Novel crispr-cas systems for genome editing

Abstract

Compositions and methods are provided for genome modification of a target sequence in the genome of a cell, using a novel Cas endonuclease. The methods and compositions employ a guide polynucleotide/endonuclease system to provide an effective system for modifying or altering target sequences within the genome of a cell or organism. Also provided are novel effectors and endonuclease systems and elements comprising such systems, such as guide polynucleotide/endonuclease systems comprising an endonuclease. Compositions and methods are also provided for guide polynucleotide/endonuclease systems comprising at least one endonuclease, optionally covalently or non-covalently linked to, or assembled with, at least one additional protein subunit, and for compositions and methods for direct delivery of endonucleases as ribonucleotide proteins.

1 . A method of editing a target polynucleotide in a cell, the method comprising:
 (a) providing the cell with a type V Cas-alpha polypeptide having DNA binding activity, wherein the Cas-alpha polypeptide comprises an N-terminal half and a C-terminal half, the C-terminal half comprising:
 (i) a tri-split RuvC domain comprising a first RuvC subdomain comprising a GxxxG amino acid motif, a second RuvC subdomain comprising an ExL amino acid motif, and a third RuvC subdomain; 
 (ii) a first zinc finger domain disposed between the second and the third RuvC subdomains, wherein the first zinc finger domain comprises a Cx n C amino acid motif or a Cx n (C or H) amino acid motif; 
 (iii) a bridge-helix domain disposed between the first and the second RuvC subdomains;
 wherein G=Glycine, E=Glutamate, C=Cysteine, H=Histidine, L=leucine, x=any amino acid; and 
 wherein n=an integer between 2 and 11; and 
 
   (b) providing the cell with at least one guide polynucleotide comprising a region of complementarity to the target polynucleotide,   wherein the Cas-alpha polypeptide is less than 800 amino acids in length, recognizes a PAM sequence on the target polynucleotide, and forms a complex with the guide polynucleotide, wherein the complex binds to the target polynucleotide; and   (c) introducing at least one nucleotide modification in the target polynucleotide via the complex, wherein the target polynucleotide is heterologous to the Cas-alpha polypeptide.   
     
     
         2 . The method of  claim 1 , further comprising: providing the cell with a donor DNA molecule or a polynucleotide modification template. 
     
     
         3 . The method of  claim 1 , wherein the cell is derived or obtained from an animal, a fungus, or a plant. 
     
     
         4 . The method of  claim 3 , wherein the plant is a dicot or a monocot. 
     
     
         5 . The method of  claim 3 , wherein the plant is maize, soybean, cotton, wheat, canola, oilseed rape, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato,  Arabidopsis , safflower, or tomato. 
     
     
         6 . The method of  claim 1 , wherein the Cas-alpha polypeptide has endonuclease activity. 
     
     
         7 . The method of  claim 1 , wherein the at least one guide polynucleotide comprises a plurality of guide polynucleotides and the Cas-alpha polypeptide is a deactivated Cas-alpha endonuclease complexed to a deaminase, and wherein the method further comprises introducing a plurality of nucleobase edits in the target polynucleotide. 
     
     
         8 . The method of  claim 1 , wherein the Cas-alpha polypeptide is complexed to a heterologous protein domain through a linker, and wherein the heterologous protein domain has methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity. 
     
     
         9 . A synthetic composition comprising:
 (a) a type V Cas-alpha polypeptide having DNA binding activity, wherein the Cas-alpha polypeptide comprises an N-terminal half and a C-terminal half, the C-terminal half comprising:
 (i) a tri-split RuvC domain comprising a first RuvC subdomain comprising a GxxxG amino acid motif, a second RuvC subdomain comprising an ExL amino acid motif, and a third RuvC subdomain; 
 (ii) a first zinc finger domain disposed between the second and the third RuvC subdomains, wherein the first zinc finger domain comprises a Cx n C amino acid motif or a Cx n (C or H) amino acid motif; 
 (iii) a bridge-helix domain disposed between the first and the second RuvC subdomains;
 wherein G=Glycine, E=Glutamate, C=Cysteine, H=Histidine, L=leucine, x=any amino acid; and 
 wherein n=an integer between 2 and 11; and 
 
   (b) at least one guide polynucleotide comprising a region of complementarity to a target polynucleotide, wherein the target polynucleotide is heterologous to the Cas-alpha polypeptide   wherein the Cas-alpha polypeptide is less than 800 amino acids in length, recognizes a PAM sequence on the target polynucleotide, and forms a complex with the guide polynucleotide, wherein the complex binds to the target polynucleotide.   
     
     
         10 . The composition of  claim 9 , wherein the GxxxG amino acid motif comprises GxDxG, wherein D=Aspartic Acid. 
     
     
         11 . The composition of  claim 9 , wherein the Cas-alpha polypeptide further comprises a second zinc finger domain disposed in tandem with the first zinc finger domain. 
     
     
         12 . The composition of  claim 11 , wherein the Cas-alpha polypeptide further comprises a third zinc finger domain disposed downstream of the third RuvC subdomain. 
     
     
         13 . The composition of  claim 12 , wherein the Cas-alpha polypeptide is Cas-alpha 10. 
     
     
         14 . The composition of  claim 9 , wherein the Cas-alpha polypeptide further comprises a second zinc finger domain disposed in the N-terminal half. 
     
     
         15 . The composition of  claim 14 , wherein the Cas-alpha polypeptide is Cas-alpha 1, Cas-alpha 2, Cas-alpha 3, or Cas-alpha 4. 
     
     
         16 . The composition of  claim 9 , wherein the Cas-alpha polypeptide is Cas-alpha 5, Cas-alpha 6, Cas-alpha 7, Cas-alpha 8, Cas-alpha 9, or Cas-alpha 11. 
     
     
         17 . The composition of  claim 9 , wherein the GxxxG amino acid motif is GVDIG, GIDRG, GIDVG, GLDVG, GVDLG, GIDLG, GVDVG, GIDIG, GIDMG, GLDLG, GVDMG, GIDFG, GIDAG, GIDLG, or GVNLG, wherein V=Valine, D=Aspartic Acid, I=Isoleucine, R=Arginine, M=Methionine, F=Phenylalanine, A=Alanine, N=Asparagine, and L=Leucine. 
     
     
         18 . The composition of  claim 9 , wherein the ExL amino acid motif is ENL, EDL, EKL, ELL, EQL, EYL, EEL, ESL, EFL, or ERL, wherein N=Asparagine, D=Aspartic Acid, K=Lysine, L=Leucine, Q=Glutamine, Y=Tyrosine, E=Glutamic Acid, S=Serine, F=Phenylalanine, and R=Arginine. 
     
     
         19 . The composition of  claim 9 , wherein the Cx n C amino acid motif is CSSC, CSKC, CAYC, CSNPNC, CSEC, CSMC, CSRC, CNNC, CSLC, CCKC, CSQC, CSNPSC, CSHC, CAKC, CSIC, CSQC, CSCC, CNRC, CHVC, CSYC, CPSC, or CHIC, wherein S=Serine, K=Lysine, A=Alanine, Y=Tyrosine, N=Asparagine, P=Proline, E=Glutamic Acid, M=Methionine, R=Arginine, L=Leucine, Q=Glutamine, H=Histidine, I=Isoleucine, and V=Valine. 
     
     
         20 . The composition of  claim 9 , wherein the Cx n (C or H) amino acid motif is CLNPTC, CLKC, CKLH, CEKC, CLNPNC, CIEC, CRAC, CLEC, CKQC, CKKC, CTKC, CKVC, CVVC, CLKC, CIKC, CVNDEC, CKEC, CVKC, CKRC, CIDC, CEKC, CTVC, CKSC, CNKC, CEFC, CSVH, CMKH, CGKC, CTKC, CLEC, CKGC, CINC, CVKC, CKNPEC, CVIC, CQQC, CLSC, CTNPEC, CQVC, CREC, CTNPQC, CPEC, CKNKEC, CKAC, CKDPNC, or CPVC, wherein S=Serine, K=Lysine, A=Alanine, Y=Tyrosine, N=Asparagine, P=Proline, E=Glutamic Acid, M=Methionine, R=Arginine, L=Leucine, Q=Glutamine, H=Histidine, I=Isoleucine, T=Threonine, F=Phenylalanine, G=Glycine, D=Aspartic Acid, and V=Valine. 
     
     
         21 . The composition of  claim 9 , wherein the Cas-alpha polypeptide comprises, in an N-terminal to C-terminal direction, the first RuvC subdomain, the bridge-helix domain, the second RuvC subdomain, the first zinc finger domain, and the third RuvC subdomain. 
     
     
         22 . The composition of  claim 9 , further comprising:
 (c) the target double-stranded DNA polynucleotide.   
     
     
         23 . The composition of  claim 9 , wherein the GxxxG amino acid motif precedes the ExL amino acid motif, the Cx n C amino acid motif, and the Cx n (C or H) amino acid motif in an N-terminal to C-terminal direction of the Cas-alpha polypeptide. 
     
     
         24 . The composition of  claim 23 , wherein the ExL amino acid motif is about 94 amino acids to about 108 amino acids from the GxxxG amino acid motif. 
     
     
         25 . The composition of  claim 23 , wherein the Cx n C amino acid motif is about 142 amino acids to about 158 amino acids from the GxxxG amino acid motif. 
     
     
         26 . The composition of  claim 23 , wherein the the Cx n (C or H) amino acid motif is about 156 amino acids to about 188 amino acids from the GxxxG amino acid motif. 
     
     
         27 . The composition of  claim 9 , wherein the guide polynucleotide is a guide RNA. 
     
     
         28 . The composition of  claim 9 , wherein the Cas-alpha polypeptide has endonuclease activity. 
     
     
         29 . The composition of  claim 9 , wherein the Cas-alpha polypeptide is a deactivated Cas-alpha endonuclease complexed to a deaminase. 
     
     
         30 . The composition of  claim 9 , wherein the Cas-alpha polypeptide is complexed to a heterologous protein domain through a linker, and wherein the heterologous protein domain has methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity.