a splice donor and/or a splice acceptor.
  • the trans-splicing system is suitable for splicing of a target nucleic acid comprising a splice donor or a splice acceptor site.
  • the trans-splicing system comprises an RNA molecule, e.g. gRNA that targets a splice acceptor site.
  • the trans-splicing system comprises an RNA molecule, e.g. gRNA that targets a splice donor site.
  • the trans-splicing system comprises is regulated or regulatable by a small molecule.
  • the small molecule is selected from abscisic acid (ABA), rapamycin (or rapalog), FK506, Cyclosporine A, FK1012, Gibberellin3-AM, FKCsA, AP1903/AP20187, and auxin.
  • the pre-mRNA is at an intron-exon junction or exon-intron junctions.
  • the trans-splicing system comprises a pre-trans-splicing (PTS) molecule, wherein the PTS molecule comprises: i) one or more guideRNAs (gRNAs) that target a pre-mRNA; ii) an intronic sequence having a splice signal; and iii) a donor sequence encoding a gene product of a gene of interest, or portion thereof.
  • the gRNA or gRNAs are within the PTS and processed by the endonuclease.
  • the gRNA or gRNAs are within the PTS and are not processed by the endonuclease.
  • the gRNA or gRNAs are not within the PTS.
  • the PTS is the repRNA.
  • the trans-splicing system comprises a repair RNA (repRNA) sequence, comprising: (a) one or more exons and/or introns; (b) a splice donor and/or splice acceptor, wherein the repRNA is suitable for trans-splicing.
  • the trans-splicing system comprises a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and/or introns; and (b) a splice donor and/or splice acceptor.
  • the repRNA comprises one or more binding motifs that direct, and/or hybridizes, the repRNA to a target nucleic acid molecule.
  • the repRNA further comprises a guide RNA (gRNA).
  • the gRNA hybridizes to the target nucleic acid molecule.
  • the gRNA directs the repRNA to a target nucleic acid molecule.
  • the repRNA is operably linked to one or more sequences that are antisense to the target nucleic acid molecule. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and/or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is not operably linked to one or more sequences that bind to, and/or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is provided in trans to one or more sequences that bind to, and/or hybridize to, the target nucleic acid molecule. In embodiments, the repRNA is operably linked to one or more sequences that bind to, and/or hybridize to, an RNA- binding polypeptide. In embodiments, the repRNA is provided in cis to one or more sequences that bind to, and/or hybridize to, a RNA-binding polypeptide.
  • the repRNA is not operably to one or more sequences that bind to, and/or hybridize to, a RNA-binding polypeptide, which is herein described as a “grepRNA”.
  • the grepRNA comprises a repair RNA and a gRNA.
  • “grepRNA” is used interchangeably with “PTS”.
  • the trans-splicing system is suitable for splicing of a target nucleic acid comprising a splice donor or a splice acceptor site.
  • the trans-splicing system comprises an RNA molecule, e.g. gRNA that targets a splice acceptor site.
  • the trans-splicing system comprises an RNA molecule, e.g. gRNA that targets a splice donor site.
  • the trans-splicing system comprises is regulated or regulatable by a small molecule.
  • the small molecule is selected from abscisic acid (ABA), rapamycin (or rapalog), FK506, Cyclosporine A, FK1012, Gibberellin3-AM, FKCsA, AP1903/AP20187, and auxin.
  • the pre-mRNA is at an intron-exon junction or exon-intron junctions.
  • the trans-splicing system comprises a pre-trans-splicing (PTS) molecule, wherein the PTS molecule comprises: i) one or more guideRNAs (gRNAs) that target a pre-mRNA; ii) an intronic sequence having a splice signal; and iii) a donor sequence encoding a gene product of a gene of interest, or portion thereof.
  • the gRNA or gRNAs are within the PTS and processed by the endonuclease.
  • the gRNA or gRNAs are within the PTS and are not processed by the endonuclease.
  • the gRNA or gRNAs are not within the PTS.
  • the endonuclease is fused to a nucleic acid-interacting domain (e.g. MCP) and the PTS is tethered by the nuclease by interacting with the nucleic acid-interacting domain.
  • one or more gRNAs hybridize to one or more pre-mRNAs.
  • the endonuclease is catalytically active.
  • the endonuclease is catalytically inactive.
  • the endonuclease (or chimera) is delivered to a cell with viral methods.
  • the endonuclease (or chimera), and gRNA(s) or PTS are delivered or deliverable with viral methods.
  • the endonuclease (or chimera), gRNA(s), and PTS are delivered or deliverable with viral methods.
  • the endonuclease (or chimera) is delivered or deliverable to a cell with non-viral methods.
  • the endonuclease (or chimera), and gRNA(s) or PTS are delivered or deliverable with non-viral methods.
  • the endonuclease (or chimera), gRNA(s), and PTS are delivered or deliverable with non-viral methods.
  • the endonuclease (or chimera) is delivered or deliverable to a cell implicated in disease.
  • the endonuclease (or chimera) mediates trans-splicing of a pre-mRNA implicated in disease.
  • the RNA molecule binds to or interacts with the present endonuclease.
  • the RNA molecule binds to or interacts with the present chimeric protein.
  • the RNA molecule binds to or interacts with the present target nucleic acid.
  • the RNA molecule is or comprises a guide ribonucleic structure configured to form a complex with the endonuclease.
  • the guide ribonucleic structure (i) comprises (a) a CRISPR RNA (crRNA) suitable for hybridizing to a target nucleic acid molecule and/or (b) a transactivating CRISPR RNA (tracrRNA) suitable for interacting with the endonuclease or (ii) lacks a (a) a crRNA suitable for hybridizing to a target nucleic acid molecule and/or (b) a tracrRNA suitable for interacting with the endonuclease.
  • crRNA CRISPR RNA
  • tracrRNA transactivating CRISPR RNA
  • the RNA molecule is or comprises a gRNA.
  • the gRNA comprises a sequence that interacts with the endonuclease.
  • the endonuclease forms a complex with the gRNA.
  • the RNA molecule contains a repeat and spacer. In embodiments, the RNA has a repeat before a spacer. In embodiments the RNA has a spacer before a repeat. In embodiments the RNA has a repeat before a spacer before a repeat. In embodiments, the RNA has multiple spacers separated by multiple repeats. In embodiments, the RNA has multiple gRNAs. In embodiments, the RNA has multiple gRNAs and is cleaved or processed to separate the gRNAs. In embodiments, the RNA has spacers that target different nucleic acid targets. In embodiments, the RNA has spacers that target the same nucleic acid target.
  • the endonuclease repeat sequences to formulate the guide RNA are selected from Table 3 below.
  • the guide RNA is or comprises a sequence of SEQ ID NO: 28 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the guide RNA is or comprises a sequence of SEQ ID NO: 28 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • the endonuclease is a polypeptide of SEQ ID NO: 1, or a fragment or a variant thereof, or a nucleic acid polypeptide of SEQ ID NO: 1, or a fragment or variant thereof.
  • the endonuclease is guided to a target nucleic acid using a guide RNA of SEQ ID NO: 28, or a fragment or variant thereof and/or the endonuclease is associated with a guide RNA of SEQ ID NO: 28, or a fragment or variant thereof.
  • the endonuclease is SEQ ID NO: 1, or a fragment or variant thereof
  • the guide RNA is SEQ ID NO: 28.
  • the RNA molecule is 10-50 nucleotides in length, e.g., 10-45, 10-40, 10-35, 10- 30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38
  • the present disclosure provides a composition comprising a nucleic acid encoding an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, in conjunction with an RNA containing a repeat having at least about 70% identity to SEQ ID NO: 28.
  • the composition has least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%%, or at least about 97%, or at least about 98%, or at least about 99%) identity to SEQ ID NO: 28, or has about 1 to about 20 nucleotide modifications (e.g. about 1 , or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).
  • the sequence comprises at least one HEPN domain, or fragments or variants thereof.
  • the sequence comprises at least two HEPN domains, or fragments or variants thereof
  • a nucleic acid disclosed herein has one or more modifications, e.g., a base modification, a backbone modification, and the like, to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).
  • a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to the 2', the 3', or the 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are suitable.
  • linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • a suitable nucleic acid modification include, but are not limited to: 2'0 methyl modified nucleotides, 2' Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5' cap (e.g., a 7-methylguanylate cap (m7G)).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • a 2’-O-Methyl modified nucleotide also referred to as 2’-O-Methyl RNA
  • 2’-O-Methyl RNA is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification.
  • Oligonucleotides can be directly synthesized that contain 2'-O-Methyl RNA. Without wishing to be bound by theory, this modification increases Tm of RNA:RNA duplexes but results in only small changes in RNA:DNA stability. It is stabile with respect to attack by single-stranded ribonucleases and is typically about 5 to about 10-fold less susceptible to DNases than DNA.
  • 2' Fluoro modified nucleotides e.g., 2' Fluoro bases
  • Tm binding affinity
  • LNA bases have a modification to the ribose backbone that locks the base in the C3'-endo position, which favors RNA A-type helix duplex geometry. Without wishing to be bound by theory, this modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an oligo at any position except the 3 '-end. Applications have been described ranging from antisense oligos to hybridization probes to SNP detection and allele specific PCR. Due to the large increase in Tm conferred by LNAs, they also can cause an increase in primer dimer formation as well as self-hairpin formation. In embodiments, the number of LNAs incorporated into a single oligo is 10 bases or less.
  • the phosphorothioate (PS) bond (e.g., a phosphorothioate linkage) substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo).
  • this modification renders the intemucleotide linkage resistant to nuclease degradation.
  • Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5'- or 3'-end of the oligo to inhibit exonuclease degradation. Including phosphorothioate bonds within the oligo (e.g., throughout the entire oligo) can help reduce attack by endonucleases as well.
  • nucleic acids optionally containing modifications, include nucleic acids containing modified backbones or non-natural internucleoside linkages.
  • nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothi oates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphorami dates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 ’-5’ linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to
  • Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e., a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
  • MMI type intemucleoside linkages are disclosed in the above referenced U.S. Pat. No.
  • Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • the present nucleic acid is in the form of or comprises a nucleic acid mimetic.
  • the term “mimetic” as it is applied to polynucleotides is intended to include polynucleotides where only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate.
  • the heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • the sugar- backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • the present nucleic acid is or comprises a mimetic based on morpholino units (morpholino nucleic acid), having heterocyclic bases attached to the morpholino ring.
  • morpholino nucleic acid morpholino nucleic acid
  • a number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid.
  • One class of linking groups has been selected to give a non-ionic oligomeric compound.
  • the non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins.
  • Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R.
  • Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506, the disclosure of which is incorporated herein by reference in its entirety. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.
  • the present nucleic acid is a mimetic is in the form of or comprises cyclohexenyl nucleic acids (CeNA).
  • CeNA cyclohexenyl nucleic acids
  • the furanose ring normally present in a DNA/RNA molecule is replaced with a cyclohexenyl ring.
  • CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595- 8602, the disclosure of which is incorporated herein by reference in its entirety).
  • the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid.
  • CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes.
  • the study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.
  • the present nucleic acid comprises Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C, 4'-C- oxymethylene linkage thereby forming a bicyclic sugar moiety.
  • LNAs Locked Nucleic Acids
  • the linkage can be a methylene (- CH2-), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of which is incorporated herein by reference in its entirety).
  • the present nucleic acid comprises one or more substituted sugar moieties.
  • Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N- alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • Suitable polynucleotides comprise a sugar substituent group selected from: Ci to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a sugar substituent group selected from: Ci to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alka
  • a suitable modification includes 2'- methoxyethoxy (2'-O-CH2 CH2OCH3, also known as 2'-O-(2 -methoxyethyl) or 2'-M0E) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety) i.e., an alkoxyalkoxy group.
  • a further suitable modification includes 2'- dimethylaminooxy ethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMA0E, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy -ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.
  • 2'- dimethylaminooxy ethoxy i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMA0E, as described in examples hereinbelow
  • 2'-dimethylaminoethoxyethoxy also known in the art as 2'-O-dimethyl-amino-ethoxy -ethyl or 2'-DMAEOE
  • 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position.
  • a suitable 2'- arabino modification is 2'-F.
  • Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • the present nucleic acid comprises one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Certain nucleobases are, without wishing to be bound by theory, useful for increasing the binding affinity of an oligomeric compound. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability and are suitable base substitutions, e.g., when combined with 2'-O-methoxyethyl sugar modifications.
  • the present compositions are chemically linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • the moieties or conjugates include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject nucleic acid.
  • the conjugate may include a Protein Transduction Domain or PTD (also known as a CPP - cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • PTD Protein Transduction Domain
  • a PTD is covalently linked to the 3' end of an exogenous polynucleotide.
  • a PTD is covalently linked to the 5' end of an exogenous polynucleotide.
  • kits comprising a container comprising the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein and with instructions for use in modulating and/or modifying a nucleic acid.
  • kits for carrying out the methods described herein.
  • the kit comprises a composition described herein, a recombinant expression vector, a delivery system, and/or a pharmaceutical composition described herein, optionally further with a reagent for reconstitution and/or dilution.
  • the present disclosure provides a method of modulating and/or modifying a nucleic acid in a cell, comprising contacting the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a method of modulating and/or modifying a nucleic acid in a subject in need thereof, comprising administering an effective amount of the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein to the subject.
  • the modulating and/or modifying is selected from one or more of cleaving, nicking, methylating, labeling, and mutating the nucleic acid. In embodiments, the modulating and/or modifying is selected from one or more of cleaving the nucleic acid; inserting a nucleic acid, editing the nucleic acid; modulating transcription from the nucleic acid; isolating the nucleic acid, binding the nucleic acid, and imaging the nucleic acid.
  • the present disclosure provides a method of disrupting, correcting, and/or replacing a gene in a cell, comprising contacting the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a method of disrupting, correcting, and/or replacing a gene in a subject in need thereof, comprising administering an effective amount of the composition of any one of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein to the subject.
  • the present disclosure provides a method of trans-splicing a nucleic acid, e.g. a pre- mRNA in a cell, the method comprising contacting the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a method of trans-splicing a nucleic acid, e.g. a pre- mRNA in a cell, in a subject in need thereof, comprising administering an effective amount of the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the trans-splicing method further comprises use of a repair template comprising a splice donor and/or a splice acceptor. In embodiments, the trans-splicing method further comprises use of a repair template lacking a splice donor and/or a splice acceptor. In embodiments, the trans-splicing method provides splicing of a target nucleic acid comprising a splice donor or a splice acceptor site. In embodiments, the trans-splicing method uses an RNA molecule, e.g. gRNA that targets a splice acceptor site. In embodiments, the trans-splicing method uses an RNA molecule, e.g.
  • the activity of at least one portion of the system used in the trans-splicing methods is regulated by a small molecule.
  • the small molecule is selected from abscisic acid (ABA), rapamycin (or rapalog), FK506, Cyclosporine A, FK1012, Gibberellin3-AM, FKCsA, AP1903/AP20187, and auxin.
  • the pre-mRNA is at an intron-exon junction or exon-intron junctions.
  • the trans-splicing method further comprises use of a pre-trans-splicing (PTS) molecule, wherein the PTS molecule comprises: i) one or more guideRNAs (gRNAs) that target a pre-mRNA; ii) an intronic sequence having a splice signal; and iii) a donor sequence encoding a gene product of a gene of interest, or portion thereof.
  • the gRNA or gRNAs are within the PTS and processed by the endonuclease.
  • the gRNA or gRNAs are within the PTS and are not processed by the endonuclease.
  • the gRNA or gRNAs are not within the PTS.
  • the endonuclease is fused to a nucleic acid-interacting domain (e.g. MCP) and the PTS is tethered by the nuclease by interacting with the nucleic acidinteracting domain.
  • one or more gRNAs hybridize to one or more pre-mRNAs.
  • the endonuclease is catalytically active.
  • the endonuclease is catalytically inactive.
  • the endonuclease (or chimera) is delivered to a cell with viral methods.
  • the endonuclease (or chimera), and gRNA(s) or PTS are delivered with viral methods.
  • the endonuclease (or chimera), gRNA(s), and PTS are delivered with viral methods.
  • the endonuclease (or chimera) is delivered to a cell with non-viral methods.
  • the endonuclease (or chimera), and gRNA(s) or PTS are delivered with non-viral methods.
  • the endonuclease (or chimera), gRNA(s), and PTS are delivered with non-viral methods.
  • the endonuclease (or chimera) is delivered to a cell implicated in disease.
  • the endonuclease (or chimera) mediates trans-splicing of a pre-mRNA implicated in disease.
  • the present disclosure provides a method of modifying a nucleic acid in a cell using a method of exon skipping, comprising contacting the cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the present disclosure provides a method of modifying a nucleic acid in a cell in a subject in need thereof, using a method of exon skipping, comprising administering an effective amount of the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein.
  • the cell is induced to skip over faulty sections of pre-mRNA molecules by interfering with mRNA splicing.
  • the methods produce a truncated, but functional, protein despite the presence of a mutation.
  • the method of exon skipping involves binding an oligonucleotide (e.g. without limitation the present RNA molecule) to a splice site in a pre-mRNA molecule.
  • the corresponding exon is skipped over, which, for example, restores a disrupted reading frame caused by the mutation.
  • the exon skipping allows translation of an internally-deleted, but substantially functional protein.
  • the present exon skipping methods comprise creating a single-strand or doublestrand break in a gene.
  • the single-strand or double-strand break causes persistent altered splicing of the gene.
  • the altered splicing results in expression of a truncated protein which lacks at least the polypeptide sequence corresponding to an exon containing the mutation.
  • the single-strand or double-strand break removes a splice acceptor site or produces a non-functional splice acceptor site in or near an exon of the gene or removes a splice donor site or produces a non-functional splice donor site in or near an exon of the gene.
  • the present disclosure provides a method of treating, ameliorating or preventing a disease or disorder in a subject, comprising (a) contacting a cell with the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein, and (b) administering an effective amount of the cell to the subject.
  • the present disclosure provides a method of treating, ameliorating or preventing a disease or disorder in a subject, comprising administering an effective amount of the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein to the subj ect.
  • the present disclosure provides use of the composition of any one of the embodiments and/or aspects disclosed herein, the nucleic acid of any one of the embodiments and/or aspects disclosed herein, the viral vector of any one of the embodiments and/or aspects disclosed herein, the lipid nanoparticle of any one of the embodiments and/or aspects disclosed herein, the cell of any one of the embodiments and/or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and/or aspects disclosed herein in the manufacture of a medicament for the treating, ameliorating or preventing of a disease or disorder.
  • the present disclosure provides a method of detecting and/or quantifying a nucleic acid in a sample, comprising contacting the sample with a composition of any one of the embodiments and/or aspects disclosed herein.
  • the nucleic acid is a target and/or reporter nucleic acid.
  • the method comprises detection of a reporter signal, the reporter signal being generated upon endonuclease cleavage.
  • the reporter signal is a fluorescent signal.
  • the endonuclease has collateral cleavage activity.
  • a composition of any one of the embodiments and/or aspects disclosed herein can be used to (i) modify (e.g., cleave, e.g., nick; methylate; and the like) 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, and the like); (v) modify a polypeptide (e.g., a histone) associated with a target nucleic acid; and the like.
  • modify e.g., cleave, e.g., nick; methylate; and the like
  • target nucleic acid DNA or RNA; single stranded or double stranded
  • the present disclosure provides a method of modifying a target nucleic acid.
  • a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with (a) a composition of the present disclosure (e.g. an endonuclease and/or a chimeric protein of any one of the embodiments and/or aspects disclosed herein); and (b) one or more (e.g., two) RNAs of any one of the embodiments and/or aspects disclosed herein.
  • a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a composition of the present disclosure (e.g.
  • the contacting step is carried out in a cell in vitro. In embodiments, the contacting step is carried out in a cell in vivo. In embodiments, the contacting step is carried out in a cell ex vivo.
  • a method of binding may result in nothing more than binding of the target nucleic acid
  • the method can have different final results (c. ., the method can result in modification of the target nucleic acid, e.g., cleavage/methylation/and the like, modulation of transcription from the target nucleic acid; modulation of translation of the target nucleic acid; genome editing; modulation of a protein associated with the target nucleic acid; isolation of the target nucleic acid; and the like).
  • Jinek et al. Science. 2012 Aug 17;337(6096): 816-21 ; Chylinski et al., RNA Biol. 2013 May;10(5):726-37; Ma et al., Biomed Res Int. 2013 ;2013:270805; Hou et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15644-9; Jinek et al., Elife. 2013;2:e00471 ; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43; Qi et al, Cell.
  • 20140349400 20140349405; 20140356867; 20140356956; 20140356958; 20140356959 20140357523; 20140357530; 20140364333; and 20140377868; each of which is hereby incorporated by reference in its entirety.
  • the present disclosure provides (but is not limited to) methods of cleaving a target nucleic acid; methods of editing a target nucleic acid; methods of modulating transcription from a target nucleic acid; methods of isolating a target nucleic acid, methods of binding a target nucleic acid, methods of imaging a target nucleic acid, methods of modifying a target nucleic acid, and the like.
  • a present polypeptide e.g. of the present endonuclease and/or chimeric protein
  • RNA encoding the present polypeptide
  • DNA encoding the present polypeptide
  • a present RNA can be provided as a RNA or as a nucleic acid encoding the RNA.
  • a method that includes contacting the target nucleic acid encompasses the introduction into the cell of any or all of the components in their active/final state (e.g., in the form of a protein(s) for a polypeptide; in the form of a protein for a chimeric polypeptide; in the form of an RNA in embodiments for the present RNA), and also encompasses the introduction into the cell of one or more nucleic acids encoding one or more of the components (e.g., nucleic acid(s) comprising nucleotide sequence(s) encoding a present polypeptide or a present chimeric polypeptide, nucleic acid(s) comprising nucleotide sequence(s) encoding guide RNA(s), nucleic acid comprising a nucleotide sequence en
  • a method that includes contacting a target nucleic acid encompasses contacting outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, or inside of a cell ex vivo.
  • the method of the present disclosure for modifying a target nucleic acid comprises introducing into a target cell a present locus, e.g., a nucleic acid comprising a nucleotide sequence encoding a present polypeptide as well as nucleotide sequences of about 1 kilobase (kb) to 5 kb in length surrounding the present-encoding nucleotide sequence from a cell e.g., in embodiments, a cell that in its natural state (the state in which it occurs in nature) comprises a present locus) comprising a present locus, where the target cell does not normally (in its natural state) comprise a present locus.
  • a present locus e.g., a nucleic acid comprising a nucleotide sequence encoding a present polypeptide as well as nucleotide sequences of about 1 kilobase (kb) to 5 kb in length surrounding the present-encoding nucleotide sequence from a cell e
  • a method of the present disclosure for modifying a target nucleic acid comprises introducing into a target cell a present locus, e.g., a nucleic acid obtained from a source cell (e.g., in embodiments, a cell that in its natural state (the state in which it occurs in nature) comprises a present locus), where the nucleic acid has a length of from 100 nucleotides (nt) to 5 kb in length e.g., from 100 nt to 500 nt, from 500 nt to 1 kb, from 1 kb to 1.5 kb, from 1.5 kb to 2 kb, from 2 kb to 2.5 kb, from 2.5 kb to 3 kb, from 3 kb to 3.5 kb, from 3.5 kb to 4
  • nt nucleotides
  • one or more spacer sequences, encoding guide sequences for the encoded crRNA(s), can be modified such that one or more target sequences of interest are targeted.
  • the method comprises introducing into a target cell: i) a present locus; and ii) a donor DNA template.
  • the target nucleic acid is in a cell- free composition in vitro.
  • the target nucleic acid is present in a target cell.
  • the target nucleic acid is present in a target cell, where the target cell is a prokaryotic cell.
  • the target nucleic acid is present in a target cell, where the target cell is a eukaryotic cell.
  • the target nucleic acid is present in a target cell, where the target cell is a mammalian cell.
  • the target nucleic acid is present in a target cell, where the target cell is a plant cell.
  • a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a present polypeptide of the present disclosure, or with a present chimeric polypeptide of the present disclosure. In embodiments, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a present polypeptide and a present guide RNA.
  • a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a present polypeptide, a first present guide RNA, and a second present guide RNA In embodiments, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a present polypeptide of the present disclosure and a present guide RNA and a donor DNA template.
  • the “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, and non-human animals (including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested)).
  • the subject is a human.
  • first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first subject could be termed a second subject, and, similarly, a second subject could be termed a first subject, without departing from the scope of the present disclosure. The first subject and the second subject are both subjects, but they are not the same subject. Furthermore, the terms “subject,” “user,” and “patient” are used interchangeably herein.
  • the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • Example 1 Guide RNA Design
  • the experiments of this example evaluated the structure of the CRISPR RNA of the Casl 3K2G system disclosed herein.
  • gRNAs were designed for the Casl3K2G system disclosed herein to target multiple sites across the coding sequence of eGFP in HEK293T cells (FIG. 2).
  • the gRNAs were tested alongside a scrambled non-targeting guide, demonstrating reprogrammability and specificity of the RNase activity of this CRISPR-Cas system towards different targets.
  • the gRNA structure for Casl3K2G system was determined to have a spacer sequence complementary to the target RNA at the 5’ end of the gRNA followed by a CRISPR repeat at the 3' end of the gRNA (see, e.g., FIG. 3).
  • the experiments of this example demonstrate the capability of the Casl3K2G system disclosed herein to target and knockdown eGFP RNA.
  • an SV40 NLS sequence was added to the N-terminus (MSPKKKRKVEAS (SEQ ID NO: 78)) and an SV40 NLS with an appended HA tag was added to the C-terminus (GSGPKKKRKVAAAYPYDVPDYA (SEQ ID NO: 77)).
  • the resulting ORFs were then mammalian codon optimized and synthesized into mammalian expression vectors. Transcription was driven by a CMV promoter, and an SV40 polyadenylation signal was used to terminate transcription.
  • the sequence of the CMV promoter is the following:
  • sequence of the SV40 polyadenylation signal is the following:
  • FIG. 4 shows clear distinction in phylogenetic placement as separate protein families for the Casl3K2G system disclosed herein. Compared to other CAS systems, the Casl3K2G system disclosed herein is novel and shows low sequence similarity to other systems. The results in FIG. 4 are reproduced in Table 4 below.
  • the Casl3K2G family is evaluated for targeted trans-splicing using a truncated GFP reporter assay.
  • the truncated GFP exons (5' GFP, 3' GFP) introduced by the splice donor and acceptor reporters are spliced together, generating a transcript that contains a complete GFP mRNA sequence capable of creating a functional GFP protein product, and thereby demonstrating successful trans-splicing.
  • HEK293T cells are transfected with either a first or second Casl3K2G gRNA targeting the splice donor (SD) reporter (Casl3K2G gRNA 1 or 2), a fusion of catalytically inactive Casl3K2G orthologue (dCasl3K2G) and MS2 (dCasl3K2G-MS2), and the splice acceptor (SA) reporter having MS2 stem loops.
  • SD splice donor
  • SA splice acceptor
  • R70007 are cultured in a humidified incubator at 5% CO2 at 37°C using high-glucose DMEM (Invitrogen) complemented with 10% FBS and 1% penicillin/streptomycin. Cell cultures are kept at low passage ( ⁇ 20) and regularly tested for mycoplasma contamination. Cells are plated in 96- well format at 15,000 cells per well with 100 uL of media. Cells are transfected 24 hours later with 25 ng gRNA plasmid (U6 driven expression), 25 ng Casl 3-RBP plasmid, 25 ng of the SD reporter, and 25 ng of the SA reporter using 0.32 uL lipofectamine and 10 uL of OptiMEM total.
  • DMEM Invitrogen
  • HEK293T cells can be transfected using a dCasl3b variant from Prevotella sp. (“dPspCasl3b”), fused to a panel of RBPs sourced from RNA bacteriophage genomes.
  • dPspCasl3b a dCasl3b variant from Prevotella sp.
  • the experiments of this example will show that cells undergo successful trans-splicing using the Casl3K2G system, which is a level of trans-splicing comparable to that achieved using the dPspCasl3b system.
  • the experiments of this example will further evaluate the dPspCasl3b systems using different RNA binding proteins. These experiments measure trans-splicing between the SD reporter encoding a 5'GFP upstream of a splice donor, and a SA reporter having two sequence motifs upstream a splice donor and exon encoding 3'GFP.
  • the SA reporters that are tested have sequence motifs recognized by RNA binding proteins (RBPs).
  • the sequence motif and corresponding RBP that will be evaluated includes: (i) a MS2 hairpin and a MS2 coat protein (“MS2”); (ii) a PP7 hairpin and a PP7 coat protein (“PP7”); (iii) a M hairpin and a phage M coat protein (“M”); (iv) a PRR1 hairpin and a PRR1 coat protein (“PRR1”); and (v) a Qbeta hairpin and Qbeta coat protein (“QB”).
  • MS2 MS2
  • PP7 a PP7 hairpin and a PP7 coat protein
  • M M hairpin and a phage M coat protein
  • PRR1 PRR1
  • QB Qbeta hairpin and Qbeta coat protein
  • HEK293T cells are transfected with the SD reporter, splice editor (i.e., PspCasl3b gRNA and a fusion of dPspCasl3b to an RBP), and an SA reporter with the corresponding RBP sequence motif.
  • Control cells were transfected with the SD reporter and SA reporter only. Trans-splicing between the SD reporter and SA reporter is expected to yield a measurable GFP signal.
  • the experiments of this example will demonstrate how each of the splice editors that is evaluated will result in a level of trans-splicing that is higher than that observed in control cells.
  • the experiments of this example will evaluate the Casl3K2G system for trans- splicing using an alternate reporter system having a target nucleic acid containing distinct gene segments from an SD reporter (the target nucleic acid (“target”)).
  • the target contains from 5' to 3': the 5' portion of GFP, a first intron from a human protein-coding gene (“gene A”), a splice acceptor, and an exon from gene A.
  • the trans-splicing template (the “template”) contains from 5' to 3': an intron containing two MS2 stem loops, a splice acceptor, and the 3' portion of GFP.
  • the constructs are designed such that successful trans-splicing between the splice donor of the target and the splice acceptor of the template yields a GFP signal.
  • HEK293T cells are transfected using lipofectamine 2000 per manufacturer’s instructions in 96-well format.
  • Each plasmid (target, template, Casl3K2G-MCP+gRNA) is one-third (33 ng) of the total DNA transfection.
  • a non-coding dummy DNA is included in conditions where less than four components are delivered, such that there would be a total of 100 ng of DNA in each transfection. Media is changed 12 hours after the transfection.
  • HEK293T cells are transfected with constructs encoding dCas!3K2G fused to a MS2 coat protein, a gRNA directed to the target, the target, and the template.
  • Control cells are transfected with a non-targeting gRNA or target only. The experiments in this example will show that the editing performance is dramatically increased using the Casl3K2G system, indicating successful trans-splicing of the target.
  • the experiments of this example demonstrate how internal exon replacement is achieved with two gRNAs, two nucleases and RBPs (in this case, PCP and MCP) in combination with, or without, a binding motif (BM) in the repRNA.
  • the experiments in this example show targeting (T) or nontargeting (NT) of two gRNAs, two nucleases and RBPs in combination with, or without, a binding motif (BM) in the repRNA to facilitate internal exon replacement.
  • grepRNA guide repair RNA
  • dCasl3K2G can be used with or without an RBP fusion.
  • internal exon replacement across two different RNA targets can be shown.
  • HEK293T cells are transfected in these experiments with pA0120 reporter plasmid (25 ng), grepRNA (50 ng), and dCas!3K2G-MCP (25 ng).
  • the experiments of this example will show targeting (T) or non-targeting (NT) of an integrated target to facilitate internal exon replacement.
  • transient transfection of the dCasl3K2G-MCP, grepRNA (targeting (T) or non-targeting (NT)), and pUC19 in HEK293FT cells are performed with an integrated USH2A target.
  • Each splice editor (SE) component receives 25 ng for a total of 100 ng/transfection/well in a 96-well plate.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La divulgation concerne des compositions et des procédés qui modifient des acides nucléiques cibles, ainsi que des procédés de détection d'acides nucléiques. Diverses compositions sont décrites, y compris des compositions comprenant des endonucléases, des systèmes d'endonucléase et des protéines chimériques possédant l'endonucléase et un domaine de modulation d'acide nucléique ou un domaine de modification d'acide nucléique.
PCT/US2023/083377 2022-12-09 2023-12-11 Endonucléases de modification génique WO2024124238A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202263386791P 2022-12-09 2022-12-09
US63/386,791 2022-12-09
US202363602526P 2023-11-24 2023-11-24
US63/602,526 2023-11-24

Publications (1)

Publication Number Publication Date
WO2024124238A1 true WO2024124238A1 (fr) 2024-06-13

Family

ID=91380335

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/083377 WO2024124238A1 (fr) 2022-12-09 2023-12-11 Endonucléases de modification génique

Country Status (1)

Country Link
WO (1) WO2024124238A1 (fr)

Similar Documents

Publication Publication Date Title
US12084692B2 (en) Guide scaffolds
US11692184B2 (en) Thermostable RNA-guided endonucleases and methods of use thereof
US20240167052A1 (en) Rna-guided nucleic acid modifying enzymes and methods of use thereof
US11371062B2 (en) RNA-guided nucleic acid modifying enzymes and methods of use thereof
US20200339967A1 (en) Cas12c compositions and methods of use
KR20240095525A (ko) 조작된 casx 억제 시스템
US20220315914A1 (en) Variant type v crispr/cas effector polypeptides and methods of use thereof
US20210380957A1 (en) Crispr-cas effector polypeptides and methods of use thereof
CN116113692A (zh) 用于植入碱基编辑细胞的组合物和方法
WO2020023529A1 (fr) Enzymes de modification d'acide nucléique guidées par arn et procédés d'utilisation de celles-ci
US20210269782A1 (en) Rna-guided effector proteins and methods of use thereof
US20240344087A1 (en) Gene-modifying endonucleases
WO2024124238A1 (fr) Endonucléases de modification génique
WO2024124237A2 (fr) Endonucléases de modification génique
US20230407276A1 (en) Crispr-cas effector polypeptides and methods of use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23901725

Country of ref document: EP

Kind code of ref document: A1