US2022364071A1PendingUtilityA1
Novel crispr enzymes and systems
Est. expiryJul 31, 2038(~12 yrs left)· nominal 20-yr term from priority
C12N 2310/128A61K 38/00C12Y 305/04004C07K 2319/80C12Q 1/6823C12N 9/22C12N 15/111C12N 9/78C07K 2319/00A61K 38/50C07K 2319/33C12N 2310/20C12Q 1/686C12Q 2563/107C12Q 1/6851C12N 15/63C12Q 2563/155C12N 15/102C12N 2310/16
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Claims
Abstract
The present disclosure provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides mutated Cas13 proteins and their use in modifying target sequences as well as mutated Cas13 nucleic acid sequences and vectors encoding mutated Cas13 proteins and vector systems or CRISPR-Cas13 systems.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . An engineered adenosine deaminase comprising one or more mutations, wherein the engineered adenosine deaminase has cytidine deaminase activity, wherein said adenosine deaminase protein or catalytic domain thereof comprises one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T based on amino acid sequence positions of hADAR2-D, and corresponding mutations in a homologous ADAR protein.
2 . An engineered adenosine deaminase comprising one or more mutations, wherein the engineered adenosine deaminase has cytidine deaminase activity.
3 . The engineered adenosine deaminase of claim 2 , wherein the engineered adenosine deaminase has adenosine deaminase activity.
4 . The engineered adenosine deaminase of claim 2 , wherein the engineered adenosine deaminase is a portion of a fusion protein.
5 . The engineered adenosine deaminase of claim 2 , wherein the fusion protein comprises a functional domain.
6 . The engineered adenosine deaminase of claim 2 , wherein the functional domain is capable of directing the engineered adenosine deaminase to bind to a target nucleic acid.
7 . The engineered adenosine deaminase of claim 2 , wherein the functional domain is a CRISPR-Cas protein of any one of claims 50 to 55 .
8 . The engineered adenosine deaminase of claim 2 , wherein the CRISPR-Cas protein is a dead form CRISPR-Cas protein or CRISPR-Cas nickase protein.
9 . The engineered adenosine deaminase of claim 2 , wherein the one or more mutations comprises: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T based on amino acid sequence positions of hADAR2-D, and corresponding mutations in a homologous ADAR protein.
10 . The engineered adenosine deaminase of claim 2 , wherein the one or more mutations comprises: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, and S661T based on amino acid sequence positions of hADAR2-D, and corresponding mutations in a homologous ADAR protein.
11 . A polynucleotide encoding the engineered adenosine deaminase of any one of claims above claims, or a catalytic domain thereof.
12 . A vector comprising the polynucleotide of claim 11 .
13 . A pharmaceutical composition comprising the engineered adenosine deaminase of any one of claims 1 - 10 or a catalytic domain thereof formulated for delivery by liposomes, nanoparticles, exosomes, microvesicles, nucleic acid nanoassemblies, a gene gun, or an implantable device.
14 . An engineered cell expressing the engineered adenosine deaminase of any one of claims 1 - 10 or a catalytic domain thereof.
15 . The engineered cell of claim 14 , wherein the cell transiently expresses the engineered adenosine deaminase or the catalytic domain thereof.
16 . The engineered cell of claim 15 , wherein the cell non-transiently expresses the engineered adenosine deaminase or the catalytic domain thereof.
17 . An engineered, non-naturally occurring system for modifying nucleotides in a target nucleic acid, comprising
a) a dead CRISPR-Cas or CRISPR-Cas nickase protein, or a nucleotide sequence encoding said dead Cas or Cas nickase protein; b) a guide molecule comprising a guide sequence that hybridizes to a target sequence and designed to form a complex with the dead CRISPR-Cas or CRISPR-Cas nickase protein; and c) a nucleotide deaminase protein or catalytic domain thereof, or a nucleotide sequence encoding said nucleotide deaminase protein or catalytic domain thereof, wherein said nucleotide deaminase protein or catalytic domain thereof is covalently or non-covalently linked to said dead CRISPR-Cas or CRISPR-Cas nickase protein or said guide molecule is adapted to link thereof after delivery.
18 . The system of claim 17 , wherein said adenosine deaminase protein or catalytic domain thereof comprises one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T based on amino acid sequence positions of hADAR2-D, and corresponding mutations in a homologous ADAR protein.
19 . The system of claim 17 , wherein said adenosine deaminase protein or catalytic domain thereof comprises mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, and S661T based on amino acid sequence positions of hADAR2-D, and corresponding mutations in a homologous ADAR protein.
20 . The system of claim 17 , wherein the CRISPR-Cas protein is Cas9, Cas12, Cas13, Cas 14, CasX, or CasY.
21 . The system of claim 17 , wherein the CRISPR-Cas protein is Cas13b.
22 . The system of claim 17 , wherein the CRISPR-Cas protein is Cas13b-t1, Cas13b-t2, or Cas13b-t3.
23 . The system of claim 17 , wherein the CRISPR-Cas is an engineered CRISPR-Cas protein of any one of claims 50 to 367 .
24 . A method for modifying nucleotide in a target nucleic acid, comprising:
delivering to said target nucleic acid the engineered adenosine deaminase of any one of claims 1 - 10 , or the system of any one of claims 17 - 23 , wherein the deaminase deaminates a nucleotide at one or more target loci on the target nucleic acid.
25 . The method of claim 24 , wherein said nucleotide deaminase protein or catalytic domain thereof has been modified to increase activity against a DNA-RNA heteroduplex.
26 . The method of claim 24 , wherein said nucleotide deaminase protein or catalytic domain thereof has been modified to reduce off-target effects.
27 . The method of claim 24 , wherein the target nucleic acid is within a cell.
28 . The method of claim 24 , wherein said cell is a eukaryotic cell.
29 . The method of claim 24 , wherein said cell is a non-human animal cell.
30 . The method of claim 24 , wherein said cell is a human cell.
31 . The method of claim 24 , wherein said cell is a plant cell.
32 . The method of claim 24 , wherein said target nucleic acid is within an animal.
33 . The method of claim 24 , wherein said target nucleic acid is within a plant.
34 . The method of claim 24 , wherein said target nucleic acid is comprised in a DNA molecule in vitro.
35 . The method of claim 24 , wherein the engineered adenosine deaminase, or one or more components of the system are delivered to the cell as a ribonucleoprotein complex.
36 . The method of claim 24 , wherein the engineered adenosine deaminase, or one or more components of the system are delivered via one or more particles, one or more vesicles, or one or more viral vectors.
37 . The method of claim 24 , wherein said one or more particles comprise a lipid, a sugar, a metal or a protein.
38 . The method of claim 24 , wherein said one or more particles comprise lipid nanoparticles.
39 . The method of claim 24 , wherein said one or more vesicles comprise exosomes or liposomes.
40 . The method of claim 24 , wherein said one or more viral vectors comprise one or more adenoviral vectors, one or more lentiviral vectors, or one or more adeno-associated viral vectors.
41 . The method of claim 24 , where said method modifies a cell, a cell line or an organism by manipulation of one or more target sequences at genomic loci of interest.
42 . The method of claim 24 , wherein said deamination of said nucleotide at said target locus of interest remedies a disease caused by a G→A or C→T point mutation or a pathogenic SNP.
43 . The method of claim 24 , wherein said disease is selected from cancer, haemophilia, beta-thalassemia, Marfan syndrome and Wiskott-Aldrich syndrome.
44 . The method of claim 24 , wherein said deamination of said nucleotide at said target locus of interest remedies a disease caused by a T→C or A→G point mutation or a pathogenic SNP.
45 . The method of claim 24 , wherein said deamination of said nucleotide at said target locus of interest inactivates a target gene at said target locus.
46 . The method of claim 24 , wherein the engineered adenosine deaminase, or one or more components of the system are delivered by liposomes, nanoparticles, exosomes, microvesicles, nucleic acid nanoassemblies, a gene gun, an implantable device, or the vector system of claim 302 .
47 . The method of claim 24 , wherein modification of the nucleotide modifies gene product encoded at the target locus or expression of the gene product.
48 . The engineered adenosine deaminase of any one of claims 1 - 10 or the system of any one of claims 17 - 23 , wherein the adenosine protein or catalytic domain thereof comprises a mutation on S375 based on amino acid sequence positions of hADAR2-D, and a corresponding mutation in a homologous ADAR protein.
49 . The engineered adenosine deaminase or the system of claim 48 , wherein the mutation on S375 is S375N.
50 . An engineered CRISPR-Cas protein comprising one or more HEPN domains and further comprising one or more modified amino acids, wherein the amino acids:
a. interact with a guide RNA that forms a complex with the engineered CRISPR-Cas protein; b. are in a HEPN active site, an inter-domain linker domain, a lid domain, a helical domain 1, a helical domain 2, or a bridge helix domain of the engineered CRISPR-Cas protein; or c. a combination thereof.
51 . The engineered CRISPR-Cas protein of claim 50 , wherein the HEPN domain comprises a RxxxxH motif.
52 . The engineered CRISPR-Cas protein of claim 51 , wherein the RxxxxH motif comprises a R{N/H/K}X 1 X 2 X 3 H sequence.
53 . The engineered CRISPR-Cas protein of claim 52 , wherein:
X 1 is R, S, D, E, Q, N, G, or Y, X 2 is independently I, S, T, V, or L, and X 3 is independently L, F, N, Y, V, I, S, D, E, or A.
54 . The engineered CRISPR-Cas protein of claim 50 , wherein the CRISPR-Cas protein is a Type VI CRISPR-Cas protein.
55 . The engineered CRISPR-Cas protein of claim 54 , wherein the Type VI CRISPR-Cas protein is a Cas13.
56 . The engineered CRISPR-Cas protein of claim 55 , wherein the Type VI CRISPR-Cas protein is Cas13a, Cas13b, Cas13c, or Cas13d.
57 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): T405, H407, K457, H500, K570, K590, N634, R638, N652, N653, K655, S658, K741, K744, N756, S757, R762, R791, K846, K857, K870, R877, K183, K193, R600, K607, K612, R614, K617, K826, K828, K829, R824, R830, Q831, K835, K836, R838, R618, D434, K431, R53, K943, R1041, Y164, R285, R287, K292, E296, N297, Q646, N647, R402, K393, N653, N652, R482, N480, D396, E397, D398, E399, K294, E400, R56, N157, H161, H452, N455, K484, N486, G566, H567, A656, V795, A796, W842, K871, E873, R874, R1068, N1069, or H1073.
58 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): H407, K457, H500, K570, K590, N634, R638, N652, N653, K655, S658, K741, K744, N756, S757, R762, R791, K846, K857, K870, R877, K183, K193, R600, K607, K612, R614, K617, K826, K828, K829, R824, R830, Q831, K835, K836, R838, R618, D434, K431, R53, K943, R1041, Y164, R285, R287, K292, E296, N297, Q646, N647, R402, K393, N653, N652, R482, N480, D396, E397, D398, E399, K294, E400, R56, N157, H161, H452, N455, K484, N486, G566, H567, W842, K871, E873, R874, R1068, N1069, or H1073.
59 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): T405, H407, K457, H500, K570, K590, N634, R638, N652, N653, K655, S658, K741, K744, N756, S757, R762, R791, K846, K857, K870, R877, K183, K193, R600, K607, K612, R614, K617, K826, K828, K829, R824, R830, Q831, K835, K836, R838, R618, D434, K431, R53, K943, R1041, Y164, R285, R287, K292, E296, N297, Q646, N647, R402, K393, N653, N652, R482, N480, D396, E397, D398, E399, K294, or E400.
60 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K393, R402, N482, T405, H407, S658, N653, A656, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, K741, R56, N157, H161, R1068, N1069, or H1073.
61 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K393, R402, N482, H407, S658, N653, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, K741, R56, N157, H161, R1068, N1069, or H1073.
62 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: W842, K846, K870, E873, or R877.
63 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1 of PbCas13b: W842, K846, K870, E873, or R877.
64 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of PbCas13b: W842, K846, K870, E873, or R877.
65 . The engineered CRISPR-Cas protein of claim 55 comprising in the bridge helix domain one or more mutation of an amino acid corresponding to the following amino acids in the bridge helix domain of PbCas13b: W842, K846, K870, E873, or R877.
66 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K393, R402, N480, N482, N652, or N653.
67 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K393, R402, N480, or N482.
68 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of PbCas13b: K393, R402, N480, or N482.
69 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: N652 or N653.
70 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of PbCas13b: N652 or N653.
71 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: T405, H407, S658, N653, A656, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, or K741.
72 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H407, S658, N653, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, or K741.
73 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, A656, K655, N652, H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, K590, R638, S757, N756, or K741.
74 . The engineered CRISPR-Cas protein of claim 55 comprising in a helical domain one or more mutation of an amino acid corresponding to the following amino acids in a helical domain of PbCas13b: S658, N653, A656, K655, N652, H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, K590, R638, S757, N756, or K741.
75 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, S757, or N756.
76 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1 of PbCas13b: H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, S757, or N756.
77 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H567, H500, R762, V795, A796, R791, G566, S757, or N756.
78 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1 of PbCas13b: H567, H500, R762, V795, A796, R791, G566, S757, or N756.
79 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K871, K857, K870, W842, E873, R877, K846, or R874.
80 . The engineered CRISPR-Cas protein of claim 55 comprising in the bridge helix domain one or more mutation of an amino acid corresponding to the following amino acids in the bridge helix domain of PbCas13b: K871, K857, K870, W842, E873, R877, K846, or R874.
81 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H567, H500, or G566.
82 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-2 of PbCas13b: H567, H500, or G566.
83 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, S757, or N756.
84 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of PbCas13b: K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, S757, or N756.
85 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: R762, V795, A796, R791, S757, or N756.
86 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of PbCas13b: R762, V795, A796, R791, S757, or N756.
87 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, A656, K655, N652, K590, R638, or K741.
88 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of PbCas13b: S658, N653, A656, K655, N652, K590, R638, or K741.
89 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: T405, H407, N486, K484, N480, H452, N455, or K457.
90 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of PbCas13b: T405, H407, N486, K484, N480, H452, N455, or K457.
91 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, K655, N652, H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, K590, R638, S757, N756, or K741.
92 . The engineered CRISPR-Cas protein of claim 55 comprising in a helical domain one or more mutation of an amino acid corresponding to the following amino acids in a helical domain of PbCas13b: S658, N653, K655, N652, H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, K590, R638, S757, N756, or K741.
93 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, S757, or N756.
94 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1 of PbCas13b: H567, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, S757, or N756.
95 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H567, H500, R762, R791, G566, S757, or N756.
96 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1 of PbCas13b: H567, H500, R762, R791, G566, S757, or N756.
97 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, S757, or N756.
98 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of PbCas13b: K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, S757, or N756.
99 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: R762, R791, S757, or N756.
100 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of PbCas13b: R762, R791, S757, or N756.
101 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, K655, N652, K590, R638, or K741.
102 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of PbCas13b: S658, N653, K655, N652, K590, R638, or K741.
103 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H407, N486, K484, N480, H452, N455, or K457.
104 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of PbCas13b: H407, N486, K484, N480, H452, N455, or K457.
105 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: R56, N157, H161, R1068, N1069, or H1073.
106 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of PbCas13b: R56, N157, H161, R1068, N1069, or H1073.
107 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: R56, N157, or H161.
108 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of PbCas13b: R56, N157, or H161.
109 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: R1068, N1069, or H1073.
110 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 2 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 2 of PbCas13b: R1068, N1069, or H1073.
111 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K393, R402, N482, T405, H407, N486, K484, N480, H452, N455, or K457.
112 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of PbCas13b: K393, R402, N482, T405, H407, N486, K484, N480, H452, N455, or K457.
113 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K393, R402, N482, H407, N486, K484, N480, H452, N455, or K457.
114 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of PbCas13b: K393, R402, N482, H407, N486, K484, N480, H452, N455, or K457.
115 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: T405, H407, S658, N653, A656, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, K741, K393, R402, or N482.
116 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: H407, S658, N653, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, K741, K393, R402, or N482.
117 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, A656, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, or K741.
118 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, or K741.
119 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: N486, K484, N480, H452, N455, or K457.
120 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of PbCas13b: N486, K484, N480, H452, N455, or K457.
121 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: K393, R402, N482, N486, K484, N480, H452, N455, or K457.
122 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of PbCas13b: K393, R402, N482, N486, K484, N480, H452, N455, or K457.
123 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, A656, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, V795, A796, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, K741, K393, R402, or N482.
124 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of PbCas13b: S658, N653, K655, N652, H567, N455, H500, K871, K857, K870, W842, E873, R877, K846, R874, R762, R791, G566, K590, R638, H452, S757, N756, N486, K484, N480, K457, K741, K393, R402, or N482.
125 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K943, or R1041.
126 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53 or Y164.
127 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K943 or R1041.
128 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K943, or R1041.
129 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of Prevotella buccae Cas13b (PbCas13b): R53 or Y164.
130 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 2 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 2 of Prevotella buccae Cas13b (PbCas13b): K943 or R1041.
131 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K943, R1041, R56, N157, H161, R1068, N1069, or H1073.
132 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, R56, N157, or H161.
133 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K943, R1041, R1068, N1069, or H1073.
134 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K943, R1041, R56, N157, H161, R1068, N1069, or H1073.
135 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of Prevotella buccae Cas13b (PbCas13b): R53, Y164, R56, N157, or H161.
136 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 2 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 2 of Prevotella buccae Cas13b (PbCas13b): K943, R1041, R1068, N1069, or H1073.
137 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, K193, K943, or R1041.
138 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, or K193.
139 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K943 or R1041.
140 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, K193, K943, or R1041.
141 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, or K193.
142 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 2 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 2 of Prevotella buccae Cas13b (PbCas13b): K943 or R1041.
143 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, K193, K943, R1041, R56, N157, H161, R1068, N1069, or H1073.
144 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, K193, R56, N157, or H161.
145 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K943, R1041, R1068, N1069, or H1073.
146 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, K193, K943, R1041, R56, N157, H161, R1068, N1069, or H1073.
147 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K183, K193, R56, N157, or H161.
148 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 2 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 2 of Prevotella buccae Cas13b (PbCas13b): K943, R1041, R1068, N1069, or H1073.
149 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K183 or K193.
150 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of Prevotella buccae Cas13b (PbCas13b): K183 or K193.
151 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K943, or R1041.
152 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella buccae Cas13b (PbCas13b): R53, Y164, K943, or R1041.
153 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, K943, or R1041; preferably R53A, R53K, R53D, or R53E; K943A, K943R, K943D, or K943E; or R1041A, R1041K, R1041D, or R1041E.
154 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella buccae Cas13b (PbCas13b): R53, K943, or R1041; preferably R53A, R53K, R53D, or R53E; K943A, K943R, K943D, or K943E; or R1041A, R1041K, R1041D, or R1041E.
155 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid Y164 of Prevotella buccae Cas13b (PbCas13b), preferably Y164A, Y164F, or Y164W.
156 . The engineered CRISPR-Cas protein of claim 55 comprising HEPN domain 1 a mutation of an amino acid corresponding to amino acid Y164 HEPN domain 1 of Prevotella buccae Cas13b (PbCas13b), preferably Y164A, Y164F, or Y164W.
157 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): T405, H407, K457, D434, K431, R402, K393, R482, N480, D396, E397, D398, or E399.
158 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of Prevotella buccae Cas13b (PbCas13b): T405, H407, K457, D434, K431, R402, K393, R482, N480, D396, E397, D398, or E399.
159 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H407 of Prevotella buccae Cas13b (PbCas13b), preferably H407Y, H407W, or H407F.
160 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R402, K393, R482, N480, D396, E397, D398, or E399.
161 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of Prevotella buccae Cas13b (PbCas13b): R402, K393, R482, N480, D396, E397, D398, or E399.
162 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K457, D434, or K431.
163 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of Prevotella buccae Cas13b (PbCas13b): K457, D434, or K431.
164 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): H500, K570, K590, N634, R638, N652, N653, K655, S658, K741, K744, N756, S757, R762, R791, K846, K857, K870, R877, R600, K607, K612, R614, K617, K826, K828, K829, R824, R830, Q831, K835, K836, R838, R618, Q646, N647, N653, or N652.
165 . The engineered CRISPR-Cas protein of claim 55 comprising in a helical domain one or more mutation of an amino acid corresponding to the following amino acids in a helical domain of Prevotella buccae Cas13b (PbCas13b): H500, K570, K590, N634, R638, N652, N653, K655, S658, K741, K744, N756, S757, R762, R791, K846, K857, K870, R877, R600, K607, K612, R614, K617, K826, K828, K829, R824, R830, Q831, K835, K836, R838, R618, Q646, N647, N653, or N652.
166 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): H500, K570, N756, S757, R762, R791, K846, K857, K870, R877, K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
167 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1 of Prevotella buccae Cas13b (PbCas13b): H500, K570, N756, S757, R762, R791, K846, K857, K870, R877, K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
168 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): H500, K570, N756, S757, R762, or R791.
169 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1 of Prevotella buccae Cas13b (PbCas13b): H500, K570, N756, S757, R762, or R791.
170 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K846, K857, K870, R877, K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
171 . The engineered CRISPR-Cas protein of claim 55 comprising in the bridge helix domain one or more mutation of an amino acid corresponding to the following amino acids in the bridge helix domain of Prevotella buccae Cas13b (PbCas13b): K846, K857, K870, R877, K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
172 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): H500 or K570.
173 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-2 of Prevotella buccae Cas13b (PbCas13b): H500 or K570.
174 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): N756, S757, R762, R791, K846, K857, K870, R877, K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
175 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of Prevotella buccae Cas13b (PbCas13b): N756, S757, R762, R791, K846, K857, K870, R877, K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
176 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): N756, S757, R762, or R791.
177 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of Prevotella buccae Cas13b (PbCas13b): N756, S757, R762, or R791.
178 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): N756, S757, R762, R791, K846, K857, K870, or R877.
179 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of Prevotella buccae Cas13b (PbCas13b): N756, S757, R762, R791, K846, K857, K870, or R877.
180 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
181 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 1-3 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 1-3 of Prevotella buccae Cas13b (PbCas13b): K826, K828, K829, R824, R830, Q831, K835, K836, or R838.
182 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K590, N634, R638, N652, N653, K655, S658, K741, K744, R600, K607, K612, R614, K617, R618, Q646, N647, N653, or N652.
183 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of Prevotella buccae Cas13b (PbCas13b): K590, N634, R638, N652, N653, K655, S658, K741, K744, R600, K607, K612, R614, K617, R618, Q646, N647, N653, or N652.
184 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): Q646 or N647.
185 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of Prevotella buccae Cas13b (PbCas13b): Q646 or N647.
186 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): N653 or N652.
187 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of Prevotella buccae Cas13b (PbCas13b): N653 or N652.
188 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K590, N634, R638, N652, N653, K655, S658, K741, or K744.
189 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of Prevotella buccae Cas13b (PbCas13b): K590, N634, R638, N652, N653, K655, S658, K741, or K744.
190 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R600, K607, K612, R614, K617, or R618.
191 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of Prevotella buccae Cas13b (PbCas13b): R600, K607, K612, R614, K617, or R618.
192 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R285, R287, K292, E296, N297, or K294.
193 . The engineered CRISPR-Cas protein of claim 55 comprising in the IDL domain one or more mutation of an amino acid corresponding to the following amino acids in the IDL domain of Prevotella buccae Cas13b (PbCas13b): R285, R287, K292, E296, N297, or K294.
194 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R285, K292, E296, or N297.
195 . The engineered CRISPR-Cas protein of claim 55 comprising in the IDL domain one or more mutation of an amino acid corresponding to the following amino acids in the IDL domain of Prevotella buccae Cas13b (PbCas13b): R285, K292, E296, or N297.
196 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): T405, H500, K570, K590, N634, R638, N652, N653, K655, S658, K741, K744, N756, S757, R762, R791, K846, K857, K870, R877, K183, K193, R600, K607, K612, R614, K617, K826, K828, K829, R824, R830, Q831, K835, K836, R838, R618, D434, K431, R285, R287, K292, E296, N297, Q646, N647, or K294.
197 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R402, K393, N653, N652, R482, N480, D396, E397, D398, or E399.
198 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53, K655, R762, or R1041; preferably R53A or R53D; K655A; R762A; or R1041E or R1041D.
199 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): N297, E296, K292, or R285; preferably N297A, E296A, K292A, or R285A.
200 . The engineered CRISPR-Cas protein of claim 55 comprising in (the central channel of) the IDL domain one or more mutation of an amino acid corresponding to the following amino acids in (the central channel of) the IDL domain of Prevotella buccae Cas13b (PbCas13b): N297, E296, K292, or R285; preferably N297A, E296A, K292A, or R285A.
201 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): Q831, K836, R838, N652, N653, R830, K655 or R762; preferably Q831A, K836A, R838A, N652A, N653A, R830A, K655A, or R762A.
202 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): N652, N653, R830, K655 or R762; preferably N652A, N653A, R830A, K655A, or R762A.
203 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K655 or R762; preferably K655A or R762A.
204 . The engineered CRISPR-Cas protein of claim 55 comprising in a helical domain one or more mutation of an amino acid corresponding to the following amino acids in a helical domain of Prevotella buccae Cas13b (PbCas13b): Q831, K836, R838, N652, N653, R830, K655 or R762; preferably Q831A, K836A, R838A, N652A, N653A, R830A, K655A, or R762A.
205 . The engineered CRISPR-Cas protein of claim 55 comprising a helical domain one or more mutation of an amino acid corresponding to the following amino acids a helical domain of Prevotella buccae Cas13b (PbCas13b): N652, N653, R830, K655 or R762; preferably N652A, N653A, R830A, K655A, or R762A.
206 . The engineered CRISPR-Cas protein of claim 55 comprising in helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in helical domain 2 of Prevotella buccae Cas13b (PbCas13b): K655 or R762; preferably K655A or R762A.
207 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R614, K607, K193, K183 or R600; preferably R614A, K607A, K193A, K183A or R600A.
208 . The engineered CRISPR-Cas protein of claim 55 comprising in the trans-subunit loop of helical domain 2 one or more mutation of an amino acid corresponding to the following amino acids in the trans-subunit loop of helical domain 2 of Prevotella buccae Cas13b (PbCas13b): Q646 or N647; preferably Q646A or N647A.
209 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R53 or R1041; preferably R53A or R53D, or R1041E or R1041D.
210 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella buccae Cas13b (PbCas13b): R53 or R1041; preferably R53A or R53D, or R1041E or R1041D.
211 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K457, D397, E398, D399, E400, T405, H407 or D434; preferably D397A, E398A, D399A, E400A, T405A, H407A, H407W, H407Y, H407F or D434A.
212 . The engineered CRISPR-Cas protein of claim 55 comprising in the LID domain one or more mutation of an amino acid corresponding to the following amino acids in the LID domain of Prevotella buccae Cas13b (PbCas13b): K457, D397, E398, D399, E400, T405, H407 or D434; preferably D397A, E398A, D399A, E400A, T405A, H407A, H407W, H407Y, H407F or D434A.
213 . The engineered CRISPR-Cas protein of claim 55 , wherein the amino acids correspond to the following amino acids of Prevotella buccae Cas13b (PbCas13b): amino acids 46-57, 73-79, 152-164, 1036-1046, and 1064-1074.
214 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R156, N157, H161, R1068, N1069, and H1073.
215 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): R285, R287, K292, K294, E296, and N297.
216 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): K826, K828, K829, R824, R830, Q831, K835, K836, and R838.
217 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella buccae Cas13b (PbCas13b): T405, H407, K457, H500, K570, K590, N634, R638, N652, N653, K655, S658, K741, K744, N756, S757, R762, R791, K846, K857, K870, and R877.
218 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid T405 of Prevotella buccae Cas13b (PbCas13b).
219 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H407 of Prevotella buccae Cas13b (PbCas13b).
220 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K457 of Prevotella buccae Cas13b (PbCas13b).
221 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H500 of Prevotella buccae Cas13b (PbCas13b).
222 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K570 of Prevotella buccae Cas13b (PbCas13b).
223 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K590 of Prevotella buccae Cas13b (PbCas13b).
224 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N634 of Prevotella buccae Cas13b (PbCas13b).
225 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R638 of Prevotella buccae Cas13b (PbCas13b).
226 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N652 of Prevotella buccae Cas13b (PbCas13b).
227 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N653 of Prevotella buccae Cas13b (PbCas13b).
228 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K655 of Prevotella buccae Cas13b (PbCas13b).
229 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid S658 of Prevotella buccae Cas13b (PbCas13b).
230 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K741 of Prevotella buccae Cas13b (PbCas13b).
231 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K744 of Prevotella buccae Cas13b (PbCas13b).
232 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N756 of Prevotella buccae Cas13b (PbCas13b).
233 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid S757 of Prevotella buccae Cas13b (PbCas13b).
234 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R762 of Prevotella buccae Cas13b (PbCas13b).
235 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R791 of Prevotella buccae Cas13b (PbCas13b).
236 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K846 of Prevotella buccae Cas13b (PbCas13b).
237 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K857 of Prevotella buccae Cas13b (PbCas13b).
238 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K870 of Prevotella buccae Cas13b (PbCas13b).
239 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R877 of Prevotella buccae Cas13b (PbCas13b).
240 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K183 of Prevotella buccae Cas13b (PbCas13b).
241 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K193 of Prevotella buccae Cas13b (PbCas13b).
242 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R600 of Prevotella buccae Cas13b (PbCas13b).
243 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K607 of Prevotella buccae Cas13b (PbCas13b).
244 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K612 of Prevotella buccae Cas13b (PbCas13b).
245 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R614 of Prevotella buccae Cas13b (PbCas13b).
246 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K617 of Prevotella buccae Cas13b (PbCas13b).
247 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K826 of Prevotella buccae Cas13b (PbCas13b).
248 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K828 of Prevotella buccae Cas13b (PbCas13b).
249 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K829 of Prevotella buccae Cas13b (PbCas13b).
250 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R824 of Prevotella buccae Cas13b (PbCas13b).
251 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R830 of Prevotella buccae Cas13b (PbCas13b).
252 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid Q831 of Prevotella buccae Cas13b (PbCas13b).
253 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K835 of Prevotella buccae Cas13b (PbCas13b).
254 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K836 of Prevotella buccae Cas13b (PbCas13b).
255 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R838 of Prevotella buccae Cas13b (PbCas13b).
256 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R618 of Prevotella buccae Cas13b (PbCas13b).
257 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid D434 of Prevotella buccae Cas13b (PbCas13b).
258 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K431 of Prevotella buccae Cas13b (PbCas13b).
259 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R53 of Prevotella buccae Cas13b (PbCas13b).
260 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K943 of Prevotella buccae Cas13b (PbCas13b).
261 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R1041 of Prevotella buccae Cas13b (PbCas13b).
262 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid Y164 of Prevotella buccae Cas13b (PbCas13b).
263 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R285 of Prevotella buccae Cas13b (PbCas13b).
264 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R287 of Prevotella buccae Cas13b (PbCas13b).
265 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K292 of Prevotella buccae Cas13b (PbCas13b).
266 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid E296 of Prevotella buccae Cas13b (PbCas13b).
267 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N297 of Prevotella buccae Cas13b (PbCas13b).
268 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid Q646 of Prevotella buccae Cas13b (PbCas13b).
269 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N647 of Prevotella buccae Cas13b (PbCas13b).
270 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R402 of Prevotella buccae Cas13b (PbCas13b).
271 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K393 of Prevotella buccae Cas13b (PbCas13b).
272 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N653 of Prevotella buccae Cas13b (PbCas13b).
273 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N652 of Prevotella buccae Cas13b (PbCas13b).
274 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R482 of Prevotella buccae Cas13b (PbCas13b).
275 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N480 of Prevotella buccae Cas13b (PbCas13b).
276 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid D396 of Prevotella buccae Cas13b (PbCas13b).
277 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid E397 of Prevotella buccae Cas13b (PbCas13b).
278 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid D398 of Prevotella buccae Cas13b (PbCas13b).
279 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid E399 of Prevotella buccae Cas13b (PbCas13b).
280 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K294 of Prevotella buccae Cas13b (PbCas13b).
281 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid E400 of Prevotella buccae Cas13b (PbCas13b).
282 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R56 of Prevotella buccae Cas13b (PbCas13b).
283 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N157 of Prevotella buccae Cas13b (PbCas13b).
284 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H161 of Prevotella buccae Cas13b (PbCas13b).
285 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H452 of Prevotella buccae Cas13b (PbCas13b).
286 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N455 of Prevotella buccae Cas13b (PbCas13b).
287 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K484 of Prevotella buccae Cas13b (PbCas13b).
288 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N486 of Prevotella buccae Cas13b (PbCas13b).
289 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid G566 of Prevotella buccae Cas13b (PbCas13b).
290 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H567 of Prevotella buccae Cas13b (PbCas13b).
291 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid A656 of Prevotella buccae Cas13b (PbCas13b).
292 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid V795 of Prevotella buccae Cas13b (PbCas13b).
293 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid A796 of Prevotella buccae Cas13b (PbCas13b).
294 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid W842 of Prevotella buccae Cas13b (PbCas13b).
295 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid K871 of Prevotella buccae Cas13b (PbCas13b).
296 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid E873 of Prevotella buccae Cas13b (PbCas13b).
297 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R874 of Prevotella buccae Cas13b (PbCas13b).
298 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid R1068 of Prevotella buccae Cas13b (PbCas13b).
299 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid N1069 of Prevotella buccae Cas13b (PbCas13b).
300 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H1073 of Prevotella buccae Cas13b (PbCas13b).
301 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Leptotrichia shahii Cas13a (LshCas13a): R597, N598, H602, R1278, N1279, or H1283.
302 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Leptotrichia shahii Cas13a (LshCas13a): R597, N598, H602, R1278, N1279, or H1283.
303 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Leptotrichia shahii Cas13a (LshCas13a): R597, N598, H602, R1278, N1279, or H1283.
304 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Leptotrichia shahii Cas13a (LshCas13a): R597, N598, or H602.
305 . The engineered CRISPR-Cas protein of claim 55 , comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of Leptotrichia shahii Cas13a (LshCas13a): R597, N598, or H602.
306 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Leptotrichia shahii Cas13a (LshCas13a): R1278, N1279, or H1283.
307 . The engineered CRISPR-Cas protein of claim 55 , comprising in HEPN domain 2 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 2 of Leptotrichia shahii Cas13a (LshCas13a): R1278, N1279, or H1283.
308 . The engineered CRISPR-Cas protein of claim 55 , comprising one or more mutation of an amino acid corresponding to the following amino acids of Porphyromonas gulae Cas13b (PguCas13b): R146, H151, R1116, or H1121.
309 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Porphyromonas gulae Cas13b (PguCas13b): R146, H151, R1116, or H1121.
310 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Porphyromonas gulae Cas13b (PguCas13b): R146, H151, R1116, or H1121.
311 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Porphyromonas gulae Cas13b (PguCas13b): R146 or H151.
312 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 1 of Porphyromonas gulae Cas13b (PguCas13b): R146 or H151.
313 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Porphyromonas gulae Cas13b (PguCas13b): R1116 or H1121.
314 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 2 one or more mutation of an amino acid corresponding to the following amino acids in HEPN domain 2 of Porphyromonas gulae Cas13b (PguCas13b): R1116 or H1121.
315 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella sp. P5-125 Cas13b (PspCas13b): H133 or H1058.
316 . The engineered CRISPR-Cas protein of claim 55 comprising one or more mutation of an amino acid corresponding to the following amino acids of Prevotella sp. P5-125 Cas13b (PspCas13b): H133 or H1058.
317 . The engineered CRISPR-Cas protein of claim 55 comprising in a HEPN domain one or more mutation of an amino acid corresponding to the following amino acids in a HEPN domain of Prevotella sp. P5-125 Cas13b (PspCas13b): H133 or H1058.
318 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H133 of Prevotella sp. P5-125 Cas13b (PspCas13b).
319 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 1 a mutation of an amino acid corresponding to amino acid H133 in HEPN domain 1 of Prevotella sp. P5-125 Cas13b (PspCas13b).
320 . The engineered CRISPR-Cas protein of claim 55 comprising a mutation of an amino acid corresponding to amino acid H1058 of Prevotella sp. P5-125 Cas13b (PspCas13b).
321 . The engineered CRISPR-Cas protein of claim 55 comprising in HEPN domain 2 a mutation of an amino acid corresponding to the amino acid H1058 in HEPN domain 2 of Prevotella sp. P5-125 Cas13b (PspCas13b).
322 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to A, P, or V, preferably A.
323 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to a hydrophobic amino acid.
324 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to an aromatic amino acid.
325 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to a charged amino acid.
326 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to a positively charged amino acid.
327 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to a negatively charged amino acid.
328 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to a polar amino acid.
329 . The engineered CRISPR-Cas protein of any of claims 57 to 321 , wherein said amino acid is mutated to an aliphatic amino acid.
330 . The engineered CRISPR-Cas protein of claim 55 , wherein said Cas13 protein is or originates from a species of the genus Alistipes, Anaerosalibacter, Bacteroides, Bacteroidetes, Bergeyella, Blautia, Butyrivibrio, Capnocytophaga, Carnobacterium, Chloroflexus, Chryseobacterium, Clostridium, Demequina, Eubacteriaceae, Eubacterium, Flavobacterium, Fusobacterium, Herbinix, Insolitispirillum, Lachnospiraceae, Leptotrichia, Listeria, Myroides, Paludibacter, Phaeodactylibacter, Porphyromonadaceae, Porphyromonas, Prevotella, Pseudobutyrivibrio, Psychroflexus, Reichenbachiella, Rhodobacter, Riemerella, Sinomicrobium, Thalassospira, Ruminococcus ; preferably Leptotrichia shahii, Listeria seeligeri, Lachnospiraceae bacterium (such as Lb MA2020, Lb NK4A179, Lb NK4A144), Clostridium aminophilum (such as Ca DSM 10710), Carnobacterium gallinarum (such as Cg DSM 4847), Paludibacter propionicigenes (such as Pp WB4), Listeria weihenstephanensis (such as Lw FSL R9-0317), Listeriaceae bacterium (such as Lb FSL M6-0635), Leptotrichia wadei (such as Lw F0279), Rhodobacter capsulatus (such as Rc SB 1003, Rc R121, Rc DE442), Leptotrichia buccalis (such as Lb C-1013-b), Herbinix hemicellulosilytica, Eubacteriaceae bacterium (such as Eb CHKCI004), Blautia . sp Marseille-P2398 , Leptotrichia sp. oral taxon 879 str. F0557 , Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1 , Pseudobutyrivibrio sp. OR37, Butyrivibrio sp. YAB3001 , Leptotrichia sp. Marseille-P3007, Bacteroides ihuae, Porphyromonadaceae bacterium (such as Pb KH3CP3RA), Listeria riparia, Insolitispirillum peregrinum, Alistipes sp. ZOR0009, Bacteroides pyogenes (such as Bp F0041), Bacteroidetes bacterium (such as Bb GWA2_31_9), Bergeyella zoohelcum (such as Bz ATCC 43767), Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Chryseobacterium carnipullorum, Chryseobacterium jejuense, Chryseobacterium ureilyticum, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium sp. 316 , Myroides odoratimimus (such as Mo CCUG 10230, Mo CCUG 12901, Mo CCUG 3837), Paludibacter propionicigenes, Phaeodactylibacter xiamenensis, Porphyromonas gingivalis (such as Pg F0185, Pg F0568, Pg JCVI SC001, Pg W4087 , Porphyromonas gulae, Porphyromonas sp. COT-052 OH4946, Prevotella aurantiaca, Prevotella buccae (such as Pb ATCC 33574), Prevotella falsenii, Prevotella intermedia (such as Pi 17, Pi ZT), Prevotella pallens (such as Pp ATCC 700821), Prevotella pleuritidis, Prevotella saccharolytica (such as Ps F0055), Prevotella sp. MA2016, Prevotella sp. MSX73, Prevotella sp. P4-76, Prevotella sp. P5-119, Prevotella sp. P5-125, Prevotella sp. P5-60 , Psychroflexus torquis, Reichenbachiella agariperforans, Riemerella anatipestifer, Sinomicrobium oceani, Fusobacterium necrophorum (such as Fn subsp. funduliforme ATCC 51357, Fn DJ-2, Fn BFTR-1, Fn subsp. funduliforme ), Fusobacterium perfoetens (such as Fp ATCC 29250), Fusobacterium ulcerans (such as Fu ATCC 49185), Anaerosalibacter sp. ND1, Eubacterium siraeum, Ruminococcus flavefaciens (such as Rfx XPD3002), or Ruminococcus albus.
331 . The engineered CRISPR-Cas protein of claim 55 , wherein said Cas13 protein is a Cas13a protein.
332 . The engineered CRISPR-Cas protein of claim 331 , wherein said Cas13a protein is or originates from a species of the genus Bacteroides, Blautia, Butyrivibrio, Carnobacterium, Chloroflexus, Clostridium, Demequina, Eubacterium, Herbinix, Insolitispirillum, Lachnospiraceae, Leptotrichia, Listeria, Paludibacter, Porphyromonadaceae, Pseudobutyrivibrio, Rhodobacter , or Thalassospira ; preferably Leptotrichia shahii, Listeria seeligeri, Lachnospiraceae bacterium (such as Lb MA2020, Lb NK4A179, Lb NK4A144), Clostridium aminophilum (such as Ca DSM 10710), Carnobacterium gallinarum (such as Cg DSM 4847), Paludibacter propionicigenes (such as Pp WB4), Listeria weihenstephanensis (such as Lw FSL R9-0317), Listeriaceae bacterium (such as Lb FSL M6-0635), Leptotrichia wadei (such as Lw F0279), Rhodobacter capsulatus (such as Rc SB 1003, Rc R121, Rc DE442), Leptotrichia buccalis (such as Lb C-1013-b), Herbinix hemicellulosilytica, Eubacteriaceae bacterium (such as Eb CHKCI004), Blautia . sp Marseille-P2398 , Leptotrichia sp. oral taxon 879 str. F0557 , Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1 , Pseudobutyrivibrio sp. OR37, Butyrivibrio sp. YAB3001 , Leptotrichia sp. Marseille-P3007, Bacteroides ihuae, Porphyromonadaceae bacterium (such as Pb KH3CP3RA), Listeria riparia , or Insolitispirillum peregrinum.
333 . The engineered CRISPR-Cas protein of claim 55 , wherein said Cas13 protein is a Cas13b protein.
334 . The engineered CRISPR-Cas protein of claim 333 , wherein said Cas13b protein is or originates from a species of the genus Alistipes, Bacteroides, Bacteroidetes, Bergeyella, Capnocytophaga, Chryseobacterium, Flavobacterium, Myroides, Paludibacter, Phaeodactylibacter, Porphyromonas, Prevotella, Psychroflexus, Reichenbachiella, Riemerella , or Sinomicrobium ; preferably Alistipes sp. ZOR0009, Bacteroides pyogenes (such as Bp F0041), Bacteroidetes bacterium (such as Bb GWA2_31_9), Bergeyella zoohelcum (such as Bz ATCC 43767), Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Chryseobacterium carnipullorum, Chryseobacterium jejuense, Chryseobacterium ureilyticum, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium sp. 316 , Myroides odoratimimus (such as Mo CCUG 10230, Mo CCUG 12901, Mo CCUG 3837), Paludibacter propionicigenes, Phaeodactylibacter xiamenensis, Porphyromonas gingivalis (such as Pg F0185, Pg F0568, Pg JCVI SC001, Pg W4087 , Porphyromonas gulae, Porphyromonas sp. COT-052 OH4946, Prevotella aurantiaca, Prevotella buccae (such as Pb ATCC 33574), Prevotella falsenii, Prevotella intermedia (such as Pi 17, Pi ZT), Prevotella pallens (such as Pp ATCC 700821), Prevotella pleuritidis, Prevotella saccharolytica (such as Ps F0055), Prevotella sp. MA2016, Prevotella sp. MSX73, Prevotella sp. P4-76, Prevotella sp. P5-119, Prevotella sp. P5-125, Prevotella sp. P5-60 , Psychroflexus torquis, Reichenbachiella agariperforans, Riemerella anatipestifer , or Sinomicrobium oceani.
335 . The engineered CRISPR-Cas protein of claim 55 , wherein said Cas13 protein is a Cas13c protein.
336 . The engineered CRISPR-Cas protein of claim 335 , wherein said Cas13c protein is or originates from a species of the genus Fusobacterium or Anaerosalibacter ; preferably Fusobacterium necrophorum (such as Fn subsp. funduliforme ATCC 51357, Fn DJ-2, Fn BFTR-1, Fn subsp. funduliforme ), Fusobacterium perfoetens (such as Fp ATCC 29250), Fusobacterium ulcerans (such as Fu ATCC 49185), or Anaerosalibacter sp. ND1.
337 . The engineered CRISPR-Cas protein of claim 55 , wherein said Cas13 protein is a Cas13d protein.
338 . The engineered CRISPR-Cas protein of claim 337 , wherein said Cas13d protein is originates from a species of the genus Eubacterium or Ruminococcus , preferably Eubacterium siraeum, Ruminococcus flavefaciens (such as Rfx XPD3002), or Ruminococcus albus.
339 . The engineered CRISPR-Cas protein of claim 50 , wherein catalytic activity of the engineered CRISPR-Cas protein is increased as compared to a corresponding wildtype CRISPR-Cas protein.
340 . The engineered CRISPR-Cas protein of claim 50 , wherein catalytic activity of the engineered CRISPR-Cas protein is decreased as compared to a corresponding wildtype CRISPR-Cas protein.
341 . The engineered CRISPR-Cas protein of claim 50 , wherein gRNA binding of the engineered CRISPR-Cas protein is increased as compared to a corresponding wildtype CRISPR-Cas protein.
342 . The engineered CRISPR-Cas protein of claim 50 , wherein gRNA binding of the engineered CRISPR-Cas protein is decreased as compared to a corresponding wildtype CRISPR-Cas protein.
343 . The engineered CRISPR-Cas protein of claim 50 , wherein specificity of the CRISPR-Cas protein is increased as compared to a corresponding wildtype CRISPR-Cas protein.
344 . The engineered CRISPR-Cas protein of claim 50 , wherein specificity of the CRISPR-Cas protein is decreased as compared to a corresponding wildtype CRISPR-Cas protein.
345 . The engineered CRISPR-Cas protein of claim 50 , wherein stability of the CRISPR-Cas protein is increased as compared to a corresponding wildtype CRISPR-Cas protein.
346 . The engineered CRISPR-Cas protein of claim 50 , wherein stability of the CRISPR-Cas protein is decreased as compared to a corresponding wildtype CRISPR-Cas protein.
347 . The engineered CRISPR-Cas protein of claim 50 , further comprising one or more mutations which inactivate catalytic activity.
348 . The engineered CRISPR-Cas protein of claim 50 , wherein off-target binding of the CRISPR-Cas protein is increased as compared to a corresponding wildtype CRISPR-Cas protein.
349 . The engineered CRISPR-Cas protein of claim 50 , wherein off-target binding of the CRISPR-Cas protein is decreased as compared to a corresponding wildtype CRISPR-Cas protein.
350 . The engineered CRISPR-Cas protein of claim 50 , wherein target binding of the CRISPR-Cas protein is increased as compared to a corresponding wildtype CRISPR-Cas protein.
351 . The engineered CRISPR-Cas protein of claim 50 , wherein target binding of the CRISPR-Cas protein is decreased as compared to a corresponding wildtype CRISPR-Cas protein.
352 . The engineered CRISPR-Cas protein of claim 50 , wherein the engineered CRISPR-Cas protein has a higher protease activity or polynucleotide-binding capability compared to a corresponding wildtype CRISPR-Cas protein.
353 . The engineered CRISPR-Cas protein of claim 50 , wherein PFS recognition is altered as compared to a corresponding wildtype CRISPR-Cas protein.
354 . The engineered CRISPR-Cas protein of claim 1 , further comprising a functional heterologous domain.
355 . The engineered CRISPR-Cas protein of claim 50 , further comprising an NLS.
356 . The engineered CRISPR-Cas protein of claim 50 , further comprising a NES.
357 . An engineered CRISPR-Cas protein comprising one or more HEPN domains and is less than 1000 amino acids in length.
358 . The engineered CRISPR-Cas protein of claim 357 , wherein the protein is less than 950, less than 900, less than 850, less than 800, less, or than 750 amino acids in size.
359 . The engineered CRISPR-Cas protein of claim 357 , wherein the HEPN domain comprises a RxxxxH motif.
360 . The engineered CRISPR-Cas protein of claim 359 , wherein the RxxxxH motif comprises a R[N/H/K]X 1 X 2 X 3 H sequence.
361 . The engineered CRISPR-Cas protein of claim 360 , wherein:
X 1 is R, S, D, E, Q, N, G, or Y, X 2 is independently I, S, T, V, or L, and X 3 is independently L, F, N, Y, V, I, S, D, E, or A.
362 . The engineered CRISPR-Cas protein of claim 357 , wherein the CRISPR-Cas protein is a Type VI CRISPR Cas protein.
363 . The engineered CRISPR Cas protein of claim 362 , wherein the Type VI CRISPR Cas protein is a Cas13a, a Cas13b, a Cas13c, or a Cas13d.
364 . The engineered CRISPR-Cas protein of claim 357 , wherein the CRISPR-Cas protein is associated with a functional domain.
365 . The engineered CRISPR-Cas protein of claim 357 , wherein the CRISPR-Cas protein comprises one or more mutations equivalent to mutations in any one of claims 57 - 329 .
366 . The engineered CRISPR-Cas protein of claim 365 , wherein the CRISPR-Cas protein comprises one or more mutations in the helical domain.
367 . The engineered CRISPR-Cas protein of claim 357 , wherein the CRISPR-Cas protein is in a dead form or has nickase activity.
368 . A polynucleotide encoding the engineered CRISPR-Cas protein of any of claims 1 to 367 .
369 . The polynucleotide according to claim 319 , which is codon optimized.
370 . A CRISPR-Cas system comprising the engineered CRISPR-Cas protein of any of claims 1 to 367 or the polynucleotide of claim 318 or 319 , and a nucleotide component capable of forming a complex with the engineered CRISPR-Cas protein and able to hybridize with a target nucleic acid sequence and direct sequence-specific binding of said complex to the target nucleic acid sequence.
371 . A vector system comprising one or more vectors, the one or more vectors comprising one or more polynucleotide molecules encoding components of the engineered CRISPR-Cas protein of claim 370 .
372 . A method of modifying a target nucleic acid comprising: introducing in a cell or organism that comprises the target nucleic acid, the engineered CRISPR-Cas protein according to any of claims 1 to 367 , the polynucleic acid according to claim 368 or 369 , the CRISPR-Cas system according to claim 370 , or the vector or vector system according to claim 371 , such that the engineered CRISPR-Cas protein modifies the target nucleic acid in the cell or organism.
373 . The method of claim 372 , wherein the engineered CRISPR-Cas system is introduced via delivery by liposomes, nanoparticles, exosomes, microvesicles, nucleic acid nanoassemblies, a gene gun, an implantable device, or the vector system of claim 371 .
374 . The method of claim 372 , wherein the engineered CRISPR-cas protein is associated with one or more functional domains.
375 . The method of claim 372 , wherein the target nucleic acid comprises a genomic locus, and the engineered CRISPR-Cas protein modifies gene product encoded at the genomic locus or expression of the gene product.
376 . The method of claim 372 , wherein the target nucleic acid is DNA or RNA and wherein one or more nucleotides in the target nucleic acid are base edited.
377 . The method of claim 372 , wherein the target nucleic acid is DNA or RNA and wherein the target nucleic acid is cleaved.
378 . The method of claim 377 , wherein the engineered CRISPR-Cas protein further cleaves non-target nucleic acid.
379 . The method of claim 377 , further comprising visualizing activity and, optionally, using a detectable label.
380 . The method of claim 377 , further comprising detecting binding of one or more components of the CRISPR-Cas system to the target nucleic acid.
381 . The method of claim 377 , wherein said cell or organisms is a eukaryotic cell or organism.
382 . The method of claim 377 , wherein said cell or organisms is an animal cell or organism.
383 . The method of claim 377 , wherein said cell or organisms is a plant cell or organism.
384 . A method for detecting a target nucleic acid in a sample comprising:
contacting a sample with:
an engineered CRISPR-Cas protein of any one of claims 50 to 367 ;
at least one guide polynucleotide comprising a guide sequence capable of binding to the target nucleic acid and designed to form a complex with the engineered CRISPR-Cas; and
a RNA-based masking construct comprising a non-target sequence;
wherein the engineered CRISPR-Cas protein exhibits collateral RNase activity and cleaves the non-target sequence of the detection construct; and
detecting a signal from cleavage of the non-target sequence, thereby detecting the target nucleic acid in the sample.
385 . The method of claim 384 , further comprising contacting the sample with reagents for amplifying the target nucleic acid.
386 . The method of claim 385 , wherein the reagents for amplifying comprises isothermal amplification reaction reagents.
387 . The method of claim 386 , wherein the isothermal amplification reagents comprise nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, or nicking enzyme amplification reagents.
388 . The method of claim 384 , wherein the target nucleic acid is DNA molecule and the method further comprises contacting the target DNA molecule with a primer comprising an RNA polymerase site and RNA polymerase.
389 . The method of claim 384 , wherein the masking construct:
suppresses generation of a detectable positive signal until the masking construct cleaved or deactivated, or masks a detectable positive signal or generates a detectable negative signal until the masking construct cleaved or deactivated.
390 . The method of claim 384 , wherein the masking construct comprises:
a. a silencing RNA that suppresses generation of a gene product encoded by a reporting construct, wherein the gene product generates the detectable positive signal when expressed; b. a ribozyme that generates the negative detectable signal, and wherein the positive detectable signal is generated when the ribozyme is deactivated; c. a ribozyme that converts a substrate to a first color and wherein the substrate converts to a second color when the ribozyme is deactivated; d. an aptamer and/or comprises a polynucleotide-tethered inhibitor; e. a polynucleotide to which a detectable ligand and a masking component are attached; f. a nanoparticle held in aggregate by bridge molecules, wherein at least a portion of the bridge molecules comprises a polynucleotide, and wherein the solution undergoes a color shift when the nanoparticle is disbursed in solution; g. a quantum dot or fluorophore linked to one or more quencher molecules by a linking molecule, wherein at least a portion of the linking molecule comprises a polynucleotide; h. a polynucleotide in complex with an intercalating agent, wherein the intercalating agent changes absorbance upon cleavage of the polynucleotide; or l. two fluorophores tethered by a polynucleotide that undergo a shift in fluorescence when released from the polynucleotide.
391 . The method of claim 390 , wherein the aptamer
a. comprises a polynucleotide-tethered inhibitor that sequesters an enzyme, wherein the enzyme generates a detectable signal upon release from the aptamer or polynucleotide-tethered inhibitor by acting upon a substrate; or b. is an inhibitory aptamer that inhibits an enzyme and prevents the enzyme from catalyzing generation of a detectable signal from a substrate or wherein the polynucleotide-tethered inhibitor inhibits an enzyme and prevents the enzyme from catalyzing generation of a detectable signal from a substrate; or c. sequesters a pair of agents that when released from the aptamers combine to generate a detectable signal.
392 . The method of claim 390 , wherein the nanoparticle is a colloidal metal.
393 . The method of claim 384 , wherein the at least one guide polynucleotide comprises a mismatch.
394 . The method of claim 384 , wherein the mismatch is up- or downstream of a single nucleotide variation on the one or more guide sequences.
395 . A cell or organism comprising the engineered CRISPR-Cas protein according to any of claims 1 to 367 , the polynucleic acid according to claim 368 or 369 , the CRISPR-Cas system according to claim 370 , or the vector or vector system according to claim 371 .Join the waitlist — get patent alerts
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