CRISPR-Cas13 system and use thereof
The present invention relates to a CRISPR-Cas13 system and use thereof, and also relates to a Cas13 protein, a fusion protein, and a guide polynucleotide. The Cas13 protein has at least 90% sequence identity compared to SEQ ID NO: 1. The fusion protein comprises the Cas13 protein fused to a protein domain and/or a polypeptide tag. The guide polynucleotide comprises a same-direction repetition sequence and a guide sequence that has been engineered to hybridize with the target RNA. The same-direction repetition sequence has at least 70% sequence identity to any of SEQ ID NOs: 3 and 80-87. The CRISPR-Cas13 system comprises the Cas13 protein that has at least 90% sequence identity to SEQ ID NO: 1, or a coding nucleic acid therefor, and the guide polynucleotide or a coding nucleic acid therefor.
1. A non-naturally occurring Cas13 protein, wherein the amino acid sequence of the Cas13 protein has at least 90% sequence identity compared to SEQ ID NO: 1, and comprises one or more mutations compared to SEQ ID NO: 1.
2. The Cas13 protein according to claim 1 , wherein
(a) the Cas13 protein comprises at least one mutation at the location corresponding to amino acid residue positions 40-91, 146-153, 158-176, 182-209, 216-253, 271-287, 341-353, 379-424, 456-477, 521-557, 575-588, 609-625, 700-721, 724-783, 796-815, 828-852 or 880-893 of the reference protein as shown in SEQ ID NO: 1;
(b) the Cas13 protein comprises one or more mutations at the location corresponding to the following amino acid residues of the reference protein as shown in SEQ ID NO: 1: R11, N34, R35, R47, R58, R63, R64, N68, N87, N265, N274, R276, R290, R294, N299, N303, R308, R314, R320, R328, N332, R341, N346, R358, N372, N383, N390, N394, R47+R290, R47+R314, R290+R314, R47+R290+R314, R308+N68, N394+N68, N87+N68, R308+N265, N394+N265, N87+N265, R308+N68+N265, N87+N68+N265, T7, A16, S260, A263, M266, N274, F288, M302, N303, L304, V305, I311, D313, H324, P326, H327, N332, N346, T353, T360, E365, A373, M380, S382, K395, Y396, D402, D411, and S418; and/or,
(c) the Cas13 protein comprises a mutation at the location corresponding to the RxxxxH motif at positions 210-215, 750-755 and/or 785-790 of the reference protein as shown in SEQ ID NO: 1.
3. The Cas13 protein according to claim 1 , wherein the Cas13 protein comprises one or more mutations at the location corresponding to the following amino acid residues of the reference protein as shown in SEQ ID NO: 1:
R11A, N34A, R35A, R47A, R58A, R63A, R64A, N68A, N87A, N265A, N274A, R276A, R290A, R294A, N299A, N303A, R308A, R314A, R320A, R328A, N332A, R341A, N346A, R358A, N372A, N383A, N390A, N394A, R47A+R290A, R47A+R314A, R290A+R314A, R47A+R290A+R314A, R308A+N68A, N394A+N68A, N87A+N68A, R308A+N265A, N394A+N265A, N87A+N265A, R308A+N68A+N265A, N87A+N68A+N265A, T7S, A16S, S260E, A263K, M2661, N274K, F288Y, M302F, N303S, L304I, V305K, I311M D313E, H324Y, P326S, H327V, N332Y, N346D, T353L, T360S, E365D, A373E, M380K, S382R, K395G, Y396D, D402L, D411E, and S418K.
4. A CRISPR-Cas13 system comprising (1) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide, wherein the guide polynucleotide comprises (i) a direct repeat sequence having at least 70% sequence identity compared to SEQ ID NO: 3, wherein the direct repeat sequence is linked to (ii) a guide sequence engineered to hybridize with a target RNA, wherein the direct repeat sequence is GGAAGATN 1 ACTCTACAAACCTGTAGN 2 GN 3 N 4 N 5 N 6 N 7 N 8 N 9 N 10 N 11 (SEQ ID NO: 277); wherein N 1 and N 3 -N 11 are independently any one selected from A, C, G, and T; and N 2 is any one selected from A and G; and (2) the Cas13 protein of claim 1 or a nucleic acid encoding the Cas13 protein of claim 1 .
5. A fusion protein comprising a Cas13 protein fused to a heterologous protein domain and/or a polypeptide tag; wherein the amino acid sequence of the Cas13 protein has at least 90% sequence identity compared to SEQ ID NO: 1.
6. The fusion protein according to claim 5 , wherein the Cas13 protein is covalently linked to the protein domain; and/or, the Cas13 protein is fused to any one or more of the following protein domains and/or polypeptide tags: a cytosine deaminase domain, an adenosine deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, an affinity domain, a subcellular localization signal, a reporter tag, and an affinity tag.
7. The fusion protein according to claim 6 , wherein the subcellular localization signal is one or more of a nuclear localization signal (NLS) and a nuclear export signal (NES).
8. The fusion protein according to claim 5 , wherein the structure of the fusion protein is NLS-Cas13 protein-SV40 NLS-nucleoplasmin NLS.
9. The fusion protein according to claim 5 , wherein the length of the amino acid sequence of the protein domain is ≥40 amino acids; and/or, the length of the amino acid sequence of the polypeptide tag is ≤40 amino acids.
10. A pharmaceutical composition comprising the fusion protein according to claim 5 .
11. A non-naturally occurring guide polynucleotide comprising (i) a direct repeat sequence having at least 70% sequence identity compared to SEQ ID NO: 3, wherein the direct repeat sequence is linked to (ii) a guide sequence engineered to hybridize with a target RNA, wherein the guide polynucleotide can form a CRISPR complex with a Cas13 protein and guide the sequence-specific binding of the CRISPR complex to the target RNA; wherein the direct repeat sequence is GGAAGATN 1 ACTCTACAAACCTGTAGN 2 GN 3 N 4 N 5 N 6 N 7 N 8 N 9 N 10 N 11 (SEQ ID NO: 277); wherein N 1 and N 3 -N 11 are independently any one selected from A, C, G, and T; and N 2 is any one selected from A and G; and wherein the target RNA is a eukaryotic RNA.
12. A method for cleaving one or more target RNA molecules; wherein the method comprises the step of contacting a fusion protein and the guide polynucleotide according to claim 11 with the target RNA; wherein the fusion protein comprises a Cas13 protein fused to a heterologous protein domain and/or a polypeptide tag; and wherein the amino acid sequence of the Cas13 protein has at least 90% sequence identity compared to SEQ ID NO: 1.
13. A method for detecting a target RNA in a nucleic acid sample suspected of comprising the target RNA, wherein the method comprises the step of contacting a fusion protein and the guide polynucleotide according to claim 11 with the nucleic acid sample; wherein the fusion protein comprises a Cas13 protein fused to a heterologous protein domain and/or a polypeptide tag; and wherein the amino acid sequence of the Cas13 protein has at least 90% sequence identity compared to SEQ ID NO: 1.
14. The guide polynucleotide according to claim 11 , wherein the direct repeat sequence has at least 80% sequence identity compared to SEQ ID NO: 3;
and/or, the guide sequence is located at the 3′ end of the direct repeat sequence;
and/or, the guide sequence comprises 15-35 nucleotides;
and/or, the guide sequence hybridizes with the target RNA with no more than one nucleotide mismatch;
and/or, the guide polynucleotide further comprises an aptamer sequence;
and/or, the guide polynucleotide comprises a nucleotide with modification;
and/or, the Cas13 protein is a non-naturally occurring Cas protein, and the amino acid sequence of the Cas13 protein has at least 90% sequence identity compared to SEQ ID NO: 1.
15. The guide polynucleotide according to claim 14 , wherein the aptamer sequence is inserted into a loop of the guide polynucleotide;
and/or, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence;
and/or, wherein the modification comprises 2′-O-methyl, 2′-O-methyl-3′-phosphorothioate, or 2′-O-methyl-3′-thioPACE.
16. The guide polynucleotide according to claim 11 , wherein the target RNA is located in the nucleus of a eukaryotic cell; and/or, the target RNA is any one or more selected from TTR RNA, SOD1 RNA, PCSK9 RNA, VEGFA RNA, VEGFR1 RNA, PTBP1 RNA, AQp1 RNA, and ANGPTL3 RNA.
17. The guide polynucleotide according to claim 16 , wherein the guide sequence is any one or more selected from the sequences as shown in SEQ ID NOs: 5-6, and SEQ ID NOs: 42-49.
18. A CRISPR-Cas13 system comprising:
(1) a Cas13 protein comprising an amino acid sequence having at least 90% sequence identity compared to SEQ ID NO: 1, or a nucleic acid encoding the Cas13 protein; and
(2) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; wherein the guide polynucleotide comprises a direct repeat sequence linked to a guide sequence, wherein the direct repeat sequence has at least 70% sequence identity to SEQ ID NO: 3, and has the structure of GGAAGATN 1 ACTCTACAAACCTGTAGN 2 GN 3 N 4 N 5 N 6 N 7 N 8 N 9 N 10 N 11 (SEQ ID NO: 277); wherein N 1 and N 3 -N 11 are independently any one selected from A, C, G, and T; and N 2 is any one selected from A and G; wherein the guide sequence is engineered to hybridize with a target RNA;
wherein the guide polynucleotide can form a CRISPR complex with the Cas13 protein and guide a sequence-specific binding of the CRISPR complex to the target RNA; wherein the Cas13 protein and the guide polynucleotide do not naturally occur together; and wherein the target RNA is located in a eukaryotic cell.
19. The CRISPR-Cas13 system according to claim 18 , wherein the target RNA is any one or more selected from TTR RNA, SOD1 RNA, PCSK9 RNA, VEGFA RNA, VEGFR1 RNA, PTBP1 RNA, AQp1 RNA, and ANGPTL3 RNA; and/or, the guide sequence is any one or more selected from the sequences as shown in SEQ ID NOs: 5-6 and SEQ ID NOs: 42-49.
20. A vector system comprising the CRISPR-Cas13 system according to claim 18 , wherein the vector system comprises one or more vectors comprising a polynucleotide sequence encoding the Cas13 protein and a polynucleotide sequence encoding the guide polynucleotide.
21. The vector system according to claim 20 , wherein
the vector is an adeno-associated viral vector comprising a DNA encoding the Cas13 protein and the guide polynucleotide;
or, the vector is a lipid nanoparticle comprising the guide polynucleotide and an mRNA encoding the Cas13 protein;
and/or, the vector is a lentiviral vector comprising the guide polynucleotide and an mRNA encoding the Cas13 protein.
22. A ribonucleoprotein complex comprising the CRISPR-Cas13 system according to claim 18 , wherein the ribonucleoprotein complex is formed from the guide polynucleotide and the Cas13 protein.
23. A viral-like particle comprising the CRISPR-Cas13 system according to claim 18 , wherein the viral-like particle comprises a ribonucleoprotein complex formed from the guide polynucleotide and the Cas13 protein; optionally, the Cas13 protein is fused to a gag protein.
24. A pharmaceutical composition comprising the CRISPR-Cas13 system according to claim 18 .
25. An in vitro composition comprising the CRISPR-Cas13 system according to claim 18 and a labeled detector RNA that is not capable of hybridization with the guide polynucleotide.
26. A method for detecting a target RNA in a nucleic acid sample suspected of comprising the target RNA, wherein the method comprises the step of contacting the CRISPR-Cas13 system according to claim 18 with the nucleic acid sample.
27. A method for
cleaving one or more target RNA molecules;
wherein the method comprises the step of contacting the CRISPR-Cas13 system according to claim 18 with the target RNA.