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WO2025072763A2 - Compositions et procédés d'édition de précision avec des dimères cas - Google Patents

Compositions et procédés d'édition de précision avec des dimères cas Download PDF

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Publication number
WO2025072763A2
WO2025072763A2 PCT/US2024/048981 US2024048981W WO2025072763A2 WO 2025072763 A2 WO2025072763 A2 WO 2025072763A2 US 2024048981 W US2024048981 W US 2024048981W WO 2025072763 A2 WO2025072763 A2 WO 2025072763A2
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sequence
nucleic acid
effector protein
retrna
rddp
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WO2025072763A3 (fr
Inventor
Paula GONCALVES CERQUEIRA
Yuchen GAO
Stepan TYMOSHENKO
Rohan Grover
Brian R. CHAIKIND
Benjamin Julius RAUCH
Aaron DELOUGHERY
Kevin Cristopher WILKINS
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Mammoth Biosciences Inc
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Mammoth Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • 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 [CRISPR]
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid

Definitions

  • Precision editing involves very precise modification of a target DNA sequence.
  • precision editing involves modifying only one or a few nucleotides of a target sequence in a genome.
  • precision editing involves inserting or deleting a short nucleotide sequence.
  • precision editing involves replacing a short nucleotide sequence with a desired nucleotide sequence.
  • the desired nucleotide sequence is encoded on a template RNA (retRNA) that serves as a template for an RNA dependent DNA polymerase (RDDP), e.g., a reverse transcriptase.
  • retRNA template RNA
  • RDDP RNA dependent DNA polymerase
  • Precision editing systems described herein generally comprise a compact Cas protein that homodimerizes, and a guide nucleic acid that guides the dimer to atarget sequence in a DNA molecule.
  • each monomer of the dimer is linked to an RDDP.
  • the dimer cuts both strands of DNA leaving staggered ends with 5’ overhangs.
  • precision editing systems described herein comprise two template RNAs (retRNAs), each containing an intended edit or desired nucleotide sequence.
  • a template RNA is designed to hybridize to single stranded DNA created at a cut site.
  • the linked RDDPs polymerize a new strand of DNA at each 3’ end of the cut DNA.
  • Endogenous repair proteins including exonucleases and single stranded break repair proteins, may be useful for repairing the DNA cut site, see FIG. 2.
  • systems comprise DNA repair proteins to enhance DNA repair and further reduce unintended edits. Fusion of DNA repair proteins to the dimerizing Cas protein may enhance repair relative to a Cas protein that doesn’t dimerize, effectively recruiting twice as much repair protein, see, e.g., FIGs. 3 and 4. Without being bound by theory, it is contemplated that polymerization at both cut ends may reduce the chances of byproducts (unintended edits), even though the dimer produces a double stranded break.
  • precision editing systems comprise a homodimerizing effector protein, an RDDP, and two guide RNAs that hybridize to opposite strands of a DNA molecule at some distance apart from each other (e.g., 10-10,000 bp).
  • the effector proteins form ribonucleoprotein (RNP) complexes with the two guide RNAs at these two different sites where they cleave the dsDNA, generating single stranded DNA (ssDNA) flaps at both sites that extend towards each other as shown in FIG. 8.
  • RNP ribonucleoprotein
  • ssDNA single stranded DNA
  • These systems also comprise two retRNAs, each of which binds to a portion of one of the ssDNA flaps.
  • the retRNAs each have a template sequence for generating a sequence of interest while the region between the two cut sites is deleted.
  • Such systems may be referred to as dual-cut dual-flap systems.
  • the present disclosure provides systems for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises: i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5’ of the second region; d) a TS template RNA (retRNA) or nucleic acid encoding the TS retRNA, wherein the TS ret
  • retRNA
  • the effector protein cuts the TS and the NTS to generate staggered ends, thereby generating single stranded DNA with a free 3 ’ end on the TS (TS ssDNA) and single stranded DNA with a free 3’ end on the NTS (NTS ssDNA).
  • TS ssDNA single stranded DNA with a free 3 ’ end on the TS
  • NTS ssDNA single stranded DNA with a free 3’ end on the NTS
  • the TS PBS hybridizes to at least a portion of the TS ssDNA
  • the NTS PBS hybridizes to at least a portion of the NTS ssDNA.
  • the RDDP polymerizes a new strand of ssDNA on the TS that is complementary to the TS template sequence, and a new strand of ssDNA on the NTS that is complementary to the NTS template sequence.
  • the system comprises a DNA repair protein.
  • the DNA repair protein is covalently linked to the effector protein.
  • the DNA repair protein is covalently linked to the RDDP.
  • the RDDP is covalently linked to the effector protein.
  • the guide RNA is covalently linked to the retRNA.
  • the TS template sequence is located 5’ of the TS PBS and the NTS template is located 5 ’ of the NTS PBS, optionally wherein the 3 ’ end of the PBS is linked to the 5’ end of the template sequence.
  • the TS retRNA, NTS retRNA, or each of the TS retRNA and NTS retRNA comprise a protein localization sequence
  • the RDDP comprises or is covalently linked to a peptide that binds the protein localization sequence, the protein localization sequence thereby localizing the RDDP to the retRNA.
  • the TS template sequence and/or NTS template sequence comprises a difference of at least one nucleotide relative to an otherwise identical sequence in the dsDNA molecule (providing the interchangeability of thymines and uracils).
  • the effector protein is a Type V Cas protein.
  • the length of the effector protein is 400 to 500, 400 to 600, 400 to 700, 400 to 800, or 400-900 linked amino acids.
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.
  • the target sequence is a sequence present in a eukaryotic organism, optionally wherein the eukaryotic organism is a mammal, optionally wherein the mammal is a human.
  • the RDDP comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 2-82.
  • the systems comprise an expression vector, wherein the expression vector comprises any combination of: the nucleic acid encoding the effector protein; the nucleic acid encoding the RDDP; the nucleic acid encoding the guide RNA; and the nucleic acids encoding the TS retRNA and NTS retRNA.
  • the expression vector is an adeno-associated viral (AAV) vector, optionally wherein the AAV vector is an scAAV vector.
  • the systems comprise a lipid or lipid nanoparticle.
  • the nucleic acid encoding the effector protein or the nucleic acid encoding the RDDP comprises a messenger RNA.
  • compositions for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises: i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5 ’ of the second region; d) a TS template RNA (retRNA) or nucleic acid encoding the TS retRNA, wherein the TS retRNA
  • the compositions comprise a lipid nanoparticle (LNP), wherein the nucleic acid encoding the effector protein comprises an mRNA, wherein the LNP contains the mRNA, optionally wherein the mRNA comprises the nucleic acid encoding the RDDP, optionally wherein the LNP comprises the guide RNA, the TS retRNA, the NTS retRNA, or a combination thereof.
  • LNP lipid nanoparticle
  • the nucleic acid encoding the effector protein comprises an mRNA
  • the LNP contains the mRNA
  • the mRNA comprises the nucleic acid encoding the RDDP
  • the LNP comprises the guide RNA, the TS retRNA, the NTS retRNA, or a combination thereof.
  • at least one of the guide RNA, the TS retRNA, and the NTS retRNA comprises a chemical modification.
  • compositions comprising the composition described herein, and a pharmaceutically acceptable excipient.
  • the present disclosure provides expression vectors for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule, the expression vector comprising: a) a first nucleotide sequence encoding an effector protein, wherein the effector protein forms a dimer with itself in a cell; b) a second nucleotide sequence encoding a RDDP; c) a third nucleotide sequence encoding a guide RNA, wherein the guide RNA comprises i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5 ’ of the second region; d) a fourth nucleotide sequence encoding a TS retRNA, wherein the TS retRNA comprises: i) a TS primer binding sequence (PBS)
  • the present disclosure provides systems for deleting a region of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a first guide RNA (gRNA) or a nucleic acid encoding the first gRNA, wherein the first gRNA comprises: i) a first scaffold sequence, and ii) a first spacer sequence that hybridizes to a first target sequence on a first strand of the dsDNA molecule, wherein the first scaffold sequence is located 5’ of the first spacer sequence, and wherein the effector protein and the first gRNA form a first RNP complex that cleaves the dsDNA molecule to form a first single stranded DNA (sDNA) molecule, the
  • the nucleic acid encoding the effector protein, the RDDP, the gRNAs, and the retRNAs are combined in a single AAV vector.
  • the first spacer sequence and the second spacer sequence hybridize to the first target sequence and the second target sequence of the dsDNA molecule respectively, wherein the first target sequence and the second target sequence are 10 to 10,000 base pairs apart.
  • the system comprises a BREX27 peptide or a nucleic acid encoding the BREX27 peptide, optionally wherein the BREX27 peptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 112.
  • the present disclosure provides systems for modifying a target strand (TS) of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA comprises: i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS of the dsDNA molecule, wherein the first region is located 5’ of the second region; and wherein the effector protein and the gRNA form a RNP complex that produces a double stranded break; and d) a TS template
  • the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.
  • the effector protein comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is D220R.
  • the effector protein is linked to RDDP to form a fusion protein.
  • the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 115, 117, 119, 122, and 123, wherein the amino acid sequence comprises a P2A peptide that results in cleavage of the fusion protein at the site of the P2A.
  • the gRNA comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 124 or 125.
  • the nucleic acid sequences encoding the effector protein, the RDDP, the gRNA, and the TS retRNAs are combined in a single AAV vector.
  • a nucleic acid sequence encoding the effector protein and a nucleic acid sequence encoding the RDDP is linked by a nucleic acid sequence encoding a P2A peptide.
  • the present disclosure provides compositions comprising an engineered guide nucleic acid or a nucleic acid encoding the same, wherein the guide nucleic acid comprises or consists of a nucleotide sequence according to SEQ ID NO: 129. In some embodiments, the present disclosure provides compositions comprising an engineered guide nucleic acid or a nucleic acid encoding the same, wherein the guide nucleic acid comprises or consists of a nucleotide sequence selected from SEQ ID NO: 130, SEQ ID NO: 131, and a combination thereof. In some embodiments, SEQ ID NO: 131 is located 5’ of SEQ ID NO: 130.
  • the nucleic acid encoding the engineered guide nucleic acid is located in a AAV vector.
  • the composition comprises an effector protein or a nucleic acid encoding the same, wherein the effector protein comprises an amino acid sequence that is at least is 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.
  • the present disclosure provides precision editing systems comprising a fused RNA, wherein the fused RNA comprises: a) a guide RNA (gRNA) comprising from 5’ to 3’: a tracrRNA sequence, a repeat sequence, and a spacer sequence; and b) a template RNA (retRNA), wherein the retRNA is covalently linked to the 5’ end of the gRNA.
  • the fused RNA comprises one or more linker nucleotides between: i) the gRNA and the template RNA; ii) the tracrRNA sequence and the repeat sequence; iii) the repeat sequence and the spacer sequence; or a combination thereof.
  • the systems comprise an effector protein or nucleic acid encoding the same, and an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP.
  • RDDP RNA-directed DNA polymerase
  • compositions comprising an engineered RDDP or a nucleic acid encoding the same, wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R; wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 56 and comprises the amino acid substitutions of N24R, N72R, N114K, and N195R; or wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 65 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R.
  • the present disclosure provides cells modified by the systems described herein, the compositions described herein, or the expression vectors described herein.
  • the cell is a eukaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell, optionally wherein the mammalian cell is a human cell.
  • the present disclosure provides methods of modifying a dsDNA molecule, the method comprising contacting a target nucleic acid with the systems described herein, the compositions described herein, or the expression vectors described herein.
  • FIG. 1A shows an exemplary precision editing system with a dimerized effector protein, RDDPs optionally linked to the effector protein, a guide RNA that localizes the effector protein to a double stranded DNA target nucleic acid, and template RNAs (retRNA) that hybridize to both strands of target DNA.
  • RDDPs optionally linked to the effector protein
  • guide RNA that localizes the effector protein to a double stranded DNA target nucleic acid
  • retRNA template RNAs
  • FIG. 2 shows how target dsDNA may be repaired after it is modified by a precision editing system described herein.
  • the target dsDNA is cut and new DNA complementary to the template sequence is synthesized at the 3’ ends of the cut dsDNA. 5’ overhangs of the cut dsDNA are removed by exogenous or endogenous exonucleases.
  • the newly synthesized single stranded DNA ends anneal to the free ends of the cut dsDNA.
  • the 3’ ends of the cut dsDNA not used in the annealing reaction can be further processed by endogenous flap endonucleases.
  • FIG. 3 shows peptides fused to each monomer of a dimerized compact Cas protein (“dimer”). Such peptides may have DNA repair modulating activity.
  • FIG. 7A - FIG. 7B illustrate editing levels of variant RDDP candidates comprising arginine and lysine mutations in FANCF (FIG. 7A) and HEK FIG. 7B) target sites.
  • FIG. 8 illustrates an exemplary dual-cut dual-flap system for precise deletion of a region of dsDNA.
  • FIG. 9 illustrates an exemplary precision editing system using a Type V Cas nuclease that creates double stranded breaks with the target strand being extended by the RT.
  • FIG. 10 illustrates part of an exemplary plasmid encoding a gRNA and a retRNA that are not fused (split) and an exemplary plasmid encoding a fused RNA (gretRNA) comprising a gRNA fused to a retRNA.
  • FIG. 11 shows the results of an experiment testing the ability of various CasM.265466-RDDP fusion proteins to generate precise edits, as assessed with a fluorescent reporter in mammalian cells.
  • % identical refers to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity.
  • the % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100.
  • computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci.
  • sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • Binding refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a CAS polypeptide/guide RNA complex and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner).
  • Binding interactions are generally characterized by a dissociation constant (KD) of less than KF 6 M, less than IO -7 M, less than IO -8 M, less than IO -9 M, less than IO 10 M, less than 10 11 M, less than 10 12 M, less than 10 13 M, less than 10 14 M, or less than 10 15 M.
  • KD dissociation constant
  • Affinity refers to the strength of binding, increased binding affinity being correlated with a lower KD.
  • binding domain it is meant a protein or nucleic acid domain that is able to bind non- covalently to another molecule.
  • a binding domain can bind to, for example, a DNA molecule (a DNA- binding domain), an RNA molecule (an RNA-binding domain) and/or a protein molecule (a proteinbinding domain).
  • a protein having a protein-binding domain it can in some cases bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more regions of a different protein or proteins.
  • catalytically inactive effector protein refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid.
  • the catalytic activity that is reduced or eliminated is often a nuclease activity but can be nickase activity.
  • the naturally-occurring effector protein may be a wild-type protein.
  • the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein.
  • cleavage refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by a complex of an effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid. Cleavage may occur within or directly adjacent to the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.
  • cleave refers to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond.
  • the result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single -stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double -stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.
  • a nick hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule
  • single strand break hydrolysis of a single phosphodiester bond on a single -stranded molecule
  • double strand break hydrolysis of two phosphodiester bonds on both sides of a double-stranded
  • nucleic acid molecule or nucleotide sequence refers to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid.
  • the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand.
  • the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end.
  • Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.
  • Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (G); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T).
  • Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic -hydroxyl.
  • Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W).
  • Amino acids may be related by amide side chains: Asn (N), Glu (Q).
  • Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M).
  • cleavage assay refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid.
  • the cleavage activity may be cis-cleavage activity.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • donor nucleic acid refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence.
  • % editing efficiency refers to the percent of target nucleic acids in a sample or population of cells exhibiting an edited target nucleic acid. Editing efficiency may also be referred to as % editing level or % edited. There are multiple approaches to evaluate % editing efficiency, including, but not limited to, next generation sequence and real time PCR.
  • target nucleic acid refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein.
  • a target nucleic acid may comprise RNA, DNA, or a combination thereof.
  • a target nucleic acid may be single-stranded (e.g., singlestranded RNA or single -stranded DNA) or double-stranded (e.g., double-stranded DNA).
  • target sequence when used in reference to a target nucleic acid, refers to a sequence of nucleotides found within a target nucleic acid. Such a sequence of nucleotides can, for example, hybridize to a respective length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.
  • a nucleotide sequence that “encodes” a particular polypeptide or protein is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and/or is translated (in the case of mRNA) into a polypeptide.
  • the term “transgene” as used herein refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell.
  • primer binding sequence refers to a portion of a retRNA and serves to bind to a primer sequence of the target nucleic acid.
  • the primer binding sequence binds to a primer sequence in the target nucleic acid that is formed after the target nucleic acid is cleaved by an effector protein.
  • the primer binding sequence is linked to the 3’ end of a retRNA. In some embodiments, the primer binding sequence is located at the 5’ end of a retRNA.
  • Primer sequence refers to a portion of the target nucleic acid that is capable of hybridizing with the primer binding sequence portion of a retRNA that is generated after cleavage of the target nucleic acid by an effector protein described herein.
  • tracrRNA trans-activating RNA
  • tracrRNA refers to a nucleic acid that comprises a first sequence that is capable of being non-covalently bound by an effector protein, and a second sequence that hybridizes to a portion of a crRNA, which may be referred to as a repeat hybridization sequence.
  • extension refers to additional nucleotides added to a nucleic acid, RNA, or DNA, or additional amino acids added to a peptide, polypeptide, or protein. Extensions may be processed during the formation of the guide RNA. In some embodiments, the extension comprises or consists of a template RNA.
  • hybridizable or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to noncovalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, "anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.
  • a nucleic acid e.g. RNA, DNA
  • anneal or “hybridize”
  • Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA] .
  • adenine (A) pairing with thymidine (T)
  • A adenine
  • U uracil
  • G guanine
  • C cytosine [DNA, RNA]
  • guanine (G) can also base pair with uracil (U).
  • G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti -codon base-pairing with codons in mRNA.
  • a guanine (G) e.g., of dsRNA duplex of a guide RNA molecule; of a guide RNA base pairing with a target nucleic acid, etc.
  • U uracil
  • A an adenine
  • a G/U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.
  • hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
  • the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences.
  • the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8).
  • the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides ormore, or 30 nucleotides or more).
  • Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.
  • nucleotide sequences While hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically hybridizable, or for hybridization to occur. Moreover, a nucleotide sequence may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.).
  • a polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize.
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
  • Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method.
  • Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482- 489), and the like.
  • the conditions appropriate for hybridization between two nucleotide sequences depend on the length of the sequence and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8).
  • complementarity e.g., complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides
  • the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
  • Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T.
  • RuvC domain refers to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain instances, of processing a pre-crRNA. In some embodiments, the RuvC domain is located near the C-terminus of the effector protein.
  • a single RuvC domain may comprise RuvC subdomains, for example a RuvCI subdomain, a RuvCII subdomain and a RuvCIII subdomain.
  • the term “RuvC” domain can also refer to a “RuvC-like” domain.
  • Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (https://www.ebi.ac.uk/interpro/).
  • a RuvC-like domain may be a domain which shares homology with a region of TnpB proteins of the IS605 and other related families of transposons.
  • effector proteins described herein may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell.
  • the effector protein is codon optimized for a human cell.
  • coding sequences of polypeptides described herein do not necessarily require a codon encoding a N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein.
  • a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell.
  • a modifying heterologous peptide such as a fusion partner protein, protein tag or NLS
  • a start codon for the heterologous peptide serves as a start codon for the effector protein as well.
  • the natural start codon encoding an amino acid residue sufficient for initiating translation e.g. , Methionine (M) or a Valine (V)
  • the natural start codon encoding an amino acid residue sufficient for initiating translation e.g. , Methionine (M) or a Valine (V)
  • the natural start codon encoding an amino acid residue sufficient for initiating translation e.g
  • the RDDP and/or effector proteins described herein comprise one or more amino acid substitutions as compared to a naturally occurring RDDP and/or effector protein.
  • the amino acid substitution is a conservative amino acid substitution.
  • a conservative amino acid substitution is the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).
  • Conservative substitutions may be made by exchanging an amino acid from one of the groups listed below (group 1 to 6) for another amino acid of the same group.
  • Amino acid residues may be divided into groups based on common side chain properties, as follows: (group 1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Vai), Leucine (Leu), Isoleucine (He); (group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gin); (group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu); (group 4) basic: Histidine (His), Lysine (Lys), Arginine (Arg); (group 5) residues that influence chain orientation: Glycine (Gly), Proline (Pro); and (group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe).
  • group 1 hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Vai
  • an effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is at a position selected from K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof.
  • the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1, with the exception of at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is a position selected from K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof.
  • the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A218K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, K58W, S209F, M295W, M298L, Y315M, D
  • the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1, with the exception of at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A218K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, K58W, S209F, M295W, M298L, Y315M, D220R/A306K and D220R/K250N and a combination thereof.
  • these engineered effector proteins demonstrate enhanced nucle
  • the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from D237A, D418A, D418N, E335A, and E335Q, and a combination thereof.
  • the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1, with the exception of at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from D237A, D418A, D418N, E335A, and E335Q, and a combination thereof.
  • these engineered effector proteins demonstrate reduced or abolished nuclease activity relative to the wild-type effector protein.
  • TABLE 1.1 provides the exemplary amino acid alterations relative to SEQ ID NO: 1 useful in compositions, systems, and methods described herein.
  • Engineered effector proteins may provide enhanced catalytic activity (e.g.
  • nuclease or nickase activity as compared to a naturally occurring nuclease or nickase.
  • Engineered effector proteins may provide enhance nucleic acid binding activity, e.g., enhanced binding of a guide nucleic acid and/or target nucleic acid, and/or may demonstrate a stronger affinity for a target nucleic acid sequence.
  • substation of positively charged amino acids is thought to increase the interaction between the effector proteins and/or RDDPs and the negatively charged target nucleic acid sequences.
  • the RDDP is an engineered RDDP.
  • Exemplary engineered RDDPs are provided in TABLE 3 below.
  • the parental RDDP is listed followed by the amino acid mutations in parentheses.
  • 2691319 (D12R-D72R-N195R) refers to an engineered RDDP based on 2691319 (SEQ ID NO: 13) and comprising the mutations D12R, D72R, and N195R.
  • the engineered RDDP comprises an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 71- 80, wherein the effector protein comprises a corresponding amino acid substitution as described in TABLE 3.
  • the engineered RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 71-80.
  • the engineered RDDP comprises an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 132- 152.
  • the engineered RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 132-152.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 13 and comprises the amino acid substitutions of D12R, N24R, D72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 50 and comprises the amino acid substitutions of D12R, N24R, D72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 51 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 52 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 53 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 54 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 55 and comprises the amino acid substitutions of E12R, N24R, S72R, andN195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 56 and comprises the amino acid substitutions ofN24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 57 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 58 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 59 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 60 and comprises the amino acid substitutions of D9R, N21R, N69R, G11 IK, and N192R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 61 and comprises the amino acid substitutions of N12R, N24R, N72R, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 62 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 63 and comprises the amino acid substitutions of D12R, N24R, N72R, G114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 64 and comprises the amino acid substitutions of D38R, N50R, N98R, N140K, and N221R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 65 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 66 and comprises the amino acid substitutions ofN12R, N24R, D72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 67 and comprises the amino acid substitutions of K12R, N24R, D72R, N114K, and N195R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 68 and comprises the amino acid substitutions of N12R, N24R, D72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 69 and comprises the amino acid substitutions of D34R, N46R, N94R, D136K, and N217R.
  • the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 70 and comprises the amino acid substitutions ofN12R, N24R, D72R, N114K, and N195R.
  • composition comprising an engineered RDDP described herein or a nucleic acid encoding the same.
  • the effector proteins are complexed with a biological tether, and comprises all or part of (e.g., DNA binding domain from) the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein.
  • a biological tether comprises all or part of (e.g., DNA binding domain from) the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein.
  • These proteins can be used to recruit RNA molecules containing a specific stem-loop structure to a locale specified by the Type V effector protein guide RNA targeting sequences.
  • a Type V effector protein variant fused to MS2 coat protein, endoribonuclease Csy4, or lambda N can be used to recruit a long non-coding RNA (IncRNA) such as XIST or HOTAIR; see, e.g., Keryer-Bibens et al., Biol.
  • IncRNA long non-coding RNA
  • an RDDP is bound to an MCP (MS2 aptamer binding protein).
  • an MCP comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to
  • a fusion protein comprises a Brex27 peptide that inhibits non- homologous end joining (NHEJ).
  • NHEJ non- homologous end joining
  • a Brex27 peptide comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG (SEQ ID NO: 112).
  • RDDPs, effector proteins and fusion proteins of the present disclosure of the present disclosure may be synthesized, using any suitable method. Additionally, nucleic acids, including mRNA encoding effector proteins and fusion proteins of the present disclosure may be synthesized using suitable methods. RDDPs, effector proteins and fusion proteins of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells. Effector proteins can be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using any suitable method.
  • Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2 nd ed (2014)).
  • One non-limiting example of a method for preparing an effector protein is to express recombinant nucleic acids encoding the effector protein in a suitable microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art.
  • an RDDP, effector protein, and/or fusion protein provided herein is an isolated effector protein.
  • an RDDP, effector protein, and/or fusion protein described herein can be isolated and purified for use in compositions, systems, and/or methods described herein. Methods described here can include the step of isolating effector proteins described herein.
  • An isolated an RDDP, effector protein, and/or fusion protein provided herein can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like.
  • the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)).
  • the methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.
  • RDDPs, effector proteins, and/or fusion proteins disclosed herein may be covalently linked or attached to a tag, e.g., a purification tag.
  • a purification tag as used herein, can be an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the protein of interest from its environment, which can be its biological source, such as a cell lysate. Attachment of the purification tag can be at the N or C terminus of the effector protein.
  • an amino acid sequence recognized by a protease or a nucleic acid encoding for an amino acid sequence recognized by a protease can be inserted between the purification tag and the effector protein, such that biochemical cleavage of the sequence with the protease after initial purification liberates the purification tag.
  • Purification and/or isolation can be through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Examples of purification tags are as described herein.
  • guide nucleic acids including guide RNA (gRNA)
  • gRNA guide RNA
  • Such nucleotide sequence may be described as a nucleotide sequence of either DNA or RNA. Due to WIPO Standard ST.26, the Us are being represented as Ts in RNA in the Sequence Listing provided herein. However, no matter the form the sequence is described, it is readily understood that such nucleotide sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the sequence that encodes a guide nucleic acid.
  • a guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the sequences described herein. Alterations can include a nucleotide substitution, or a deletion, or an insertion.
  • a guide nucleic acid of the present disclosure may comprise one or more of the following: a) an RNA nucleobase; b) a DNA nucleobase; c) a modified nucleobase; d) a modified sugar; and e) a modified backbone. Modified nucleobases, sugars and backbones are described in greater detail herein.
  • a guide nucleic acid may be chemically synthesized or recombinantly produced by any suitable methods. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism or cell.
  • a guide nucleic acid is a nucleic acid molecule, at least a portion of which may be bound by an effector protein, thereby forming a ribonucleoprotein complex (RNP).
  • Another portion of the guide nucleic acid molecule can comprise a spacer region which is complementary to at least a portion of the target nucleic acid sequence.
  • the guide nucleic acid imparts activity or sequence selectivity to the effector protein.
  • guide nucleic acids can bring the effector protein into proximity of a target nucleic acid.
  • the guide nucleic acid spacer region may hybridize to a target nucleic acid or a portion thereof.
  • a guide nucleic acid and an effector protein form an RNP
  • at least a portion of the RNP binds spacer region, recognizes, and/or hybridizes to a target nucleic acid.
  • a RNP can hybridize, via the spacer region, to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and/or recognize a target nucleic acid or sequence contained therein.
  • a guide nucleic acid comprises a first region that is capable of being non-covalently bound by an effector protein and a second region that hybridizes to a target nucleic acid.
  • the first region may comprise or be referred to as a protein binding sequence.
  • the first region is located 5’ to the second region.
  • the second region is located 5’ to the first region.
  • the first region and second region are linked either by a covalent bond (e.g., a phosphodiester bond) or linker (e.g., one or more nucleotides).
  • the protein binding sequence may comprise a repeat sequence, an intermediary sequence, a handle sequence, or a combination thereof.
  • the first region comprises a repeat sequence. In some embodiments, the first region comprises an intermediary sequence and a repeat sequence. In some embodiments, the first region comprises a handle sequence. In some embodiments, an effector protein binds to at least a portion of the first region. In some embodiments, the second region comprises a spacer sequence, wherein the spacer sequence can interact in a sequence-specific manner with (e.g. , has complementarity with, or can hybridize to a target sequence in) a target nucleic acid.
  • the guide nucleic acid comprises a repeat region that interacts with the effector protein.
  • the term, “repeat region” may be used interchangeably herein with the term, “repeat sequence.”
  • an effector protein interacts with a repeat region.
  • an effector protein does not interact with a repeat region.
  • the repeat region is adjacent to the spacer region.
  • the repeat region is followed by the spacer region in the 5’ to 3’ direction. Exemplary repeat region sequences for exemplary effector proteins provided herein are shown in TABLE 1.
  • the spacer sequence is 15-50 linked nucleotides in length. In some embodiments, the spacer sequence is 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides in length. In some embodiments, the spacer sequence is 18-24 linked nucleotides in length. In some embodiments, the spacer sequence is at least 15 linked nucleotides in length. In some embodiments, the spacer sequence is at least 16, 18, 20, or 22 linked nucleotides in length.
  • the hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence.
  • an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • an intermediary sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure).
  • compositions, systems and methods described herein comprise the nucleic acid, wherein the nucleic acid comprises a handle sequence.
  • the handle sequence comprises an intermediary sequence.
  • the intermediary sequence is at the 3 ’-end of the handle sequence.
  • the intermediary sequence is at the 5’- end of the handle sequence.
  • the handle sequence further comprises one or more of linkers and repeat sequences.
  • the linker comprises a sequence of 5’-GAAA-3.’
  • the intermediary sequence is 5’ to the repeat sequence.
  • the intermediary sequence is 5’ to the linker.
  • the intermediary sequence is 3’ to the repeat sequence.
  • the intermediary sequence is 3’ to the linker.
  • the repeat sequence is 3’ to the linker.
  • the repeat sequence is 5’ to the linker.
  • the length of the handle sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides.
  • the length of a handle sequence in an sgRNA is not greater than 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 70, or about 50 to about 69 linked nucleotides.
  • the length of the crRNA is about 20 to about 120 linked nucleotides. In some embodiments, the length of a crRNA is about 20 to about 100, about 30 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a crRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. sgRNA
  • an sgRNA comprises a handle sequence and a spacer sequence.
  • a handle sequence is 5’ to a spacer sequence in an sgRNA.
  • an sgRNA comprises a linked handle sequence and spacer sequence.
  • a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • a handle sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • an sgRNA comprises an intermediary sequence, a repeat sequence, and a spacer sequence.
  • an intermediary sequence is 5’ to a repeat sequence in an sgRNA.
  • an sgRNA comprises a linked intermediary sequence and repeat sequence.
  • an intermediary sequence and a repeat sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond).
  • an intermediary sequence and a repeat sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • a repeat sequence is 5’ to a spacer sequence in an sgRNA.
  • a sgRNA comprises a linked repeat sequence and spacer sequence.
  • a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g, covalently linked, such as through a phosphodiester bond)
  • a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • compositions and systems described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 1, 86, and 87; and a guide nucleic acid that comprises an sgRNA.
  • the sgRNA comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 124 and 125.
  • the sgRNA consists of a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 124 and 125.
  • compositions, systems and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA-crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of the target nucleic acid.
  • the effector protein is transactivated by the tracrRNA.
  • activity of the effector protein requires binding to a tracrRNA molecule.
  • a repeat hybridization sequence is at the 3’ end of a tracrRNA.
  • a repeat hybridization sequence may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides.
  • the length of the repeat hybridization sequence is 1 to 20 linked nucleotides.
  • systems, compositions, and methods comprise a crRNA or a use thereof.
  • a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence.
  • the repeat sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)).
  • the repeat sequence and the spacer sequence are connected by a linker.
  • systems, compositions, and methods comprise a tracrRNA or a use thereof. In some embodiments, systems, compositions, and methods do not comprise a tracrRNA or a use thereof.
  • a tracrRNA and/or tracrRNA-crRNA duplex may form a secondary structure that facilitates the binding of an effector protein to a tracrRNA or a tracrRNA-crRNA.
  • the secondary structure modifies activity of the effector protein on a target nucleic acid.
  • the secondary structure comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length.
  • the secondary structure comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a secondary structure comprising multiple stem regions.
  • nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • the secondary structure comprises at least two, at least three, at least four, or at least five stem regions. In some embodiments, the secondary structure comprises one or more loops. In some embodiments, the secondary structure comprises at least one, at least two, at least three, at least four, or at least five loops.
  • compositions, systems and methods described herein comprise a template RNA (retRNA), wherein the template RNA comprises a primer binding sequence and a template sequence.
  • the primer binding sequence hybridizes to a primer sequence on the non-target strand of the target dsDNA molecule.
  • the primer binding sequence hybridizes to a primer sequence on the target strand of the target dsDNA molecule.
  • the spacer sequence is complementary to the target sequence on the target strand of the dsDNA molecule, and the primer binding sequence and/or the template sequence is complementary to a primer sequence on the non-target strand of the target dsDNA molecule.
  • the spacer sequence is complementary to the target sequence on the non-target strand of the dsDNA molecule, and the primer binding sequence and/or the template sequence is complementary to a primer sequence on the target strand of the target dsDNA molecule.
  • the primer binding sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long.
  • the template sequence is at least 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 or 35 nucleotides long.
  • at least a portion of the PBS is complimentary to at least a portion of the target nucleic acid sequence that is 5’ of the nucleotide at position 13 relative to the PAM sequence. Such embodiments are particularly useful when used in a system comprising a CasM.265466 effector protein.
  • the template sequence may comprise one or more nucleotides having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when a spacer sequence of the guide RNA and the target sequence are aligned for maximum identity.
  • the one or more nucleotides may be contiguous.
  • the one or more nucleotides may not be contiguous.
  • the one or more nucleotides may each independently be selected from guanine, adenine, cytosine and thymine.
  • the retRNA comprises a secondary structure that can be bound by a peptide or protein that is part of or linked to the RDDP.
  • the secondary structure comprises an aptamer.
  • the aptamer is an MS2 aptamer (See Said et al (November 2009). “In vivo expression and purification of aptamer-tagged small RNA regulators”. Nucleic Acids Research. 37 (20): el33; and Johansson et al (1997). “RNA recognition by the MS2 phage coat protein”. Seminars in Virology. 8 (3): 176-185).
  • dsDNA double stranded DNA
  • dsDNA comprises a target strand (TS) and a non-target strand (NTS), wherein the guide RNA hybridizes to the target strand.
  • TS target strand
  • NTS non-target strand
  • the portion of the target strand to which the guide RNA hybridizes may be referred to as the target sequence of the target strand.
  • the portion of the non-target strand that is complementary to the target sequence of the target strand may be referred to as the target sequence of the non-target strand.
  • target sequence alone may be used to refer to the target sequence of the target strand and/or the target sequence of the non-target strand.
  • a target strand comprises a target sequence
  • at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand.
  • the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand
  • at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand.
  • a target nucleic acid comprises a protospacer adjacent motif (PAM) as described herein that is located on the non-target strand.
  • PAM protospacer adjacent motif
  • Such a PAM described herein is adjacent (e.g., within 1, 2, 3, 4, 5, 10, 20, 25 nucleotides) to the 5’ or 3’ end of the target sequence on the non-target strand of the double stranded DNA molecule.
  • such a PAM described herein is directly adjacent to the 5’or 3’ end of a target sequence on the non-target strand of the double stranded DNA molecule.
  • such a PAM described herein is directly adjacent to the 5’ end of a target sequence on the non-target strand of the double stranded DNA molecule.
  • an effector protein described herein, or a multimeric complex (e.g., dimer) thereof recognizes a PAM on a target nucleic acid.
  • the PAM comprises a PAM sequence set forth in TABLE 1.
  • An effector protein or fusion protein of the present disclosure may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid.
  • PAM protospacer adjacent motif
  • cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nucleotides of a 5’ or 3’ terminus of a PAM sequence.
  • a target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer region.
  • the guide nucleic acid can bind to a target sequence, wherein the target sequence is a nucleotide sequence found in a eukaryotic organism.
  • the nucleotide sequence found in a eukaryotic organism may be at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 50 nucleotides.
  • the target sequence may be found in a gene associated with a disease. In some embodiments, the gene is selected from TABLE 4.
  • the target sequence may be found in a bacterium, a virus, a parasite, a protozoon, a fungus or other agents responsible for a disease.
  • the target sequence is within an exon of any one of the genes set forth in TABLE 4. In some embodiments, the target sequence spans the junction of two exons. In some embodiments, the target sequence is located within about 1 to about 300 nucleotides, about 10 to about 250, about 20 to about 200, about 30 to about 150, about 40 to about 100, or about 50 nucleotides of the 5’ untranslated region (UTR). In some embodiments, the target sequence is located within about 1 to about 300 nucleotides, about 10 to about 250, about 20 to about 200, about 30 to about 150, about 40 to about 100, or about 50 nucleotides of the 3’ UTR.

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Abstract

Sont divulguées des systèmes, une composition et des méthodes de modification d'une molécule d'ADN double brin (ADNdb) cible dans une cellule. En général, des systèmes, des compositions et des procédés comprennent une protéine associée à CRISPR (Cas) de dimérisation compacte, une ADN polymérase dépendante de l'ARN, et un ou plusieurs acides nucléiques guides, ou leurs utilisations.
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