CRISPR-Cas13 system and its uses

Abstract

The present invention relates to a CRISPR-Cas13 system and its use, and further 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 direct repeat sequence and a guide sequence engineered to hybridize to a target RNA, the direct repeat sequence having at least 70% sequence identity compared to any one of SEQ ID NOs: 3, 80-87. The CRISPR-Cas13 system comprises a Cas13 protein or a nucleic acid encoding the same having at least 90% sequence identity compared to SEQ ID NO: 1, and a guide polynucleotide or a nucleic acid encoding the same.

Description

[Technical Field]

[0001] The present disclosure relates to the field of CRISPR gene editing, and in particular to the CRISPR-Cas13 system and uses thereof. [Background technology]

[0002] CRISPR-Cas13 is an RNA targeting and editing system based on the bacterial immune system that protects bacteria from viruses. The CRISPR-Cas13 system is similar to the CRISPR-Cas9 system, but unlike the Cas9 protein, which targets DNA, the Cas13 protein targets RNA.

[0003] CRISPR-Cas13 belongs to the Type VI CRISPR-Cas13 system, which contains a single effector protein, Cas13. Currently, CRISPR-Cas13 can be classified into multiple subtypes (e.g., Cas13a, Cas13b, Cas13c, and Cas13d) based on phylogenetic analysis. However, there is still an urgent need to discover novel Cas13 systems that are compact in size (e.g., suitable for AAV delivery), have high editing efficiency in mammalian cells (e.g., RNA targeting / cleavage activity), and / or have low cytotoxicity (e.g., cell dormancy or apoptosis due to collateral RNA degradation). Summary of the Invention

[0004] To solve the technical problem of the lack of a Cas13 system in existing technologies that is compact in size, has high editing efficiency in mammalian cells, and / or has low cytotoxicity, the present disclosure provides a CRISPR-Cas13 system and uses thereof that target a target RNA.

[0005] One aspect of the present disclosure relates to a Cas13 protein having an amino acid sequence with at least 90% sequence identity compared to SEQ ID NO:1.

[0006] In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide comprising direct repeats linked to a guide sequence engineered to hybridize to a target RNA.

[0007] In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of specifically binding to a target RNA sequence.

[0008] In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide comprising direct repeats linked to a guide sequence engineered to direct sequence-specific binding of the CRISPR complex to a target RNA.

[0009] In some embodiments, the amino acid sequence of the Cas13 protein has at least 95% sequence identity compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein has at least 96% sequence identity compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein has at least 97% sequence identity compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein has at least 98% sequence identity compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein has at least 99% sequence identity compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein has at least 99.5% sequence identity compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein is set forth in SEQ ID NO: 1.

[0010] The Cas13 protein (i.e., C13-2 protein) whose sequence is SEQ ID NO: 1 in this disclosure was identified based on bioinformatics analysis of prokaryotic genomes and metagenomes in the CNGB database (China National Gene Bank) and subsequent activity validation. In some embodiments, the Cas13 protein described in this disclosure is derived from a species containing a genome that shares an average nucleotide identity (ANI) of ≥ 95% with the genome designated as CNA0009596 in the CNGB database.

[0011] Average nucleotide identity (ANI) is an index used to assess the similarity of all orthologous protein-encoding genes between two genomes at the nucleic acid level. In the case of bacteria / archaea, a threshold of ANI=95% is generally used as a criterion for determining whether or not a species is the same species (Richter M, Rossello-Mora R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19126-31). Therefore, the present disclosure defines species using the above threshold. All species with an ANI value of ≥ 95% relative to the reference genome are considered to be the same species, and the Cas13 protein therein is homologous to and functionally similar to the proteins protected by the present disclosure and falls within the scope of the present disclosure. ANI analysis tools include programs such as FastANI and JSpecies.

[0012] In some embodiments, the amino acid sequence of the Cas13 protein comprises one, two, three, four, five, six, seven or more mutations, such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof, relative to SEQ ID NO:1.

[0013] In some embodiments, the Cas13 protein comprises one or more mutations in the catalytic domain, resulting in reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises a mutation in the catalytic domain, resulting in reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises one or more mutations in one or two HEPN domains, resulting in substantial loss of RNA cleavage activity. In some embodiments, the Cas13 protein comprises a mutation in any one HEPN domain, resulting in substantial loss of RNA cleavage activity.

[0014] The term "substantially lacking RNA cleavage activity" refers to retaining only ≦50%, ≦40%, ≦30%, ≦20%, ≦10%, ≦5%, or ≦1% of the RNA cleavage activity compared to the wild-type Cas13 protein, or having no detectable RNA cleavage activity.

[0015] In some embodiments, the RxxxxH motif (where x represents any amino acid, and RxxxxH may also be written as Rx4H or R4xH) of the Cas13 protein comprises one or more mutations and substantially lacks RNA cleavage activity.

[0016] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215, the RxxxxH motifs at positions 750-755, and / or the RxxxxH motifs at positions 785-790 of the reference protein set forth in SEQ ID NO:1.

[0017] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to the RxxxxH motif at positions 210-215 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to the RxxxxH motif at positions 750-755 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to the RxxxxH motif at positions 785-790 of the reference protein set forth in SEQ ID NO: 1.

[0018] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215 and 750-755 of the reference protein set forth in SEQ ID NO:1.

[0019] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215 and 785-790 of the reference protein set forth in SEQ ID NO:1.

[0020] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 750-755 and 785-790 of the reference protein set forth in SEQ ID NO:1.

[0021] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215, the RxxxxH motifs at positions 750-755, and the RxxxxH motifs at positions 785-790 of the reference protein set forth in SEQ ID NO:1.

[0022] In some embodiments, the RxxxxH motif is mutated to AxxxxH, RxxxxA, or AxxxxA. In some embodiments, the RxxxxH motif is mutated to AxxxxH. In some embodiments, the RxxxxH motif is mutated to RxxxxA. In some embodiments, the RxxxxH motif is mutated to AxxxxA.

[0023] In some embodiments, the Cas13 protein comprises 1, 2, 3, 4, 5, or 6 mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein is mutated to A (alanine) at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein set forth in SEQ ID NO: 1.

[0024] In some embodiments, the amino acid sequence of the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210 and H215 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein comprises mutations at positions corresponding to amino acid residues R750 and H755 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the Cas13 protein comprises mutations at positions corresponding to amino acid residues R785 and H790 of the reference protein set forth in SEQ ID NO: 1.

[0025] In some embodiments, the amino acid sequence of the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R750, and H755 of the reference protein set forth in SEQ ID NO:1.

[0026] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R750, H755, R785, and H790 of the reference protein set forth in SEQ ID NO:1.

[0027] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R785, and / or H790 of the reference protein set forth in SEQ ID NO:1.

[0028] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and H790 of a reference protein set forth in SEQ ID NO:1.

[0029] In some embodiments, the corresponding positions of R210, R750, or R785 are mutated to A. In some embodiments, the corresponding positions of H215, H755, or H790 are mutated to A. In some embodiments, the corresponding positions of R210, H215, R750, H755, R785, and H790 are all mutated to A.

[0030] In some embodiments, the Cas13 protein is obtained by introducing a mutation into the RxxxxH motif at positions 210 to 215, the RxxxxH motif at positions 750 to 755, and / or the RxxxxH motif at positions 785 to 790 of the sequence set forth in SEQ ID NO:1.

[0031] In some embodiments, the Cas13 protein is obtained by introducing 1, 2, 3, 4, 5, or 6 mutations at positions R210, H215, R750, H755, R785, and / or H790 of the sequence set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein is obtained by mutating positions R210, H215, R750, H755, R785, and / or H790 of the sequence set forth in SEQ ID NO: 1 to A (alanine).

[0032] In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R785, and H790 of the sequence set forth in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R750, and H755 of the sequence set forth in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R750, H755, R785, and H790 of the sequence set forth in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R750, H755, R785, and H790 of the sequence set forth in SEQ ID NO: 1 to A.

[0033] In some embodiments, the Cas13 protein contains at least one mutation at a position corresponding to amino acid residues 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 a reference protein set forth in SEQ ID NO:1.

[0034] In some embodiments, compared to the reference protein set forth in SEQ ID NO: 1, the Cas13 protein has amino acid residues at positions corresponding to the following amino acid residues in the reference protein set forth 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+R and any one or more mutations at: 290+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, S418.

[0035] In some embodiments, relative to the reference protein set forth in SEQ ID NO:1, the Cas13 protein comprises any one or more mutations at positions corresponding to the following amino acid residues in the reference protein set forth in SEQ ID NO:1: R11, N34, R35, R47, R58, R63, R64, N68, N87, N265, N274, R276, R290, R294, N299, N303, R308, R314.

[0036] In some embodiments, relative to the reference protein set forth in SEQ ID NO:1, the Cas13 protein comprises any one or more mutations at positions corresponding to the following amino acid residues in the reference protein set forth in SEQ ID NO:1: R47+R290, R47+R314, R290+R314, R47+R290+R314, N394+N265, N87+N265, A263, M266, N274, F288, V305, I311, D313, H324, T360, E365, A373, M380, D402, D411, S418.

[0037] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R11 of the reference protein set forth in SEQ ID NO:1.

[0038] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N34 of the reference protein set forth in SEQ ID NO:1.

[0039] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R35 of the reference protein set forth in SEQ ID NO:1.

[0040] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R47 of the reference protein set forth in SEQ ID NO:1.

[0041] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R58 of the reference protein set forth in SEQ ID NO:1.

[0042] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R63 of the reference protein set forth in SEQ ID NO:1.

[0043] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R64 of the reference protein set forth in SEQ ID NO:1.

[0044] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N68 of the reference protein set forth in SEQ ID NO:1.

[0045] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N87 of the reference protein set forth in SEQ ID NO:1.

[0046] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N265 of the reference protein set forth in SEQ ID NO:1.

[0047] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N274 of the reference protein set forth in SEQ ID NO:1.

[0048] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R276 of the reference protein set forth in SEQ ID NO:1.

[0049] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R290 of the reference protein set forth in SEQ ID NO:1.

[0050] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R294 of the reference protein set forth in SEQ ID NO:1.

[0051] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N299 of the reference protein set forth in SEQ ID NO:1.

[0052] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N303 of the reference protein set forth in SEQ ID NO:1.

[0053] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R308 of the reference protein set forth in SEQ ID NO:1.

[0054] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R314 of the reference protein set forth in SEQ ID NO:1.

[0055] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R320 of the reference protein set forth in SEQ ID NO:1.

[0056] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R328 of the reference protein set forth in SEQ ID NO:1.

[0057] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N332 of the reference protein set forth in SEQ ID NO:1.

[0058] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R341 of the reference protein set forth in SEQ ID NO:1.

[0059] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N346 of the reference protein set forth in SEQ ID NO:1.

[0060] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue R358 of the reference protein set forth in SEQ ID NO:1.

[0061] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N372 of the reference protein set forth in SEQ ID NO:1.

[0062] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N383 of the reference protein set forth in SEQ ID NO:1.

[0063] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N390 of the reference protein set forth in SEQ ID NO:1.

[0064] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N394 of the reference protein set forth in SEQ ID NO:1.

[0065] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R47 and R290 of the reference protein set forth in SEQ ID NO:1.

[0066] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R47 and R314 of the reference protein set forth in SEQ ID NO:1.

[0067] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R290 and R314 of the reference protein set forth in SEQ ID NO:1.

[0068] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R47, R290, and R314 of the reference protein set forth in SEQ ID NO:1.

[0069] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R308 and N68 of the reference protein set forth in SEQ ID NO:1.

[0070] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues N394 and N68 of the reference protein set forth in SEQ ID NO:1.

[0071] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues N87 and N68 of the reference protein set forth in SEQ ID NO:1.

[0072] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R308 and N265 of the reference protein set forth in SEQ ID NO:1.

[0073] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues N394 and N265 of the reference protein set forth in SEQ ID NO:1.

[0074] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues N87 and N265 of the reference protein set forth in SEQ ID NO:1.

[0075] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R308, N68, and N265 of the reference protein set forth in SEQ ID NO:1.

[0076] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues N87, N68, and N265 of the reference protein set forth in SEQ ID NO:1.

[0077] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue T7 of the reference protein set forth in SEQ ID NO:1.

[0078] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue A16 of the reference protein set forth in SEQ ID NO:1.

[0079] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue S260 of the reference protein set forth in SEQ ID NO:1.

[0080] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue A263 of the reference protein set forth in SEQ ID NO:1.

[0081] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue M266 of the reference protein set forth in SEQ ID NO:1.

[0082] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N274 of the reference protein set forth in SEQ ID NO:1.

[0083] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue F288 of the reference protein set forth in SEQ ID NO:1.

[0084] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue M302 of the reference protein set forth in SEQ ID NO:1.

[0085] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N303 of the reference protein set forth in SEQ ID NO:1.

[0086] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue L304 of the reference protein set forth in SEQ ID NO:1.

[0087] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue V305 of the reference protein set forth in SEQ ID NO:1.

[0088] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue 1311 of the reference protein set forth in SEQ ID NO:1.

[0089] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue D313 of the reference protein set forth in SEQ ID NO:1.

[0090] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue H324 of the reference protein set forth in SEQ ID NO:1.

[0091] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue P326 of the reference protein set forth in SEQ ID NO:1.

[0092] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue H327 of the reference protein set forth in SEQ ID NO:1.

[0093] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N332 of the reference protein set forth in SEQ ID NO:1.

[0094] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue N346 of the reference protein set forth in SEQ ID NO:1.

[0095] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue T353 of the reference protein set forth in SEQ ID NO:1.

[0096] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue T360 of the reference protein set forth in SEQ ID NO:1.

[0097] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue E365 of the reference protein set forth in SEQ ID NO:1.

[0098] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue A373 of the reference protein set forth in SEQ ID NO:1.

[0099] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue M380 of the reference protein set forth in SEQ ID NO:1.

[0100] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue S382 of the reference protein set forth in SEQ ID NO:1.

[0101] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue K395 of the reference protein set forth in SEQ ID NO:1.

[0102] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue Y396 of the reference protein set forth in SEQ ID NO:1.

[0103] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue D402 of the reference protein set forth in SEQ ID NO:1.

[0104] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue D411 of the reference protein set forth in SEQ ID NO:1.

[0105] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to amino acid residue S418 of the reference protein set forth in SEQ ID NO:1.

[0106] In some embodiments, compared to the comparison protein set forth in SEQ ID NO: 1, the Cas13 protein is mutated to the same amino acid residue at the position corresponding to the mutation site in the comparison protein set forth in SEQ ID NO: 1 in Table 24 or Table 29.

[0107] In some embodiments, compared to the comparative protein set forth in SEQ ID NO: 1, the Cas13 protein comprises the same mutation at a position corresponding to the mutation site in the comparative protein set forth in SEQ ID NO: 1 in Table 24 or Table 29.

[0108] In some embodiments, the Cas13 protein is located at the following positions in the sequence set forth 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+N6 8, 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.

[0109] In some embodiments, the Cas13 protein is obtained by introducing any one or more mutations into the following positions in the sequence set forth in SEQ ID NO: 1: N34, R64, N68, N265, R276, R294, N299, R314, R47+R290, R47+R314, R290+R314, R47+R290+R314, N394+N265, N87+N265, A263, M266, N274, F288, V305, I311, D313, H324, T360, E365, A373, M380, D402, and D411.

[0110] In some embodiments, the Cas13 protein is obtained by introducing any one or more mutations in Table 24 or Table 29 into the sequence set forth in SEQ ID NO:1.

[0111] In some embodiments, the Cas13 protein is obtained by sequence deletion at positions corresponding to amino acid residues 91-120, 141-180, 211-240, 331-360, 351-400, 431-460, 461-500, 511-550, 611-640, 631-660, 661-690, 691-760, 821-860, or 861-890 of the reference protein set forth in SEQ ID NO:1.

[0112] In some embodiments, the Cas13 protein comprises one or more amino acid deletions at positions corresponding to amino acid residues 348-350, 521-556, or 883-893 of the reference protein set forth in SEQ ID NO:1.

[0113] In some embodiments, the sequence deletion is a deletion of <300, <200, <150, <100, <90, <80, <70, <60, <50, <40, <30, <20, or <10 amino acid residues.

[0114] In some embodiments, the Cas13 protein is obtained by deletion of a sequence at positions 91 to 120, 141 to 180, 211 to 240, 331 to 360, 351 to 400, 431 to 460, 461 to 500, 511 to 550, 611 to 640, 631 to 660, 661 to 690, 691 to 760, 821 to 860, or 861 to 890 of the sequence set forth in SEQ ID NO:1.

[0115] The sequence deletion is the deletion of one or more amino acid residues.

[0116] Another aspect of the present disclosure relates to fusion proteins.

[0117] In some embodiments of the present disclosure, the fusion protein comprises a Cas13 protein or a functional fragment thereof described herein, and any one or more of 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 fused to the Cas13 protein or a functional fragment thereof.

[0118] In some embodiments of the present disclosure, the fusion protein comprises a Cas13 protein or a functional fragment thereof described herein, and any one or more of 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 subcellular localization signal, a reporter tag, and an affinity tag fused to the Cas13 protein or a functional fragment thereof.

[0119] In some embodiments of the present disclosure, the fusion protein comprises a Cas13 protein or functional fragment thereof described herein fused to a homologous or heterologous protein domain and / or polypeptide tag, and in some embodiments, the fusion does not alter the native function of the Cas13 protein and / or functional fragment thereof.

[0120] "Not altering the original function of the Cas13 protein or a functional fragment thereof" means that the fusion protein still has the ability to recognize, bind to, and / or cleave a target RNA when used in combination with a gRNA. The ability of the fusion protein to recognize, bind to, or cleave a target RNA when used in combination with a gRNA may be increased or decreased compared to the ability of the Cas13 protein to recognize, bind, or cleave a target RNA when used in combination with a gRNA, but as long as the fusion protein can effectively recognize, bind, or cleave a target RNA when used in combination with a gRNA, "not altering the original function of the Cas13 protein" applies.

[0121] In some embodiments, the fusion protein comprises a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag. In some embodiments, the fusion protein comprises a functional fragment of a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag. For example, in some embodiments, the functional fragment is a fragment obtainable by deleting a subsequence of the nuclease domain of the Cas13 protein. In some embodiments, the functional fragment is a fragment obtainable by deleting sequences corresponding to HEPN-1_I, HEPN-1_II, HEPN-2, NTD, Helical-1, and / or Helical-2 in the C13-2 portion of the Cas13 protein. In some embodiments, the functional fragment is a fragment obtainable by deleting sequences corresponding to HEPN-1_I, HEPN-1_II, and / or HEPN-2 in the C13-2 portion of the Cas13 protein. In some embodiments, the functional fragment is a fragment obtainable by deleting a sequence corresponding to HEPN-1_I at C13-2 of the Cas13 protein. In some embodiments, the functional fragment is a fragment obtainable by deleting a sequence corresponding to HEPN-1_II at C13-2 of the Cas13 protein. In some embodiments, the functional fragment is a fragment obtainable by deleting a sequence corresponding to HEPN-2 at C13-2 of the Cas13 protein. In some embodiments, the functional fragment is a fragment obtainable by deleting a sequence corresponding to the NTD and HEPN-1_I at C13-2 of the Cas13 protein.

[0122] In some embodiments, the Cas13 protein or functional fragment thereof is fused to any one or more selected from 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.

[0123] In some embodiments, the Cas13 protein or functional fragment thereof is fused to a subcellular localization signal, which in some embodiments is selected from a nuclear localization signal (NLS), a nuclear export signal (NES), a chloroplast localization signal, or a mitochondrial localization signal.

[0124] In some embodiments, the Cas13 protein or functional fragment thereof is fused to any one or more selected from 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.

[0125] In some embodiments, the Cas13 protein or functional fragment thereof is fused to a subcellular localization signal, which in some embodiments is selected from a nuclear localization signal (NLS), a nuclear export signal (NES), a chloroplast localization signal, or a mitochondrial localization signal.

[0126] In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous nuclear localization signal (NLS). In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous nuclear export signal (NES).

[0127] In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked to a protein domain and / or polypeptide tag. In some embodiments, the Cas13 protein or functional fragment thereof is directly covalently linked to a protein domain and / or polypeptide tag. In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked to a protein domain and / or polypeptide tag via a linking sequence, and further, in some embodiments, the linking sequence is an amino acid sequence.

[0128] In some embodiments, the Cas13 protein or functional fragment thereof of the fusion protein is linked to a cognate or heterologous protein domain and / or polypeptide tag via a rigid linker peptide sequence. In some embodiments, the Cas13 protein portion of the fusion protein is linked to a cognate or heterologous protein domain and / or polypeptide tag via a flexible linker peptide sequence. In some embodiments, the rigid linker peptide sequence is A(EAAAK)3A (SEQ ID NO: 279). In some embodiments, the flexible linker peptide sequence is (GGGGS)3 (SEQ ID NO: 280).

[0129] In some embodiments, the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of specifically binding to a target RNA sequence. In some embodiments, the fusion protein comprises a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag, and the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of specifically binding to a target RNA sequence. In some embodiments, the fusion protein comprises a functional fragment of a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag, and the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of specifically binding to a target RNA sequence.

[0130] In some embodiments, the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a direct repeat linked to a guide sequence engineered to direct sequence-specific binding of a CRISPR complex to a target RNA. In some embodiments, the fusion protein comprises a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag, and the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a direct repeat linked to a guide sequence engineered to direct sequence-specific binding of a CRISPR complex to a target RNA. In some embodiments, the fusion protein comprises a functional fragment of a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag, and the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide comprising a direct repeat linked to a guide sequence engineered to direct sequence-specific binding of a CRISPR complex to a target RNA.

[0131] In some embodiments, the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of sequence-specific binding and cleavage of a target RNA. In some embodiments, the fusion protein comprises a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag, and the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of specifically binding to a target RNA sequence. In some embodiments, the fusion protein comprises a functional fragment of a Cas13 protein described herein fused to a homologous or heterologous protein domain and / or polypeptide tag, and the fusion protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of specifically binding to a target RNA sequence.

[0132] In some embodiments, the structure of the fusion protein is NLS-Cas13 protein-SV40 NLS-nucleoplasmin NLS.

[0133] Another aspect of the present disclosure relates to a guide polynucleotide comprising (i) a direct repeat sequence having at least 50% sequence identity to any one of SEQ ID NO:3 and SEQ ID NOs:80-87, wherein the direct repeat sequence is linked to (ii) a homologous or heterologous guide sequence engineered to hybridize to a target RNA, wherein the guide polynucleotide is capable of forming a CRISPR complex with a Cas13 protein and directing sequence-specific binding of the CRISPR complex to the target RNA.

[0134] In some embodiments, the Cas protein is Cas13a, Cas13b, Cas13c, or Cas13d. In some embodiments, the amino acid sequence of the Cas13 protein has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:1.

[0135] In some embodiments, the direct repeat sequence has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has at least 80% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has at least 85% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has at least 90% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has at least 95% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has 100% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87.

[0136] In some embodiments, the direct repeat sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to any one of SEQ ID NOs: 3, 81, 82, 84, and 87.

[0137] In some embodiments, the direct repeat sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to any one of SEQ ID NOs: 3 and 87.

[0138] In some embodiments, the base at position 26 of the direct repeat sequence corresponding to SEQ ID NO: 3 is an A.

[0139] In some embodiments, the direct repeat sequence is 10 N 11 (SEQ ID NO: 277), wherein N1 and N3 to N 11 is arbitrarily selected from A, C, G, and T; and N2 is arbitrarily selected from A and G.

[0140] In some embodiments, the direct repeat sequence is 12 ACTCTACAAACCTGTAGN 13 GN 14 N 15 N 16 N 17 N 18 N 19 N 20 N 21 N 22 (SEQ ID NO: 278), wherein N 12 , N 13 , N 19 and N 21 is arbitrarily selected from A and G, and N 14 is arbitrarily selected from A and T, and N 15 and N 16 is arbitrarily selected from C and T, and N 17 and N 18 is arbitrarily selected from G and T, and N 20 and N 22 is arbitrarily selected from C and G.

[0141] In some embodiments, the guide sequence is located at the 3' end of the direct repeat sequence. In some embodiments, the guide sequence is located at the 5' end of the direct repeat sequence.

[0142] In some embodiments, the guide sequence comprises 15-35 nucleotides. In some embodiments, the guide sequence hybridizes to the target RNA with no more than one nucleotide mismatch. In some embodiments, the direct repeat sequence comprises 25-40 nucleotides.

[0143] In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop of the guide polynucleotide. In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

[0144] In some embodiments, the guide polynucleotide comprises modified nucleotides, in some embodiments, the modifications comprise 2'-O-methyl, 2'-O-methyl-3'-thiophosphate, or 2'-O-methyl-3'-thioPACE.

[0145] In some embodiments, the target RNA of the guide polynucleotide is located in the nucleus of a eukaryotic cell.

[0146] In some embodiments, the target RNA is optionally selected from TTR RNA, SOD1 RNA, PCSK9 RNA, VEGFA RNA, VEGFR1 RNA, PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA. Optionally, in some embodiments, the target RNA is optionally selected from VEGFA RNA, PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA. Furthermore, the guide sequence is optionally selected from the sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NOs:42-49 (used to target AQp1 RNA, PTBP1 RNA, and ANGPTL3 RNA, respectively). Preferably, the guide sequence is optionally selected from the sequences set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:43, and SEQ ID NOs:45-47.

[0147] In some embodiments, the Cas13 protein is Cas13a, Cas13b, Cas13c, or Cas13d. In some embodiments, the Cas13 protein is Cas13d. In some embodiments, the Cas13 protein has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:1.

[0148] Another aspect of the present disclosure relates to a CRISPR-Cas13 system comprising a Cas13 protein or fusion protein described herein, or a nucleic acid encoding said Cas13 protein or fusion protein, and a guide polynucleotide, or a nucleic acid encoding said guide polynucleotide, wherein said guide polynucleotide comprises direct repeats linked to a guide sequence engineered to hybridize to a target RNA, and wherein said guide polynucleotide is capable of forming a CRISPR complex with said Cas13 protein or fusion protein and directing sequence-specific binding of the CRISPR complex to the target RNA.

[0149] In some embodiments, the target RNA is optionally selected from TTR RNA, SOD1 RNA, PCSK9 RNA, VEGFA RNA, VEGFR1 RNA, PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA. Optionally, in some embodiments, the target RNA is optionally selected from VEGFA RNA, PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA. In some embodiments, the guide sequence is optionally selected from the sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NOs:42-49 (used to target AQp1 RNA, PTBP1 RNA, and ANGPTL3 RNA, respectively). In some embodiments, the guide sequence is optionally selected from the sequences set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:43, and SEQ ID NOs:45-47.

[0150] In some embodiments, the direct repeat sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to any one of SEQ ID NOs: 3, 81, 82, 84, and 87.

[0151] In some embodiments, the fusion protein comprises a Cas13 protein described herein, or a functional fragment thereof, fused to a homologous or heterologous protein domain and / or polypeptide tag.

[0152] In some embodiments, the Cas13 protein or functional fragment thereof portion of the fusion protein is fused to a homologous or heterologous nuclear localization signal (NLS). In some embodiments, the Cas13 protein or functional fragment thereof portion of the fusion protein is fused to a homologous or heterologous nuclear export signal (NES).

[0153] In some embodiments, the Cas13 protein contains a mutation in the catalytic domain that reduces RNA cleavage activity. In some embodiments, the Cas13 protein contains a mutation in one or two HEPN domains that substantially lacks RNA cleavage activity. In some embodiments, "substantially lacking RNA cleavage activity" refers to retaining only ≦50%, ≦40%, ≦30%, ≦20%, ≦10%, ≦5%, or ≦1% of the RNA cleavage activity compared to wild-type Cas13 protein, or having no detectable RNA cleavage activity.

[0154] In some embodiments, the Cas13 protein or functional fragment thereof portion of the fusion protein is directly linked to the homologous or heterologous protein domain and / or polypeptide tag via a covalent bond, hi some embodiments, the Cas13 protein portion of the fusion protein is linked to the homologous or heterologous protein domain and / or polypeptide tag via a peptide sequence.

[0155] In some embodiments, the protein domain comprises 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, or a splicing factor domain. In some embodiments, the Cas13 protein is covalently linked to an affinity tag or a reporter tag.

[0156] In some embodiments, the Cas13 protein has at least 95% sequence identity compared to SEQ ID NO: 1. In some embodiments, the Cas13 protein has at least 97% sequence identity compared to SEQ ID NO: 1. In some embodiments, the Cas13 protein has at least 98% sequence identity compared to SEQ ID NO: 1. In some embodiments, the Cas13 protein has at least 99% sequence identity compared to SEQ ID NO: 1. In some embodiments, the Cas13 protein has at least 99.5% sequence identity compared to SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises the sequence set forth in SEQ ID NO: 1.

[0157] In some embodiments, the Cas13 protein is derived from a species comprising a genome having an average nucleotide identity (ANI) of ≧95% with the genome designated in the CNGB database as number CNA0009596.

[0158] In some embodiments, the Cas13 protein, when used for RNA cleavage, does not have a protospacer adjacent sequence (PFS) requirement.

[0159] In some embodiments, the guide sequence is located at the 3' end of the direct repeat sequence. In some embodiments, the guide sequence is located at the 5' end of the direct repeat sequence.

[0160] In some embodiments, the guide sequence comprises 15 to 35 nucleotides, hi some embodiments, the guide sequence hybridizes to the target RNA with no more than one nucleotide mismatch.

[0161] In some embodiments, the direct repeat sequence comprises 25 to 40 nucleotides.

[0162] In some embodiments, the direct repeat sequence has at least 80% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has at least 90% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has at least 95% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence has 100% sequence identity to SEQ ID NO:3 and any one of SEQ ID NOs:80-87. In some embodiments, the direct repeat sequence is selected from SEQ ID NO:3 and any one of SEQ ID NOs:80-87.

[0163] In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop of the guide polynucleotide. In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

[0164] In some embodiments, the CRISPR-Cas13 system comprises a fusion protein comprising an adaptor protein and a homologous or heterologous protein domain, or a nucleic acid encoding the fusion protein, wherein the adaptor protein is capable of binding to the aptamer sequence.

[0165] In some embodiments, the adaptor protein comprises MS2 phage coat protein, PP7 phage coat protein, or Qβ phage coat protein. In some embodiments, the protein domain comprises a cytosine deaminase domain, an adenosine deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, an affinity domain, a reporter tag, and an affinity tag.

[0166] In some embodiments, the guide polynucleotide comprises modified nucleotides, hi some embodiments, the modified nucleotides comprise 2'-O-methyl, 2'-O-methyl-3'-thiophosphate, or 2'-O-methyl-3'-thioPACE.

[0167] In some embodiments, the Cas13 protein or fusion protein and the guide polynucleotide do not co-exist in nature.

[0168] Another aspect of the present disclosure relates to a CRISPR-Cas13 system comprising any one of a Cas13 protein, a fusion protein thereof, or a nucleic acid encoding the same, and a guide polynucleotide described herein or a nucleic acid encoding the same.

[0169] Another aspect of the present disclosure relates to a vector system comprising the CRISPR-Cas13 system described herein, the vector system comprising one or more vectors comprising a polynucleotide sequence encoding a Cas13 protein or fusion protein described herein and a polynucleotide sequence encoding a guide polynucleotide.

[0170] Another aspect of the present disclosure relates to an adeno-associated virus (AAV) vector comprising the CRISPR-Cas13 system described herein, wherein the AAV vector comprises a DNA sequence encoding the Cas13 protein or fusion protein described herein and the guide polynucleotide.

[0171] Another aspect of the present disclosure relates to a lipid nanoparticle comprising a CRISPR-Cas13 system as described herein, wherein the lipid nanoparticle comprises a guide polynucleotide as described herein and an mRNA encoding a Cas13 protein or fusion protein as described herein.

[0172] Another aspect of the present disclosure relates to a lentiviral vector comprising a CRISPR-Cas13 system described herein, the lentiviral vector comprising a guide polynucleotide described herein and an mRNA encoding a Cas13 protein or fusion protein described herein. In some embodiments, the lentiviral vector is pseudotyped with a homologous or heterologous envelope protein, such as VSV-G. In some embodiments, the mRNA encoding the Cas13 protein or fusion protein is linked to an aptamer sequence.

[0173] Another aspect of the present disclosure relates to a ribonucleoprotein complex comprising a CRISPR-Cas13 system as described herein, wherein the ribonucleoprotein complex is formed from a guide polynucleotide as described herein and a Cas13 protein or fusion protein as described herein.

[0174] Another aspect of the present disclosure relates to a virus-like particle comprising a CRISPR-Cas13 system described herein, wherein the virus-like particle comprises a ribonucleoprotein complex formed from a guide polynucleotide described herein and a Cas13 protein or fusion protein described herein. In some embodiments, the Cas13 protein or fusion protein is fused to a gag protein.

[0175] Another aspect of the present disclosure relates to a eukaryotic cell comprising the CRISPR-Cas13 system described herein. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell.

[0176] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein.

[0177] Another aspect of the present disclosure relates to pharmaceutical compositions comprising the CRISPR-Cas13 system described herein.

[0178] Another aspect of the present disclosure relates to an in vitro composition comprising a CRISPR-Cas13 system described herein and a labeled detector RNA that cannot hybridize to or be targeted by a guide polynucleotide described herein.

[0179] Another aspect of the present disclosure relates to an isolated nucleic acid encoding a Cas13 protein or fusion protein described herein.

[0180] Another aspect of the present disclosure relates to isolated nucleic acids encoding the guide polynucleotides described herein.

[0181] Another aspect of the present disclosure relates to a CRISPR-Cas13 system comprising any one of the Cas13 proteins or nucleic acids encoding same, and a guide polynucleotide described herein or an isolated nucleic acid encoding same.

[0182] Another aspect of the present disclosure relates to the use of the CRISPR-Cas13 system described herein in the detection of a target RNA in a nucleic acid sample suspected of containing the target RNA, or in the preparation of a reagent for detecting a target RNA in a nucleic acid sample suspected of containing the target RNA.

[0183] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the preparation of a reagent for achieving any one of the following, or any one of the following schemes:

[0184] Cleavage of one or more target RNA molecules or nicking of one or more target RNA molecules, activating or upregulating one or more target RNA molecules, activating or inhibiting translation of one or more target RNA molecules, inactivating one or more target RNA molecules, visualizing, labeling or detecting one or more target RNA molecules, binding to one or more target RNA molecules, transporting one or more target RNA molecules, and masking one or more target RNA molecules.

[0185] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in cleaving one or more target RNA molecules or in the preparation of a reagent for cleaving one or more target RNA molecules.

[0186] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in binding one or more target RNA molecules.

[0187] Another aspect of the present disclosure relates to the use of the CRISPR-Cas13 system described herein in the preparation of a reagent for binding or cleaving one or more target RNA molecules.

[0188] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in cleaving or editing a target RNA in a mammalian cell, wherein said editing is base editing.

[0189] Another aspect of the present disclosure relates to the use of the CRISPR-Cas13 system described herein in the preparation of a reagent for cleaving or editing a target RNA in a mammalian cell, wherein the editing is a base edit.

[0190] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the activation or upregulation of one or more target RNA molecules, or in the preparation of a reagent for activating or upregulating one or more target RNA molecules.

[0191] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in inhibiting the translation of one or more target RNA molecules, or in the preparation of a reagent for inhibiting the translation of one or more target RNA molecules.

[0192] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the inactivation of one or more target RNA molecules or in the preparation of a reagent for inactivating one or more target RNA molecules.

[0193] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the visualization, labeling, or detection of one or more target RNA molecules, or in the preparation of a reagent for visualizing, labeling, or detecting one or more target RNA molecules.

[0194] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the delivery of one or more target RNA molecules or in the preparation of a reagent for delivery of one or more target RNA molecules.

[0195] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in masking one or more target RNA molecules or in preparing a reagent for masking one or more target RNA molecules.

[0196] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the diagnosis, treatment, or prevention of a disease or condition associated with a target RNA.

[0197] Another aspect of the present disclosure relates to the use of the CRISPR-Cas13 system described herein in the diagnosis, treatment, or prevention of a disease or condition associated with a target RNA.

[0198] Another aspect of the present disclosure relates to methods of diagnosing, treating, or preventing a disease or condition associated with a target RNA, comprising administering to a subject in need thereof, or a sample from a subject in need thereof, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a CRISPR-Cas13 system described herein, or an isolated nucleic acid described herein.

[0199] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the preparation of a medicament for diagnosing, treating, or preventing a disease or condition associated with a target RNA.

[0200] Another aspect of the present disclosure relates to the use of the CRISPR-Cas13 system described herein in the preparation of a medicament for diagnosing, treating, or preventing a disease or condition associated with a target RNA. Based on common knowledge in the art, the above preferred conditions can be arbitrarily combined to obtain each preferred example of the present disclosure.

[0201] All reagents and materials used in this disclosure can be obtained commercially. [Brief explanation of the drawings]

[0202] [Figure 1] 1 shows the CRISPR locus of the CRISPR-Cas13 system, including the CRISPR array and the coding sequence for the C13-2 protein. [Figure 2] The structure of a C13-2 guide polynucleotide, consisting of a direct repeat sequence and a guide sequence, is shown. In this figure, the scaffold sequence is the same as the direct repeat sequence, and the guide sequence consists of a variable number of nucleotides, with N representing any nucleotide. The direct repeat sequence (SEQ ID NO: 3) was predicted using RNAfold to obtain its secondary structure. As shown in the figure, a stem-loop structure is visible, with multiple complementary base pairs in the stem region. [Figure 3] Expression and purification of recombinant C13-2 protein. [Figure 4] Figure 1 shows that C13-2 has high activity in down-regulating PTBP1 (polypyrimidine tract binding protein 1) RNA in 293T cells. [Figure 5] This shows that compared to CasRx and shRNA, C13-2 has higher activity in down-regulating AQp1 (aquaporin 1) RNA in 293T cells. [Figure 6] This shows that compared to C13-113 and C13-114, C13-2 has higher activity in down-regulating PTBP1 RNA in 293T cells. [Figure 7]Figure 2 shows that C13-2 can down-regulate ANGPTL3 (angiopoietin-like 3) RNA in 293T cells. [Figure 8] The C13-2 domain predicted by computational methods is shown. In the figure, positions 1 to 95 are the NTD domain, positions 96 to 255 are the HEPN-1_I domain, positions 256 to 417 are the Helical-1 domain, positions 418 to 504 are the HEPN-1_II domain, positions 505 to 651 are the Helical-2 domain, and positions 652 to 893 are the HEPN-2 domain. [Figure 9] 1 shows the editing effect of targeting VEGFA RNA using different DRs. [Figure 10] 1 shows the editing effect of targeting PTBP1 RNA using different DRs. [Figure 11] Figure 1 shows a comparison of the efficacy of C13-2 and known Cas13 tools targeting VEGFA RNA. [Figure 12] 1 shows a comparison of the efficacy of C13-2 and known Cas13 tools targeting PTBP1 RNA. [Figure 13] A sequence peak diagram of dC13-2 after single base editing is shown. [Figure 14] Figure 1 shows VEGFA RNA levels after targeted editing of mutants by qPCR detection. [Figure 15] VEGFA RNA levels after editing as detected by RNAseq are shown. [Figure 16] 1 shows the editing efficiency of mutants for AR RNA as detected by qPCR. [Figure 17] AR RNA levels after editing measured by RNAseq are shown. [Figure 18] The results of sequence comparison of the direct repeat sequences DRrc, DR-hf2, and DR2rc are shown. [Figure 19] 1 shows the RNA secondary structure of the direct repeat sequence DR-hf2 (SEQ ID NO: 87) predicted by RNAfold. DETAILED DESCRIPTION OF THE INVENTION

[0203] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited to the scope of the examples. In the following examples, experimental methods for which specific conditions are not described are selected according to conventional methods and conditions or product instructions.

[0204] As used herein, the term "sequence identity" (or "percent identity") refers to the sequence match between two polypeptides or two nucleic acids. If a position in both compared sequences is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine, or if a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, if six out of ten positions in two sequences are identical, the two sequences have 60% sequence identity. Typically, comparisons are performed when two sequences are compared to maximize sequence identity. Such comparisons can be performed using publicly available and commercially available comparison algorithms and programs, such as, but not limited to, Clustal Ω, MAFFT, Probcons, T-Coffee, Probalign, and BLAST, which can be reasonably selected for use by those skilled in the art. Those skilled in the art can determine appropriate parameters for comparing sequences, including, for example, any algorithms needed to achieve a better or optimal comparison of the entire length of the compared sequences, and any algorithms needed to achieve a better or optimal comparison locally of the compared sequences.

[0205] As used herein, the term "guide polynucleotide" refers to a molecule that forms a CRISPR complex with a Cas protein in a CRISPR-Cas system and guides the CRISPR complex to a target sequence. Generally, a guide polynucleotide comprises a scaffold sequence linked to a guide sequence that can hybridize to a target sequence. The scaffold sequence usually comprises a direct repeat sequence and, in some cases, a tracrRNA sequence. When the scaffold sequence does not comprise a tracrRNA sequence, the guide polynucleotide comprises a guide sequence and a direct repeat sequence, and in this case, the guide polynucleotide is also referred to as a crRNA.

[0206] CRISPR-Cas13 system

[0207] Class 2 CRISPR-Cas systems confer multiple adaptive immune mechanisms to microorganisms. Herein, we present an analysis of prokaryotic genomes and metagenomes to identify previously uncharacterized RNA-guided, RNA-targeted CRISPR-Cas13 systems, including C13-2 (also known as CasRfg.4), classified as a Type VI system. Engineered CRISPR-Cas13 systems based on C13-2 have robust activity in human cells. C13-2 can also be flexibly packaged into AAV vectors as a compact, single-effector Cas13 enzyme. Our results demonstrate that C13-2 is a programmable RNA-binding module for efficient targeting of cellular RNAs, thereby providing a versatile platform for transcriptome engineering and therapeutic and diagnostic methods.

[0208] As described in Example 1, a CRISPR-Cas13 system containing C13-2 was identified based on bioinformatics analysis of prokaryotic genomes and metagenomes in the NCBI GenBank and CNGB databases, and its target RNA cleavage activity in human cells was subsequently verified experimentally. The CRISPR-Cas13 system containing C13-2 is a Type VI CRISPR-Cas system. Figure 1 shows the CRISPR locus of the CRISPR-Cas13 system containing C13-2. The protein sequence of wild-type C13-2 is SEQ ID NO: 1. The wild-type DNA coding sequence of C13-2 is SEQ ID NO: 9. Figure 8 shows the computationally predicted domains of C13-2, including the NTD, HEPN-1_I, HEPN-1_II, and HEPN-2. The NTD is the N-terminal domain, and the helical-1 and helical-2 domains are located between HEPN-1_I and HEPN-1_II, and between HEPN-1_II and HEPN-2, respectively. The direct repeat sequence of the C13-2 guide polynucleotide is SEQ ID NO: 3. Figure 2 shows the RNA secondary structure of the direct repeat sequence of the C13-2 guide polynucleotide predicted by RNAfold. The engineered CRISPR-Cas13 system described herein can efficiently knock down endogenous target RNAs in human cells, paving the way for RNA-targeting applications as part of a transcriptome engineering toolbox. In some embodiments, C13-2-mediated knockdown across multiple endogenous transcripts can achieve greater efficiency and / or specificity than CasRx-, PspCas13b-, Cas13X.1-, and / or Cas13Y.1-mediated knockdown.

[0209] Accordingly, one aspect of the present disclosure relates to a CRISPR-Cas13 system, composition, or kit comprising a Cas13 protein or fusion protein having at least 90% sequence identity compared to SEQ ID NO: 1, or a nucleic acid encoding said Cas13 protein, and a guide polynucleotide, or a nucleic acid encoding said guide polynucleotide, wherein said guide polynucleotide comprises direct repeats linked to a guide sequence engineered to hybridize to a target RNA, and said guide polynucleotide is capable of forming a CRISPR complex with said Cas13 protein and directing sequence-specific binding of the CRISPR complex to the target RNA.

[0210] In some embodiments, the guide polynucleotide comprises direct repeats linked to a guide sequence engineered to hybridize to a target RNA, wherein the guide polynucleotide is capable of forming a CRISPR complex with the Cas13 protein and directing the CRISPR complex to sequence-specifically bind and cleave the target RNA.

[0211] In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein and / or the polynucleotide sequence encoding a guide polynucleotide is operably linked to a regulatory sequence. In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein is operably linked to a regulatory sequence. In some embodiments, the polynucleotide sequence encoding a guide polynucleotide is operably linked to a regulatory sequence. In some embodiments, the regulatory sequence of the polynucleotide sequence encoding a Cas13 protein or fusion protein is the same as or different from the regulatory sequence of the polynucleotide sequence encoding a guide polynucleotide.

[0212] In some embodiments, the Cas13 proteins described herein have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity compared to SEQ ID NO: 1. In some embodiments, the Cas13 proteins described herein have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the protein sequence encoded by SEQ ID NO: 9. When the CRISPR-Cas13 system comprises a fusion protein comprising the Cas13 protein and a protein domain and / or polypeptide tag, the percentage sequence identity between the Cas13 portion of the fusion protein and a reference sequence is calculated.

[0213] In some embodiments, the Cas13 proteins described herein are derived from species containing a genome that has an average nucleotide identity (ANI) of ≧95% with the genome designated in the CNGB database as number CNA0009596.

[0214] In some embodiments, the Cas13 proteins described herein comprise one or more (e.g., one or two) native HEPN domains, each of which comprises an RX4H amino acid motif (where X represents any amino acid and the subscript "4" represents four consecutive amino acids). In some embodiments, a first catalytic RX4H motif is located at amino acid positions 210-215 of SEQ ID NO:1, a second catalytic RX4H motif is located at amino acid positions 785-790 of SEQ ID NO:1, and a third RX4H motif is located at amino acid positions 750-755 of SEQ ID NO:1. In some embodiments, the Cas13 proteins described herein comprise one or more mutant HEPN domains. In some embodiments, the mutant Cas13 proteins are capable of processing their guide polynucleotides but are unable to cleave target RNAs.

[0215] In some embodiments, the Cas13 proteins described herein, when used for RNA cleavage, do not have a Protospacer Flanking Sequence (PFS) requirement.

[0216] The CRISPR-Cas13 systems described herein can be introduced into cells (or cell-free systems) in a variety of ways, including, but not limited to, (i) as Cas13 mRNA and guide polynucleotide, (ii) as part of a single vector or plasmid or as separate vectors or plasmids, (iii) as individual Cas13 proteins and guide polynucleotides, or (iv) as an RNP complex of Cas13 proteins and guide polynucleotides.

[0217] In some embodiments, the CRISPR-Cas13 system, composition, or kit comprises a nucleic acid molecule encoding the Cas13 protein, wherein the coding sequence is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR-Cas13 system, composition, or kit comprises a nucleic acid molecule encoding the Cas13 protein, wherein the coding sequence is codon-optimized for expression in mammalian cells. In some embodiments, the CRISPR-Cas13 system, composition, or kit comprises a nucleic acid molecule encoding the Cas13 protein, wherein the coding sequence is codon-optimized for expression in human cells.

[0218] In some embodiments, the nucleic acid molecule encoding the Cas13 protein is a plasmid. In some embodiments, the nucleic acid molecule encoding the Cas13 protein is part of a viral vector genome, such as the DNA genome of an AAV vector flanked by ITRs. In some embodiments, the nucleic acid molecule encoding the Cas13 protein is mRNA.

[0219] Guide polynucleotide

[0220] In some embodiments, the guide polynucleotide of the CRISPR-Cas13 system is a guide RNA. In some embodiments, the guide polynucleotide is a chemically modified guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least one chemically modified nucleotide. In some embodiments, the guide polynucleotide is a hybrid RNA-DNA guide. In some embodiments, the guide polynucleotide is a hybrid RNA-LNA (locked nucleic acid) guide.

[0221] In some embodiments, the guide polynucleotide comprises at least one guide sequence (also called a spacer sequence) linked to at least one direct repeat (DR). In some embodiments, the guide sequence is located at the 3' end of the direct repeat. In some embodiments, the guide sequence is located at the 5' end of the direct repeat.

[0222] In some embodiments, the guide sequence comprises at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, or at least 30 nucleotides. In some embodiments, the guide sequence comprises 60 or fewer nucleotides, 55 or fewer nucleotides, 50 or fewer nucleotides, 45 or fewer nucleotides, 40 or fewer nucleotides, 35 or fewer nucleotides, or 30 or fewer nucleotides. In some embodiments, the guide sequence comprises 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, 30-35 nucleotides, or 35-40 nucleotides.

[0223] In some embodiments, the guide sequence has sufficient complementarity to the target RNA sequence to hybridize to the target RNA and direct sequence-specific binding of the CRISPR-Cas13 complex to the target RNA. In some embodiments, the guide sequence has 100% complementarity to the target RNA (or region of the RNA being targeted), although the guide sequence may have less than 100% complementarity to the target RNA, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementarity.

[0224] In some embodiments, the guide sequence is engineered to hybridize to the target RNA with no more than two nucleotide mismatches. In some embodiments, the guide sequence is engineered to hybridize to the target RNA with no more than one nucleotide mismatch. In some embodiments, the guide sequence is engineered to hybridize to the target RNA with or without mismatches.

[0225] In some embodiments, the direct repeat sequence comprises at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, or at least 36 nucleotides. In some embodiments, the direct repeat sequence comprises no more than 60 nucleotides, no more than 55 nucleotides, no more than 50 nucleotides, no more than 45 nucleotides, no more than 40 nucleotides, or no more than 35 nucleotides. In some embodiments, the direct repeat sequence comprises 20-25 nucleotides, 25-30 nucleotides, 30-35 nucleotides, or 35-40 nucleotides.

[0226] In some embodiments, the direct repeat sequence is modified to replace at least one complementary base pair in the stem region shown in Figure 2 with a different complementary base pair. In some embodiments, the direct repeat sequence is modified to change the number of complementary base pairs in the stem region shown in Figure 2. In some embodiments, the direct repeat sequence is modified to change the number of nucleotides in the loop region shown in Figure 2 (e.g., 3, 4, or 5 nucleotides in the loop). In some embodiments, the direct repeat sequence is modified to change the nucleotide sequence in the loop region. In some embodiments, an aptamer sequence is inserted or added to the end of the direct repeat sequence. In some embodiments, the direct repeat sequence has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 3 and any one of SEQ ID NOs: 80-87.

[0227] In some embodiments, the CRISPR-Cas13 system, composition, or kit comprises at least two, at least three, at least four, at least five, at least 10, or at least 20 different guide polynucleotides. In some embodiments, the guide polynucleotides target at least two, at least three, at least four, at least five, at least 10, or at least 20 different target RNA molecules, or target at least two, at least three, at least four, at least five, at least 10, or at least 20 different regions of one or more target RNA molecules.

[0228] In some embodiments, the guide polynucleotide comprises a constant direct repeat sequence located upstream of a variable guide sequence. In some embodiments, multiple guide polynucleotides are part of an array (which may be part of a vector, such as a viral vector or a plasmid). For example, a guide array comprising the sequence DR-spacer-DR-spacer-DR-spacer may contain three unique unprocessed guide polynucleotides (one for each DR-spacer sequence). When introduced into a cell or cell-free system, the array is processed by the Cas13 protein into three separate mature guide polynucleotides. This allows for multiplexing, such as delivering multiple guide polynucleotides into a cell or system to target multiple target RNAs or multiple regions within a single target RNA.

[0229] The ability of guide polynucleotide to induce the sequence-specific binding of CRISPR complex to target RNA can be evaluated by any suitable assay.For example, sufficient components of CRISPR system to form a CRISPR complex comprising the guide polynucleotide to be tested can be provided to a host cell with corresponding target RNA molecule, for example, by transfection of a vector encoding the components of the CRISPR complex, and then evaluate the preferential cleavage within the target sequence.Similarly, the cleavage of target RNA sequence can be evaluated in vitro by providing the components of the CRISPR complex comprising the target RNA, the guide polynucleotide to be tested, and a control guide polynucleotide that is different from the guide polynucleotide to be tested, and comparing the ability to bind to target RNA or the cleavage rate of target RNA between the guide polynucleotide to be tested and the control guide polynucleotide.

[0230] Cas13 mutants

[0231] In some embodiments, the Cas13 proteins provided herein comprise one or more mutations, such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof, relative to the wild-type C13-2 protein (SEQ ID NO: 1). In some instances, the Cas13 protein has the following sequence relative to the wild-type C13-2 protein (SEQ ID NO: 1): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, The guide polynucleotide comprises 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 amino acid changes (e.g., insertions, deletions, or substitutions) but retains the ability to bind to a target RNA molecule complementary to the guide sequence of the guide polynucleotide and / or retains the ability to process guide array RNA transcripts into guide polynucleotide molecules. In some instances, the Cas13 protein has the following sequence relative to the wild-type C13-2 protein (SEQ ID NO: 1): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 amino acid changes (e.g., insertions, deletions or substitutions) but retain the ability to bind to a target RNA molecule complementary to the guide sequence of the guide polynucleotide.In some instances, the Cas13 protein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type C13-2 protein (SEQ ID NO: 1), but retains the ability to bind to a target RNA molecule complementary to the guide sequence of a guide polynucleotide and / or retains the ability to process guide array RNA transcripts into guide polynucleotide molecules.

[0232] In some embodiments, the Cas13 protein comprises one or more mutations in the catalytic domain, resulting in reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises a mutation in the catalytic domain, resulting in reduced RNA cleavage activity. In some embodiments, the Cas13 protein comprises one or more mutations in one or two HEPN domains, resulting in substantial lack of RNA cleavage activity. In some embodiments, the Cas13 protein comprises a mutation in one or two HEPN domains, resulting in substantial lack of RNA cleavage activity. In some embodiments, "substantially lacking RNA cleavage activity" refers to retaining only ≦50%, ≦40%, ≦30%, ≦20%, ≦10%, ≦5%, or ≦1% of the RNA cleavage activity compared to wild-type Cas13 protein, or having no detectable RNA cleavage activity.

[0233] In some embodiments, the Cas13 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 1. In some embodiments, the Cas13 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the protein sequence encoded by SEQ ID NO: 9. When the CRISPR-Cas13 system comprises a fusion protein of the Cas13 with a protein domain and / or polypeptide tag, the percentage of sequence identity between the Cas13 portion of the fusion protein and a reference sequence is calculated.

[0234] In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide that is capable of specifically binding to a target RNA sequence.

[0235] In some embodiments, the Cas13 protein is capable of forming a CRISPR complex with a guide polynucleotide comprising direct repeats linked to a guide sequence engineered to direct sequence-specific binding of the CRISPR complex to a target RNA.

[0236] One type of modification or mutation involves substituting amino acids with amino acid residues having similar biochemical properties, i.e., conservative substitutions (e.g., conservative substitutions of 1 to 4, 1 to 8, 1 to 10, or 1 to 20 amino acids). Typically, conservative substitutions have little or no effect on the activity of the resulting protein or peptide. For example, conservative substitutions are amino acid substitutions in the Cas13 protein that do not substantially affect binding of the Cas13 protein to a target RNA molecule complementary to the guide sequence of the gRNA molecule and / or the process of processing the guide array RNA transcript into a gRNA molecule. Alanine scanning can be used to identify which amino acid residues in the Cas13 protein tolerate amino acid substitutions. In one example, substitution of alanine or other conservative amino acids with 1 to 4, 1 to 8, 1 to 10, or 1 to 20 naturally occurring amino acids results in a change in the ability of the variant Cas13 protein to modify gene expression in a CRISPR-Cas system of no more than 25%, e.g., no more than 20%, e.g., no more than 10%. Examples of amino acids that may be substituted and are considered conservative substitutions include Ala for Ser, Arg for Lys, Asn for Gln or His, Asp for Glu, Cys for Ser, Gln for Asn, Glu for Asp, Gly for Pro, His for Asn or Gln, Ile for Leu or Val, Leu for Ile or Val, Lys for Arg or Gln, Met for Leu or Ile, Phe for Met, Leu, or Tyr, Ser for Thr, Thr for Ser, Trp for Tyr, Tyr for Trp or Phe, and Val for Ile or Leu.

[0237] More substantial changes can be made by using less conservative substitutions, for example, by selecting residues that differ more in their effect on maintaining (a) the structure of the polypeptide backbone in the region where the substitution occurs, e.g., a helical or folded conformation, (b) the charge or hydrophobicity of the region interacting with the target site, or (c) the bulk of the side chain. In general, substitutions expected to produce the greatest changes in polypeptide function are (a) substitutions between a hydrophilic residue (e.g., serine or threonine) and a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine), (b) substitutions between cysteine ​​or proline and any other residue, (c) substitutions between a residue with an electropositive side chain (e.g., lysine, arginine, or histidine) and a residue with an electronegative side chain (e.g., glutamic acid or aspartic acid), or (d) substitutions between a residue with a bulky side chain (e.g., phenylalanine) and a residue with no side chain (e.g., glycine).

[0238] In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 40-91 of SEQ ID NO:1 (i.e., the region of SEQ ID NO:1 from amino acid 40 to amino acid 91, inclusive). In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 146-153 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 158-176 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 182-209 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 216-253 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 271-287 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 341-353 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 379-424 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 456-477 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 521-557 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 575-588 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 609-625 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 700-721 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 724-783 of SEQ ID NO:1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 796-815 of SEQ ID NO:1.In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 828-852 of SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises one or more mutations at amino acid positions 880-893 of SEQ ID NO: 1.

[0239] In some embodiments, the Cas13 protein comprises one or more amino acid deletions at amino acid positions 348-350 of SEQ ID NO: 1 (i.e., amino acids 348, 349, and 350 in the sequence of SEQ ID NO: 1). In some embodiments, the Cas13 protein comprises one or more amino acid deletions at amino acid positions 521-556 of SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises one or more amino acid deletions at amino acid positions 883-893 of SEQ ID NO: 1.

[0240] In some embodiments, the RxxxxH motif (where x represents any amino acid, and RxxxxH may also be written as Rx4H or R4xH) of the Cas13 protein comprises one or more mutations and substantially lacks RNA cleavage activity.

[0241] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215, the RxxxxH motifs at positions 750-755, and / or the RxxxxH motifs at positions 785-790 of the reference protein set forth in SEQ ID NO:1.

[0242] In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to the RxxxxH motif at positions 210-215 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to the RxxxxH motif at positions 750-755 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises a mutation at a position corresponding to the RxxxxH motif at positions 785-790 of the reference protein set forth in SEQ ID NO: 1.

[0243] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215 and 750-755 of the reference protein set forth in SEQ ID NO:1.

[0244] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215 and 785-790 of the reference protein set forth in SEQ ID NO:1.

[0245] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 750-755 and 785-790 of the reference protein set forth in SEQ ID NO:1.

[0246] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to the RxxxxH motifs at positions 210-215, the RxxxxH motifs at positions 750-755, and the RxxxxH motifs at positions 785-790 of the reference protein set forth in SEQ ID NO:1.

[0247] In some embodiments, the RxxxxH motif is mutated to AxxxxH, RxxxxA, or AxxxxA. In some embodiments, the RxxxxH motif is mutated to AxxxxH. In some embodiments, the RxxxxH motif is mutated to RxxxxA. In some embodiments, the RxxxxH motif is mutated to AxxxxA.

[0248] In some embodiments, the Cas13 protein comprises 1, 2, 3, 4, 5, or 6 mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein is mutated to A (alanine) at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein set forth in SEQ ID NO: 1.

[0249] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210 and H215 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R750 and H755 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R785 and H790 of the reference protein set forth in SEQ ID NO: 1.

[0250] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R750, and H755 of the reference protein set forth in SEQ ID NO:1.

[0251] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R750, H755, R785, and H790 of the reference protein set forth in SEQ ID NO:1.

[0252] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R785, and / or H790 of the reference protein set forth in SEQ ID NO:1.

[0253] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and H790 of a reference protein set forth in SEQ ID NO:1.

[0254] In some embodiments, the corresponding positions of R210, R750, or R785 are mutated to A. In some embodiments, the corresponding positions of H215, H755, or H790 are mutated to A. In some embodiments, the corresponding positions of R210, H215, R750, H755, R785, and H790 are all mutated to A.

[0255] In some embodiments, the Cas13 protein is obtained by introducing a mutation into the RxxxxH motif at positions 210 to 215, the RxxxxH motif at positions 750 to 755, and / or the RxxxxH motif at positions 785 to 790 of the sequence set forth in SEQ ID NO:1.

[0256] In some embodiments, the Cas13 protein is obtained by introducing 1, 2, 3, 4, 5, or 6 mutations at positions R210, H215, R750, H755, R785, and / or H790 of the sequence set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein is obtained by mutating positions R210, H215, R750, H755, R785, and / or H790 of the sequence set forth in SEQ ID NO: 1 to A (alanine).

[0257] In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R785, and H790 of the sequence set forth in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R750, and H755 of the sequence set forth in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R750, H755, R785, and H790 of the sequence set forth in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R750, H755, R785, and H790 of the sequence set forth in SEQ ID NO: 1 to A.

[0258] In some embodiments, the Cas13 protein comprises at least one mutation at a position corresponding to amino acid residues 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 a reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises one or more amino acid deletions at a position corresponding to amino acid residues 348-350, 521-556, or 883-893 of a reference protein set forth in SEQ ID NO: 1.

[0259] In some embodiments, compared to the reference protein set forth in SEQ ID NO: 1, the Cas13 protein has amino acid residues at positions corresponding to the following amino acid residues in the reference protein set forth 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+R and any one or more mutations at: 290+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, S418.

[0260] In some embodiments, compared to the comparative protein set forth in SEQ ID NO: 1, the Cas13 protein is mutated to the same amino acid residue at a position corresponding to the mutation site in the comparative protein set forth in SEQ ID NO: 1 in Table 24. In some embodiments, compared to the comparative protein set forth in SEQ ID NO: 1, the Cas13 protein comprises the same mutation at a position corresponding to the mutation site in the comparative protein set forth in SEQ ID NO: 1 in Table 24.

[0261] In some embodiments, the Cas13 protein is located at the following positions in the sequence set forth 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+N 68, 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, S418.

[0262] In some embodiments, the Cas13 protein is obtained by introducing any one or more mutations in Table 24 into the sequence set forth in SEQ ID NO:1.

[0263] In some embodiments, the Cas13 protein is obtained by sequence deletion at positions corresponding to amino acid residues 91-120, 141-180, 211-240, 331-360, 351-400, 431-460, 461-500, 511-550, 611-640, 631-660, 661-690, 691-760, 821-860, or 861-890 of the reference protein set forth in SEQ ID NO:1.

[0264] In some embodiments, the sequence deletion is a deletion of <300, <200, <150, <100, <90, <80, <70, <60, <50, <40, <30, <20, or <10 amino acid residues.

[0265] In some embodiments, the Cas13 protein is obtained by deletion of a sequence at positions 91 to 120, 141 to 180, 211 to 240, 331 to 360, 351 to 400, 431 to 460, 461 to 500, 511 to 550, 611 to 640, 631 to 660, 661 to 690, 691 to 760, 821 to 860, or 861 to 890 of the sequence set forth in SEQ ID NO:1.

[0266] Subcellular localization signals (also called localization signals)

[0267] In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least one homologous or heterologous subcellular localization signal. Exemplary subcellular localization signals include organelle localization signals, such as a nuclear localization signal (NLS), a nuclear export signal (NES), or a mitochondrial localization signal.

[0268] In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least one homologous or heterologous NLS. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two NLSs. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least three NLSs. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least one N-terminal NLS and at least one C-terminal NLS. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two C-terminal NLSs. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two N-terminal NLSs.

[0269] In some embodiments, the NLSs are independently selected from the group consisting of SPKKKRKVEAS (SEQ ID NO: 53), GPKKKRKVAAA (SEQ ID NO: 54), PKKKRKV (SEQ ID NO: 55), KRPAATKKA GQA KKKK (SEQ ID NO: 56), PAAKRVKLD (SEQ ID NO: 57), RQRRNELKRSP (SEQ ID NO: 58), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 59), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 60), VSRKRPRP (SEQ ID NO: 61), PPKKARED (SEQ ID NO: 62), POPKKKPL (SEQ ID NO: 63), SALIKKKKKMAP (SEQ ID NO: 64), DRLRR (SEQ ID NO: 65), PKQKKRK (SEQ ID NO: 66), RKLKKKIKKL (SEQ ID NO: 67), REKKKFLKRR (SEQ ID NO: 68), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 69), RKCLQAGMNLEARKTKK (SEQ ID NO: 70) and PAAKKKKLD (SEQ ID NO: 71).

[0270] In some embodiments, the Cas13 protein or functional fragment thereof is fused to the same or a different NES. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two NESs. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least three NESs. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least one N-terminal NES and at least one C-terminal NES. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two C-terminal NESs. In some embodiments, the Cas13 protein or functional fragment thereof is fused to at least two N-terminal NESs.

[0271] In some embodiments, the NESs are independently selected from an adenovirus type 5 E1B NES, an HIV Rev NES, a MAPK NES, and a PTK2 NES.

[0272] In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous NLS and NES, and a cleavable linker is present between the NLS and the NES. In some embodiments, the NES facilitates production of delivery particles (e.g., virus-like particles) comprising the Cas13 protein or functional fragment thereof in a producer cell line. In some embodiments, cleavage of the linker in a target cell exposes the NLS, which can facilitate nuclear localization of the Cas13 protein or functional fragment thereof in the target cell.

[0273] Protein domains and polypeptide tags

[0274] In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked or fused to a homologous or heterologous protein domain and / or polypeptide tag. In some embodiments, the Cas13 protein or functional fragment thereof is fused to a homologous or heterologous protein domain and / or polypeptide tag.

[0275] In some embodiments, the protein domains and polypeptide tags are optionally selected from a cytosine deaminase domain, an adenosine deaminase domain, a translation activation domain, a translation 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.

[0276] In some embodiments, the protein domain comprises a cytosine deaminase domain, an adenosine deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a ribonuclease domain, a splicing factor domain, a reporter domain, and an affinity domain. In some embodiments, the polypeptide tag comprises a reporter tag and an affinity tag.

[0277] In some embodiments, the length of the amino acid sequence of the protein domain is ≥ 40 amino acids, ≥ 50 amino acids, ≥ 60 amino acids, ≥ 70 amino acids, ≥ 80 amino acids, ≥ 90 amino acids, ≥ 100 amino acids, ≥ 150 amino acids, ≥ 200 amino acids, ≥ 250 amino acids, ≥ 300 amino acids, ≥ 350 amino acids, or ≥ 400 amino acids.

[0278] Exemplary protein domains include domains capable of RNA cleavage (e.g., a PIN endonuclease domain, a NYN domain, an SMR domain from SOT1, or an RNase domain from Staphylococcal nuclease), domains that can affect RNA stability (e.g., tristetraprolin (TTP), or domains from UPF1, EXOSC5, and STAU1), domains capable of nucleotide or ribonucleotide editing (e.g., cytidine deaminase, PPR proteins, adenosine deaminases, ADAR family proteins, or APOBEC family proteins), domains capable of translational activation (e.g., domains of eIF4E and other translation initiation factors, yeast poly(A) binding protein, or GLD2), domains capable of translational inhibition (e.g., Pumilio or FBF domains), and domains capable of RNA cleavage (e.g., a domain from Pumilio or FBF domains). These include domains capable of RNA methylation (e.g., domains from m6A methyltransferase factors (e.g., METTL14, METTL3, or WTAP)), domains capable of RNA demethylation (e.g., human alkylation repair homolog 5), domains that can affect splicing (e.g., the RS-rich domain of SRSF1, the Gly-rich domain of hnRNP A1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1), domains capable of affinity purification or immunoprecipitation, and domains capable of proximity-based protein labeling and identification (e.g., biotin ligases (e.g., BirA) or peroxidases (e.g., APEX2) for biotinylating proteins that interact with target RNAs).

[0279] In some embodiments, the protein domain comprises an adenosine deaminase domain. In some embodiments, the Cas13 protein with a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to an adenosine deaminase domain to direct A-to-I deaminase activity of RNA transcripts in mammalian cells. An engineered adenosine deaminase domain based on ADAR2 for targeting A-to-I RNA editing is described in Cox et al., Science 358(6366):1019-1027(2017), incorporated herein by reference in its entirety. In other embodiments, the adenosine deaminase domain is covalently linked or fused to an adaptor protein capable of binding to an aptamer sequence inserted or added within the guide polynucleotide, thereby allowing the adenosine deaminase domain to be non-covalently linked to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

[0280] In some embodiments, the protein domain comprises a cytosine deaminase domain. In some embodiments, the Cas13 protein with a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to a cytosine deaminase domain to direct C-to-U deaminase activity of RNA transcripts in mammalian cells. A cytosine deaminase domain evolved from ADAR2 to target C-to-U RNA editing is described in Abudayyeh et al., Science 365(6451):382-386(2019), incorporated herein by reference in its entirety. In other embodiments, the cytosine deaminase domain is covalently linked or fused to an adaptor protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby allowing the cytosine deaminase domain to be non-covalently linked to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

[0281] In some embodiments, the protein domain comprises a splicing factor domain. In some embodiments, the Cas13 protein having a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to a splicing factor domain to direct alternative splicing of a target RNA in mammalian cells. Splicing factor domains for targeting alternative splicing are described in Konermann et al., Cell 173(3):665-676 (2018), the entire contents of which are incorporated herein by reference. Non-limiting examples of splicing factor domains include the RS-rich domain of SRSF1, the Gly-rich domain of hnRNPA1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1. In other embodiments, the splicing factor domain is covalently linked or fused to an adaptor protein that can bind to an aptamer sequence inserted or added to the guide polynucleotide, thereby allowing the splicing factor domain to be non-covalently linked to the Cas13 protein, or functional fragment thereof, complexed with the guide polynucleotide.

[0282] In some embodiments, the protein domain comprises a translational activation domain. In some embodiments, the Cas13 protein with a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to a translational activation domain to activate or increase expression of a target RNA. Non-limiting examples of translational activation domains include domains of eIF4E and other translation initiation factors, yeast poly(A) binding protein, or GLD2. In other embodiments, the translational activation domain is covalently linked or fused to an adaptor protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby allowing the translational activation domain to be non-covalently linked to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

[0283] In some embodiments, the protein domain comprises a translational repression domain. In some embodiments, the Cas13 protein with a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to a translational repression domain to inhibit or reduce expression of a target RNA. Non-limiting examples of translational repression domains include Pumilio or FBF PUF proteins, deadenylase, CAF1, and Argonaute proteins. In other embodiments, the translational repression domain is covalently linked or fused to an adaptor protein capable of binding to an aptamer sequence inserted or added within the guide polynucleotide, thereby allowing the translational repression domain to be non-covalently linked to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

[0284] In some embodiments, the protein domain comprises an RNA methylation domain. In some embodiments, the Cas13 protein with a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to an RNA methylation domain for methylation of target RNA. Non-limiting examples of RNA methylation domains include m6A domains such as METTL14, METTL3, or WTAP. In other embodiments, the RNA methylation domain is covalently linked or fused to an adaptor protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby allowing the RNA methylation domain to be non-covalently linked to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

[0285] In some embodiments, the protein domain comprises an RNA demethylation domain. In some embodiments, the Cas13 protein with a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to an RNA demethylation domain for demethylating a target RNA. Non-limiting examples of RNA demethylation domains include human alkylation repair homolog 5 or ALKBH5. In other embodiments, the RNA demethylation domain is covalently linked or fused to an adaptor protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby allowing the RNA demethylation domain to be non-covalently linked to the Cas13 protein or functional fragment thereof complexed with the guide polynucleotide.

[0286] In some embodiments, the protein domain comprises a ribonuclease domain. In some embodiments, the Cas13 protein having a mutated HEPN domain, the catalytically inactive Cas13 protein, or a functional fragment thereof is covalently linked or fused to a ribonuclease domain to cleave a target RNA. Non-limiting examples of ribonuclease domains include a PIN endonuclease domain, a NYN domain, an SMR domain from SOT1, or an RNase domain from Staphylococcus aureus nuclease.

[0287] In some embodiments, the protein domain comprises an affinity domain and / or a reporter domain. In some embodiments, the Cas13 protein or functional fragment thereof is covalently linked or fused to a reporter domain, such as a fluorescent protein. Non-limiting examples of reporter domains include GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, and BFP.

[0288] In some embodiments, the Cas13 protein is covalently linked or fused to a polypeptide tag. In some embodiments, the polypeptide tag is exemplified by a small polypeptide sequence. In some embodiments, the length of the amino acid sequence of the polypeptide tag is ≦50 amino acids, ≦40 amino acids, ≦30 amino acids, ≦25 amino acids, ≦20 amino acids, ≦15 amino acids, ≦10 amino acids, or ≦5 amino acids. In some embodiments, the Cas13 protein is covalently linked or fused to an affinity tag, such as a purification tag. Non-limiting examples of affinity tags include an HA tag, a His tag (e.g., 6-His), a Myc tag, an E tag, an S tag, a calmodulin tag, a FLAG tag, a GST tag, a MBP tag, a Halo tag, or biotin.

[0289] In some embodiments, the C13-2 inactivating mutant (R210A+H215A+R750A+H755A+R785A+H790A) is fused to a protein domain and / or polypeptide tag.

[0290] In some embodiments, the Cas13 protein is fused to an ADAR.

[0291] In some embodiments, the C13-2 inactivating mutant (R210A+H215A+R750A+H755A+R785A+H790A) is fused to a cytosine deaminase or an adenine deaminase.

[0292] In some embodiments, the C13-2 inactivating mutant (R210A+H215A+R750A+H755A+R785A+H790A) is covalently linked to the cytosine deaminase or adenine deaminase directly, via the rigid linker peptide sequence A(EAAAK)3A, or via the flexible linker peptide sequence (GGGGS)3.

[0293] Aptamer Sequence

[0294] In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop of the guide polynucleotide. In some embodiments, the aptamer sequence is inserted into a tetraloop of the guide polynucleotide. An exemplary tetraloop of a guide polynucleotide is shown in Figure 2. In some embodiments, the aptamer sequence is added to the end of the guide polynucleotide.

[0295] Insertion of an aptamer sequence into a guide polynucleotide of a CRISPR-Cas system is described in Konermann et al., Nature 517:583-588 (2015), which is incorporated herein by reference in its entirety. In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

[0296] Adaptor protein

[0297] In some embodiments, the CRISPR-Cas13 system further comprises a fusion protein, or a nucleic acid encoding the fusion protein, comprising an adaptor protein and a homologous or heterologous protein domain and / or polypeptide tag, wherein the adaptor protein is capable of binding to an aptamer sequence.

[0298] Fusion proteins of adaptor proteins and protein domains are described in Konermann et al., Nature 517:583-588 (2015), which is incorporated herein by reference in its entirety. In some embodiments, the adaptor protein comprises MS2 phage coat protein (MCP), PP7 phage coat protein (PCP), or Qβ phage coat protein (QCP). In some embodiments, the protein domain comprises 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, an affinity domain, or a reporter domain.

[0299] Modified Guide Polynucleotides

[0300] In some embodiments, the guide polynucleotide comprises a modified nucleotide. In some embodiments, the modified nucleotide comprises 2'-O-methyl, 2'-O-methyl-3'-thiophosphate, or 2'-O-methyl-3'-thioPACE. In some embodiments, the guide polynucleotide is a chemically modified guide polynucleotide. Chemically modified guide polynucleotides are described in Hendel et al., Nat. Biotechnol. 33(9):985-989 (2015), which is incorporated herein by reference in its entirety.

[0301] In some embodiments, the guide polynucleotide is a hybrid RNA-DNA guide, a hybrid RNA-LNA (locked nucleic acid) guide, a hybrid DNA-LNA guide, or a hybrid DNA-RNA-LNA guide. In some embodiments, the direct repeat sequence comprises one or more ribonucleotides substituted with corresponding deoxyribonucleotides. In some embodiments, the guide sequence comprises one or more ribonucleotides substituted with corresponding deoxyribonucleotides. Hybrid RNA-DNA guide polynucleotides are described in WO2016 / 123230, the entire contents of which are incorporated herein by reference.

[0302] Vector System

[0303] Another aspect of the present disclosure relates to a vector system comprising the CRISPR-Cas13 system described herein, the vector system comprising one or more vectors comprising a polynucleotide sequence encoding the Cas13 protein or fusion protein and a polynucleotide sequence encoding the guide polynucleotide.

[0304] In some embodiments, the vector system comprises at least one plasmid or viral vector (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes simplex virus). In some embodiments, the polynucleotide sequence encoding the Cas13 protein or fusion protein and the polynucleotide sequence encoding the guide polynucleotide are located on the same vector. In some embodiments, the polynucleotide sequence encoding the Cas13 protein or fusion protein and the polynucleotide sequence encoding the guide polynucleotide are located on multiple vectors.

[0305] In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein and / or the polynucleotide sequence encoding a guide polynucleotide is operably linked to a regulatory sequence. In some embodiments, the polynucleotide sequence encoding a Cas13 protein or fusion protein is operably linked to a regulatory sequence. In some embodiments, the polynucleotide sequence encoding a guide polynucleotide is operably linked to a regulatory sequence. In some embodiments, the regulatory sequence of the polynucleotide sequence encoding a Cas13 protein or fusion protein is the same as or different from the regulatory sequence of the polynucleotide sequence encoding a guide polynucleotide. In some embodiments, the regulatory sequence is optionally selected from a promoter, an enhancer, an internal ribosome entry site (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). In some embodiments, the regulatory sequence includes a regulatory sequence that allows a nucleotide sequence to be constitutively expressed in many types of host cells and a regulatory sequence that allows a nucleotide sequence to be expressed only in specific host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a tissue of interest, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a specific cell type (e.g., lymphocytes). Regulatory sequences can also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell-type-specific. In some embodiments, the regulatory sequence is an enhancer element, such as the WPRE, CMV enhancer, the R-U5 segment in the LTR of HTLV-1, the SV40 enhancer, or the intron sequence between rabbit β-globin exons 2 and 3.

[0306] In some embodiments, the vector comprises a pol III promoter (e.g., U6 and H1 promoters), a pol II promoter (e.g., the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), an SV40 promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, or an EF1α promoter), or a pol III promoter and a pol II promoter.

[0307] In some embodiments, the promoter is a constitutive promoter that is continuously active and not regulated by an external signal or molecule. Suitable constitutive promoters include, but are not limited to, the CMV, RSV, SV40, EF1α, CAG, and β-actin promoters. In some embodiments, the promoter is an inducible promoter that is regulated by an external signal or molecule (e.g., a transcription factor).

[0308] In some embodiments, the promoter is a tissue-specific promoter that can be used to drive tissue-specific expression of the Cas13 protein or fusion protein. Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and its mutants, and the SPc5-12 synthetic promoter. Suitable immune cell-specific promoters include, but are not limited to, the B29 promoter (B cells), CD14 promoter (monocytes), CD43 promoter (leukocytes and platelets), CD68 (macrophages), and SV40 / CD43 promoter (leukocytes and platelets). Suitable blood cell-specific promoters include, but are not limited to, the CD43 promoter (leukocytes and platelets), CD45 promoter (hematopoietic cells), INF-β (hematopoietic cells), WASP promoter (hematopoietic cells), SV40 / CD43 promoter (leukocytes and platelets), and SV40 / CD45 promoter (hematopoietic cells). Suitable pancreas-specific promoters include, but are not limited to, the elastase-1 promoter. Suitable endothelial cell-specific promoters include, but are not limited to, the Fit-1 promoter and the ICAM-2 promoter. Suitable neural tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astrocytes), the SYN1 promoter (neurons), and the NSE / RU5' (mature neurons). Suitable kidney-specific promoters include, but are not limited to, the NphsI promoter (podocytes). Suitable bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts). Suitable lung-specific promoters include, but are not limited to, the SP-B promoter (lung). Suitable liver-specific promoters include, but are not limited to, the SV40 / Alb promoter. Suitable cardiac-specific promoters include, but are not limited to, the α-MHC promoter.

[0309] AAV vectors

[0310] Another aspect of the present disclosure relates to an adeno-associated viral (AAV) vector comprising a CRISPR-Cas13 system described herein, wherein the adeno-associated viral (AAV) vector comprises DNA encoding a Cas13 protein or fusion protein and a guide polynucleotide described herein.

[0311] Delivery of CRISPR-Cas systems via AAV vectors is described in Maeder et al., Nature Medicine 25:229-233 (2019), which is incorporated herein by reference in its entirety. In some embodiments, the AAV vector comprises a ssDNA genome that includes the coding sequence for a Cas13 protein or fusion protein and a guide polynucleotide flanked by ITRs.

[0312] In some embodiments, the CRISPR-Cas13 system described herein is packaged into an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh74. In some embodiments, the CRISPR-Cas13 system described herein is packaged into an AAV vector containing an engineered capsid with tissue tropism, such as an engineered muscle-tropic capsid. Engineering AAV capsids with tissue tropism through directed evolution is described in Tabeboldbar et al., Cell 184:4919-4938 (2021), the entire contents of which are incorporated herein by reference.

[0313] lipid nanoparticles

[0314] Another aspect of the present disclosure relates to a lipid nanoparticle (LNP) comprising a CRISPR-Cas13 system as described herein, wherein the LNP comprises a guide polynucleotide as described herein and an mRNA encoding a Cas13 protein or fusion protein as described herein.

[0315] LNP delivery using the CRISPR-Cas system is described in Gillmore et al., N. Engl. J. Med., 385:493-502 (2021), incorporated herein by reference in its entirety. In some embodiments, in addition to an RNA payload (Cas13 mRNA and guide polynucleotide), the lipid nanoparticle (LNP) comprises four components: a cationic or ionizable lipid, cholesterol, a helper lipid, and a PEG lipid. In some embodiments, the cationic or ionizable lipid comprises cKK-E12, C12-200, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, FTT5, Moderna SM-102, and Intellia LP01. In some embodiments, the PEG lipid comprises PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159. In some embodiments, the helper lipid comprises DSPC. The components of LNPs are described in Paunovska et al., Nature Reviews Genetics 23:265-280 (2022), which is incorporated herein by reference in its entirety.

[0316] Lentiviral vectors

[0317] Another aspect of the present disclosure relates to a lentiviral vector comprising a CRISPR-Cas13 system described herein, wherein the lentiviral vector comprises a guide polynucleotide described herein and an mRNA encoding a Cas13 protein or fusion protein described herein. In some embodiments, the lentiviral vector is pseudotyped with a homologous or heterologous envelope protein, such as VSV-G. In some embodiments, the mRNA encoding the Cas13 protein or fusion protein is linked to an aptamer sequence.

[0318] RNP complex

[0319] Another aspect of the present disclosure relates to a ribonucleoprotein complex comprising a CRISPR-Cas13 system described herein, wherein the ribonucleoprotein complex is formed from a guide polynucleotide and a Cas13 protein or fusion protein described herein. In some embodiments, the ribonucleoprotein complex can be delivered to eukaryotic, mammalian, or human cells by microinjection or electroporation. In some embodiments, the ribonucleoprotein complex can be packaged into a virus-like particle and delivered to a mammalian or human subject in vivo.

[0320] virus-like particles

[0321] Another aspect of the present disclosure relates to a virus-like particle (VLP) comprising a CRISPR-Cas13 system as described herein, wherein the virus-like particle comprises a guide polynucleotide as described herein and a Cas13 protein or fusion protein, or a ribonucleoprotein complex formed from the guide polynucleotide and the Cas13 protein or fusion protein.

[0322] Engineered VLPs are described in Banskota et al. Cell 185(2):250-265 (2022), Mangeot et al., Nature Communications 10(1):1-15 (2019), Campbell, et al., Molecular Therapy 27:151-163 (2019), Campbell, et al., Molecular Therapy,27 (2019): 151-163, and Mangeot et al. Molecular Therapy, 19(9):1656-1666 (2011), which are incorporated by reference in their entireties. In some embodiments, the engineered virus-like particles (VLPs) are pseudotyped with homologous or heterologous envelope proteins, such as VSV-G. In some embodiments, the Cas13 protein is fused to a gag protein (e.g., MLVgag) via a cleavable linker, and cleavage of the linker in the target cell exposes an NLS between the linker and the Cas13 protein. In some embodiments, the fusion protein comprises (e.g., from 5' to 3') a gag protein (e.g., MLVgag), one or more NESs, a cleavable linker, one or more NLSs, and Cas13, as described in Banskota et al. Cell 185(2):250-265(2022). In some embodiments, the Cas13 protein is fused to a first dimerization domain that can dimerize or heterodimerize with a second dimerization domain fused to a membrane protein, as described in Campbell, et al., Molecular Therapy 27:151-163 (2019), wherein the presence of a ligand promotes the dimerization and concentrates the Cas13 protein or fusion protein into a VLP.

[0323] cell

[0324] Another aspect of the present disclosure relates to cells comprising the CRISPR-Cas13 system described herein. The cells (e.g., that can be used to produce a cell-free system) can be eukaryotic or prokaryotic. Examples of such cells include, but are not limited to, bacteria, archaea, plant, fungal, yeast, insect, and mammalian cells, such as Lactobacillus, Lactococcus, Bacillus (e.g., Bacillus subtilis), Escherichia (e.g., Escherichia coli), Clostridium, Saccharomyces, or Pichia (e.g., Saccharomyces cerevisiae or Pichia pastoris), Kluyveromyces lactis, Salmonella typhimurium, Drosophila cells, Caenorhabditis elegans cells, Xenopus cells, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian cell lines (e.g., HeLa cells, myeloid cell lines, and lymphoid cell lines).

[0325] In some embodiments, the cell is a prokaryotic cell, such as a bacterial cell, such as E. coli. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell or a human cell. In some embodiments, the cell is a primary eukaryotic cell, a stem cell, a tumor / cancer cell, a circulating tumor cell (CTC), a blood cell (e.g., a T cell, a B cell, a NK cell, a Treg, etc.), a hematopoietic stem cell, a specialized immune cell (e.g., a tumor-infiltrating lymphocyte or a tumor-inhibitory lymphocyte), or a stromal cell in the tumor microenvironment (e.g., a cancer-associated fibroblast, etc.). In some embodiments, the cell is a brain or neuronal cell of the central or peripheral nervous system (e.g., a neuron, an astrocyte, a microglia, a retinal ganglion cell, a rod / cone cell, etc.).

[0326] target RNA molecule

[0327] The CRISPR-Cas13 systems, compositions, or kits described herein can be used to target one or more target RNA molecules, such as target RNA molecules present in a biological sample, an environmental sample (e.g., a soil, air, or water sample), or the like. In some embodiments, the target RNA is a coding RNA, such as a pre-mRNA or a mature mRNA. In some embodiments, the target RNA is a nuclear RNA. In some embodiments, the target RNA is an RNA transcript located in the nucleus of a eukaryotic cell. In some embodiments, the target RNA is a non-coding RNA, such as a functional RNA, siRNA, microRNA, snRNA, snoRNA, piRNA, scaRNA, tRNA, rRNA, lncRNA, or lincRNA.

[0328] In some embodiments, in addition to targeting a target RNA molecule, the CRISPR-Cas13 systems, compositions, or kits described herein perform one or more of the following functions on the target RNA: cleaving one or more target RNA molecules or nicking one or more target RNA molecules, activating or upregulating one or more target RNA molecules, activating or inhibiting translation of one or more target RNA molecules, inactivating one or more target RNA molecules, visualizing, labeling, or detecting one or more target RNA molecules, binding to one or more target RNA molecules, editing one or more target RNA molecules, transporting one or more target RNA molecules, and masking one or more target RNA molecules. In some examples, the CRISPR-Cas13 systems, compositions, or kits described herein modify one or more target RNA molecules, where the modification of the one or more target RNA molecules comprises one or more of RNA base substitution, RNA base deletion, RNA base insertion, target RNA cleavage, RNA methylation, and RNA demethylation. In some embodiments, the CRISPR-Cas13 systems, compositions, or kits described herein are capable of targeting one or more target RNA molecules. In some embodiments, the CRISPR-Cas13 systems, compositions, or kits described herein are capable of binding to one or more target RNA molecules. In some embodiments, the CRISPR-Cas13 systems, compositions, or kits described herein are capable of cleaving one or more target RNA molecules. In some embodiments, the CRISPR-Cas13 systems, compositions, or kits described herein are capable of activating the translation of one or more target RNA molecules. In some embodiments, the CRISPR-Cas13 systems, compositions, or kits described herein are capable of inhibiting the translation of one or more target RNA molecules. In some embodiments, the CRISPR-Cas13 systems, compositions, or kits described herein are capable of detecting one or more target RNA molecules.In some embodiments, the CRISPR-Cas13 systems, compositions, or kits described herein are capable of editing one or more target RNA molecules.

[0329] In some embodiments, the target RNA is AQp1 RNA. Knocking down the level of Aqp1 RNA using the CRISPR-Cas13 system described herein reduces aqueous humor production and intraocular pressure, and can be used to treat diseases such as glaucoma. In some embodiments, the target RNA is AQp1 RNA, and the guide sequence of the guide polynucleotide is SEQ ID NO:5.

[0330] In some embodiments, the target RNA is PTBP1 RNA.Knockdown of the level of PTBP1 RNA using the CRISPR-Cas13 system described herein can promote the transdifferentiation of brain astrocytes into neurons, which can be used to treat diseases such as Parkinson's disease.In some embodiments, the target RNA is PTBP1 RNA, and the guide sequence of the guide polynucleotide is SEQ ID NO:6.

[0331] In some embodiments, the target RNA is VEGFA RNA.By using the CRISPR-Cas13 system described herein to reduce the level of VEGFA RNA, the formation of choroidal neovascularization can be prevented, and can be used to treat diseases such as age-related macular degeneration.

[0332] In some embodiments, the target RNA is ANGPTL3 RNA. Knocking down ANGPTL3 RNA levels using the CRISPR-Cas13 system described herein can lower blood lipids, such as low-density lipoprotein cholesterol (LDL-C), and can be used to treat atherosclerotic cardiovascular diseases, such as hyperlipidemia and familial hypercholesterolemia. In some embodiments, the target RNA is ANGPTL3 RNA, and the guide sequence of the guide polynucleotide is selected from any one or more of SEQ ID NOs: 42-49.

[0333] Therapeutic Applications

[0334] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein. The pharmaceutical composition may, for example, comprise a Cas13 protein or fusion protein described herein and an AAV vector encoding the guide polynucleotide. The pharmaceutical composition may, for example, comprise a lipid nanoparticle comprising a guide polynucleotide described herein and mRNA encoding the Cas13 protein or fusion protein. The pharmaceutical composition may, for example, comprise a lentiviral vector comprising a guide polynucleotide described herein and mRNA encoding the Cas13 protein or fusion protein. The pharmaceutical composition may, for example, comprise a virus-like particle comprising a guide polynucleotide and a Cas13 protein or fusion protein described herein, or a ribonucleoprotein complex formed from the guide polynucleotide and a Cas13 protein or fusion protein.

[0335] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in cleaving or editing a target RNA in a mammalian cell.

[0336] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in any one of: cleaving one or more target RNA molecules or nicking one or more target RNA molecules; activating or upregulating one or more target RNA molecules; activating or inhibiting translation of one or more target RNA molecules; inactivating one or more target RNA molecules; visualizing, labeling, or detecting one or more target RNA molecules; binding to one or more target RNA molecules; transporting one or more target RNA molecules; and masking one or more target RNA molecules.

[0337] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in modifying one or more target RNA molecules in a mammalian cell, wherein the modification of the one or more target RNA molecules comprises one or more of RNA base substitution, RNA base deletion, RNA base insertion, target RNA cleavage, RNA methylation, and RNA demethylation.

[0338] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the diagnosis, treatment, or prevention of a disease or condition associated with a target RNA. In some embodiments, the disease or condition is Parkinson's disease. In some embodiments, the disease or condition is Parkinson's disease and the target RNA is PTBP1 RNA. In some embodiments, the disease or condition is glaucoma. In some embodiments, the disease or condition is glaucoma and the target RNA is AQp1 RNA. In some embodiments, the disease or condition is amyotrophic lateral sclerosis. In some embodiments, the disease or condition is amyotrophic lateral sclerosis and the target RNA is superoxide dismutase 1 (SOD1) RNA. In some embodiments, the disease or condition is age-related macular degeneration and the target RNA is VEGFA RNA. In some embodiments, the disease or condition is age-related macular degeneration and the target RNA is VEGFA RNA or VEGFR1 RNA. In some embodiments, the disease or condition is elevated plasma LDL cholesterol levels. In some embodiments, the disease or condition is elevated plasma LDL cholesterol levels and the target RNA is PCSK9 RNA or ANGPTL3 RNA.

[0339] Another aspect of the present disclosure relates to the use of a CRISPR-Cas13 system described herein, a Cas13 protein described herein, a fusion protein described herein, a guide polynucleotide described herein, a nucleic acid described herein, a vector system described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a eukaryotic cell described herein in the preparation of a medicament for diagnosing, treating, or preventing a disease or condition associated with a target RNA. In some embodiments, the disease or condition is Parkinson's disease. In some embodiments, the disease or condition is glaucoma. In some embodiments, the disease or condition is amyotrophic lateral sclerosis. In some embodiments, the disease or condition is age-related macular degeneration. In some embodiments, the disease or condition is elevated plasma LDL cholesterol levels. In some embodiments, the disease or condition is Parkinson's disease and the target RNA is PTBP1 RNA. In some embodiments, the disease or condition is glaucoma and the target RNA is AQp1 RNA. In some embodiments, the disease or condition is amyotrophic lateral sclerosis and the target RNA is superoxide dismutase 1 (SOD1) RNA. In some embodiments, the disease or condition is age-related macular degeneration and the target RNA is VEGFA RNA or VEGFR1 RNA. In some embodiments, the disease or condition is elevated plasma LDL cholesterol levels and the target RNA is PCSK9 RNA or ANGPTL3 RNA.

[0340] In some embodiments, the pharmaceutical composition is delivered to a human subject in vivo. The pharmaceutical composition can be delivered by any effective route. Exemplary administration routes include intravenous infusion, intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, subcutaneous injection, intradermal injection, intraventricular injection, intravascular injection, intracerebellar injection, intraocular injection, subretinal injection, intravitreal injection, intracameral injection, intratympanic injection, intranasal administration, and inhalation.

[0341] In some embodiments, the RNA targeting method results in editing of the sequence of the target RNA. For example, by using a Cas13 protein or fusion protein with an unmutated HEPN domain and a guide polynucleotide containing a guide sequence specific to the target RNA, the target RNA can be cleaved at a precise location or nicked (e.g., if the target RNA is present as a double-stranded nucleic acid molecule, cleaving one of the single strands) in the target RNA. In some instances, this method is used to reduce expression of the target RNA, thereby reducing translation of the corresponding protein. This method can be used in cells that do not require increased RNA expression. In one example, the RNA is associated with a disease such as cystic fibrosis, Huntington's disease, Tay-Sachs disease, Fragile X syndrome, Fragile X-associated tremor / ataxia syndrome, muscular dystrophy, myotonic dystrophy, spinal muscular atrophy, spinocerebellar ataxia, age-related macular degeneration, or familial ALS. In another example, the RNA is associated with cancer (e.g., lung cancer, breast cancer, colon cancer, liver cancer, pancreatic cancer, prostate cancer, bone cancer, brain cancer, skin cancer (e.g., melanoma), or kidney cancer). Examples of target RNAs include, but are not limited to, those associated with cancer (e.g., PD-L1, BCR-ABL, Ras, Raf, p53, BRCA1, BRCA2, CXCR4, β-catenin, HER2, and CDK4). Editing such target RNAs can produce a therapeutic effect.

[0342] In some embodiments, the RNA is expressed in immune cells. For example, the target RNA can encode a protein that leads to the inhibition of a desired immune response (e.g., tumor infiltration). Knocking down such RNA can promote this necessary immune response (e.g., PD1, CTLA4, LAG3, TIM3). In another example, the target RNA encodes a protein that leads to the activation of an undesired immune response, such as in the case of autoimmune diseases such as multiple sclerosis, Crohn's disease, lupus, and rheumatoid arthritis.

[0343] Diagnostic Applications

[0344] Another aspect of the present disclosure relates to an in vitro composition comprising a CRISPR-Cas13 system described herein and a labeled detector RNA that cannot hybridize to a guide polynucleotide described herein.

[0345] Another aspect of the present disclosure relates to the use of the CRISPR-Cas13 system described herein in the detection of a target RNA in a nucleic acid sample suspected of containing the target RNA.

[0346] In some embodiments, methods for detecting target RNA include a Cas13 protein or fusion protein fused to a fluorescent protein or other detectable label and a guide polynucleotide containing a guide sequence specific for the target RNA. Binding of the Cas13 protein or fusion protein to the target RNA can be visualized by microscopy or other imaging methods. In another example, an RNA aptamer sequence can be added or inserted into the guide polynucleotide, such as MS2, PP7, Qβ, and other aptamers. Because the Cas13-guide-target RNA complex is labeled through the aptamer interaction, introducing a protein that specifically binds to these aptamers, such as MS2 phage coat protein fused to a fluorescent protein or other detectable label, can be used to detect the target RNA.

[0347] In some embodiments, the method for detecting a target RNA in a cell-free system results in the generation of a detectable label or enzymatic activity. For example, by using a Cas13 protein, a guide polynucleotide containing a guide sequence specific to the target RNA, and a detectable label, the target RNA is recognized by Cas13. When Cas13 binds to the target RNA, its RNase activity is triggered, resulting in cleavage of the target RNA and the detectable label.

[0348] In some embodiments, the detectable label is RNA linked to a fluorescent probe and a quencher. The complete detectable RNA is linked to the fluorescent probe and quencher, suppressing fluorescence. After the detectable RNA is cleaved by Cas13, the fluorescent probe is released from the quencher and exhibits fluorescent activity. This method can be used to determine whether a target RNA is present in a lysed cell sample, a lysed tissue sample, a blood sample, a saliva sample, an environmental sample (e.g., water, soil, or air sample), or other lysed cell or acellular sample. This method can also be used to detect pathogens such as viruses and bacteria, and to diagnose conditions such as cancer.

[0349] In some embodiments, detection of target RNA is useful for diagnosing disease and / or pathological conditions, or the presence of viral or bacterial infection. For example, Cas13-mediated detection of non-coding RNA such as PCA3 can be used to diagnose prostate cancer when detected in a patient's urine. In another example, Cas13-mediated detection of lncRNA-AA174084, a biomarker for gastric cancer, can be used to diagnose gastric cancer. [Example]

[0350] Experimental Example 1: Screening of C13-2 protein 1. CRISPR and gene annotation The software was used to predict whole genome proteins against microbial genomes from the NCBI Genbank and CNGB (China National Gene Bank) databases, and then the software was used to predict CRISPR arrays on the genomes. 2. Protein Preliminary Screening Clustering was used to remove redundant proteins, while excluding proteins with amino acid sequence lengths less than 800 aa (amino acids) or greater than 1400 aa. 3. Obtaining CRISPR-associated proteins Protein sequences within 10 kb upstream and downstream of the CRISPR array were compared with known Cas13 sequences, and proteins with an e value greater than 1*e-5 were eliminated. Then, the sequences were compared with the NCBI NR library and the EBI patent library to eliminate highly similar proteins. Through experimental validation, the C13-2 protein (SEQ ID NO: 1, 893 aa) was finally obtained. The C13-2 protein is also known as the CasRfg.4 protein. The sources of the genomic sequences of the C13-2 proteins are listed in Table 1.

[0351] Table 1. Sources of genomic sequences for C13-2 proteins

[0352] [Table 1]

[0353] The native (wild-type) DNA coding sequence for the C13-2 protein is SEQ ID NO:9.

[0354] The locus structure of the C13-2 protein, including the CRISPR array and the coding sequence of C13-2, is shown in Figure 1.

[0355] The direct repeat (DR) sequence or scaffold sequence of the gRNA used in combination with C13-2 is: It may also be 5'-GGAAGAUAACUCUACAAACCUGUAGGGUUCUGAGAC-3' (SEQ ID NO: 3).

[0356] The RNA secondary structures of the above direct repeat sequences obtained using RNAfold prediction are shown in Figure 2.

[0357] Experimental Example 2: Preparation, isolation and purification of C13-2 protein

[0358] (1) Vector construction 1. The pET28a vector plasmid was taken and double-digested with BamHI and XhoI. The linearized vector was recovered by agarose gel electrophoresis. A DNA fragment containing the protein coding sequence (which can encode the C13-2 protein and nuclear localization signal) was inserted into the cloning region of the pET28a vector by homologous recombination. The reaction solution was transformed into Stbl3 competent strain, plated on kanamycin sulfate-resistant LB plates, and cultured overnight at 37°C. Afterwards, clones were selected for sequencing and identification. The constructed recombinant vector was designated C13-2-pET28a (SEQ ID NO: 10) and was used to express the C13-2 recombinant protein (SEQ ID NO: 11), which has the following structure: His tag-NLS-Cas13-SV40 NLS-nucleoplasmin NLS. 2. Positive clones with the correct sequence were cultured overnight, and the plasmids were extracted and transformed into the expression strain RIPL-BL21(DE3), which was then coated onto kanamycin sulfate-resistant LB plates and cultured overnight at 37°C.

[0359] (2) Protein Expression 1. Kanamycin sulfate resistance Monoclonal inoculation was carried out in 5 ml of LB medium and cultured overnight at 37°C. 2. The strain was inoculated into 500 ml of kanamycin sulfate-resistant TB medium at a volume ratio of 1:100 and cultured at 220 rpm and 37°C until the OD value reached 0.6. IPTG was added to a final concentration of 0.2 mM and induced at 16°C for 24 hours. 3. The cells were collected by centrifugation, washed with 15 ml of PBS, and then collected by centrifugation. Lysis buffer was added and the cells were disrupted by ultrasound. The mixture was then centrifuged at 10,000 g for 30 minutes to obtain a supernatant containing the recombinant protein. The supernatant was then filtered through a 0.45 μm filter membrane and purified using a column.

[0360] (3) Protein purification The purified C13-2 recombinant protein was purified by MAC (Ni Sepharose 6 Fast Flow, CYTIVA) and HITRAP HEPARIN HP (CYTIVA). The purified C13-2 recombinant protein was visualized as a band after SDS-PAGE electrophoresis (shown in Figure 3).

[0361] Experimental Example 3: Verification of exogenous gene editing efficiency 1. Synthesis of EGFP-targeting vectors to be tested and control vectors The sequence of the synthesized exogenous EGFP expression vector is shown in SEQ ID NO: 13, and the plasmid structure is CMV-EGFP. The full-length sequence of the synthesized EGFP-targeting C13-2 protein validation vector plasmid is shown in SEQ ID NO: 14, and the plasmid structure is CMV-C13-2-U6-gRNA. EGFP was used as an exogenous reporter gene, and its nucleic acid sequence (720 bp) is shown in SEQ ID NO:12. The guide sequence targeting EGFP is ugccguucuucugcuugucggccaugauau (SEQ ID NO: 4).

[0362] 2. Transfection of 293T cells with the vectors to be validated The exogenous EGFP expression vector and the validation vector plasmid of C13-2 protein targeting EGFP were transfected into 293T cells in a 24-well plate at a ratio of 1:2 (166 ng:334 ng). The transfection method is as follows:

[0363] 1.293T cells were digested with trypsin (trypsin 0.25%, EDTA, Thermo, 25200056), and the cells were counted and diluted to 2 × 10 5 Spread the cells in a 24-well plate at 500 μL per well. 2. For each transfection sample, prepare complexes according to the following procedure: a. To each well of the 24-well plate containing the cells, add 50 μL of serum-free Opti-MEM I (Thermo, 11058021) serum-reduced medium, dilute the 500 ng of plasmid DNA described above, and mix gently. b. Gently mix Lipofectamine 2000 (Thermo, 11668019) before use, then dilute 1 μL of Lipofectamine 2000 in 50 μL of Opti-MEM I medium in each well. Incubate at room temperature for 5 minutes. Note: Continue performing step c within 25 minutes. c. After 5 minutes of incubation, combine the diluted DNA with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature (the solution may appear cloudy). Note: Allow the complex to stabilize for 6 hours at room temperature. Add the complex to 293T cells, mix, and detect using flow cytometry after 48 hours.

[0364] 3. Detecting the Downregulation Effect of EGFP Expression by Flow Cytometry A description of the cells and plasmids used is given in Table 2:

[0365] Table 2. Experimental grouping

[0366] [Table 2]

[0367] 48 hours after transfection, the cells were digested with trypsin (trypsin 0.25%, EDTA, Thermo), centrifuged at 300g for 5 minutes, and the supernatant was removed. The cells in each well were resuspended in 500 μL of PBS and analyzed by flow cytometry to detect EGFP fluorescence. After removing cell debris by FCS-A and SSC-A gating, data were collected by flow cytometry.

[0368] The Mean-FITC-A results of the FITC channel were collected and recorded, and the downregulation amplitude was calculated according to the following formula:

[0369] The GFP fluorescence of the EGFP group was defined as a, and the GFP fluorescence of the other groups as x. The downregulation magnitude (%) = (ax) ÷ a × 100. The blank control group was not included in the comparison. The downregulation magnitude results are shown in Table 3. When C13-2 targeted EGFP, its expression was downregulated by 64.49%.

[0370] Table 3. GFP fluorescence detection results by flow cytometry

[0371] [Table 3]

[0372] Experimental Example 4: Verification of endogenous gene editing efficiency 1. Construction of vectors targeting AQp1 and PTBP1 The sequence of the synthesized expression vector C13-2-BsaI plasmid is shown in SEQ ID NO:15.

[0373] The target nucleic acids selected for the experiment were AQp1 (Aquaporin 1) and PTBP1 (Polypyrimidine Tract Binding Protein 1). The 293T cell line, which highly expresses AQp1, was used to validate AQp1, and the 293T cell line was used to validate PTBP1.

[0374] Construction of a 293T cell line (293T-AQp1 cells) overexpressing AQp1: A vector, Lv-AQp1-T2a-GFP (SEQ ID NO: 16), was constructed to overexpress the AQp1 gene and the EGFP gene. AQp1 and EGFP were separated by a 2A peptide. 293T cells were transduced with the Lv-AQp1-T2a-GFP plasmid-packaged lentivirus to form a cell line stably overexpressing the AQp1 gene.

[0375] The gRNA guide sequence targeting AQp1 is AGGGCAGAACCGAUGCUGAUGAAGAC (SEQ ID NO: 5). The gRNA guide sequence targeting PTBP1 is: GUGGUUGGAGAACUGGAUGUAGAUGGGCUG (SEQ ID NO: 6).

[0376] Primer annealing was used to obtain a fragment targeting the target site, and the primers are shown below:

[0377] Targeting PTBP1 Upstream primer: 5'-AGACGTGGTTGGAGAACTGGATGTAGATGGGCTG-3' (SEQ ID NO: 22) Downstream primer: 5'-AAAACAGCCCATCTACATCCAGTTCTCCAACCAC-3' (SEQ ID NO: 23) Targeting AQp1 Upstream primer: 5'-AGACAGGGCAGAACCGATGCTGATGAAGAC-3' (SEQ ID NO: 24) Downstream primer: 5'-AAAAGTCTTCATCAGCATCGGTTCTGCCCT-3' (SEQ ID NO: 25)

[0378] The primer annealing reaction system is shown in the table below. After incubation at 95°C for 5 minutes in the PCR device, it was immediately removed and incubated on ice for 5 minutes to anneal the primers together and form double-stranded DNA with sticky ends.

[0379] Oligo-F (10 μM) 2 μl Oligo-R (10 μM) 2 μl 10x endonuclease reaction buffer* 2 μl Deionized water up to 20 μl

[0380] The synthesized C13-2-BsaI vector plasmid was digested with BsaI endonuclease, and the annealed product and the purified scaffold were ligated with T4 DNA ligase. After transforming E. coli, positive clones were selected and the plasmid was extracted to obtain the C13-2 vector (C13-2 vector plasmid targeting AQP1 or PTBP1) used in the C13-2 experiments described below. The C13-2 vector structure is CMV-C13-2-U6-gRNA, which can be used to express the C13-2 protein and gRNA targeting AQP1 or PTBP1.

[0381] The following control vectors were prepared using standard methods:

[0382] The sequence of the CasRx-AQp1 plasmid (a positive control vector for CasRx targeting AQp1) is shown in SEQ ID NO: 17, and the plasmid structure is CMV-CasRx-U6-gRNA, which contains a sequence encoding CasRx (the amino acid sequence of which is shown in SEQ ID NO: 2).

[0383] The sequence of the shRNA-AQp1 plasmid (a positive control vector for shRNA targeting AQp1) is shown in SEQ ID NO: 19 and is used to express the shRNA molecule, the sequence of which is CCACGACCCUCUUUGUCUUCACUCGAGUGAAGACAAAGAGGGUCGUGG (SEQ ID NO: 7).

[0384] The sequence of the shRNA-PTBP1 plasmid (a positive control vector for shRNA targeting PTBP1) is shown in SEQ ID NO: 20 and is used to express the shRNA molecule, the sequence of which is CAGCCCAUCUACAUCCAGUUCCUCGAGGAACUGGAUGUAGAUGGGCUG (SEQ ID NO: 8).

[0385] The sequence of the CasRx-blank plasmid (a positive control vector capable of expressing CasRx and gRNA, but in which the gRNA does not target AQp1 and PTBP1) is shown in SEQ ID NO: 21, and the plasmid structure is CMV-CasRx-U6-gRNA.

[0386] 2. Transfection of the tested vectors into 293T cells and 293T-AQp1 cells 293T-AQp1 cells were transfected with 500 ng of either a control plasmid or a vector plasmid targeting AQP1 in a 24-well plate. 293T cells were transfected with 500 ng of either a control plasmid or a vector plasmid targeting PTBP1 in a 24-well plate.

[0387] The transfection method is as follows: 1. Digest the cells with trypsin (trypsin 0.25%, EDTA, Thermo Scientific), count the cells, and separate them into 2 x 10 5 Spread the cells in a 24-well plate at 500 μL per well. 2. For each transfection sample, prepare complexes according to the following procedure. a. Add 50 μL of serum-free Opti-MEM I (Thermo) serum-reduced medium to each well of the 24-well plate containing the cells, dilute the above-mentioned plasmid DNA, and mix gently. b. Gently mix Lipofectamine 2000 (Thermo, 11668019) before use, then dilute 1.8 μL of Lipofectamine 2000 in 50 μL of Opti-MEM I medium in each well. Incubate at room temperature for 5 minutes. Note: Continue performing step c within 25 minutes. c. After a 5-minute incubation, combine the diluted DNA with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature (the solution may appear cloudy). Note: Allow the complex to stabilize for 6 hours at room temperature. The complex is added to the cells and mixed.

[0388] 3. qPCR Detection of Target Gene RNA Forty-eight hours after transfection, cells were subjected to RNA extraction using the SteadyPure Universal RNA Extraction Kit AG21017, and RNA concentration was detected using an ultra-microvolume spectrophotometer. The RNA products were reverse-transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR Reverse Transcription Kit, and the reverse-transcribed products were detected using the SYBR Green Premix Pro Taq HS qPCR Kit.

[0389] The primers used for qPCR are as follows:

[0390] Detection of PTBP1: Upstream primer: 5'-ATTGTCCCAGATATAGCCGTTG-3' (SEQ ID NO: 26) Downstream primer: 5'-GCTGTCATTTCCGTTTGCTG-3' (SEQ ID NO: 27) Detection of AQp1: Upstream primer: 5'-GCTCTTCTGGAGGGCAGTGG-3' (SEQ ID NO: 28) Downstream primer: 5'-CAGTGTGACAGCCGGGTTGAG-3' (SEQ ID NO: 29) Detection of internal reference GAPDH: Upstream primer: 5'-CCATGGGGAAGGTGAAGGTC-3' (SEQ ID NO: 30) Downstream primer: 5'-GAAGGGGTCATTGATGGCAAC-3' (SEQ ID NO: 31)

[0391] Assemble the reaction according to the SYBR Green Premix Pro Taq HS qPCR Kit instructions and run it on the QuantStudio TM 5 Detection was performed using a Real-Time PCR System.

[0392] In this experiment, the relative quantification method, 2 -△△CtThe target RNA was calculated using the method below: △Ct=Ct(AQp1)-Ct(GAPDH) or Ct(PTBP1)-Ct(GAPDH) △△Ct = △Ct (sample to be tested, such as C13-2) - △Ct (CasRx-blank or C13-2-BsaI) 2 -△△Ct =2^(-△△Ct)

[0393] The RNA levels of AQp1 and PTBP1 were calculated according to the calculation method described above. The results of the validation experiment targeting PTBP1 are shown in Table 4 and Figure 4. To validate targeting of AQp1, three independent biological replicate experiments were performed (the same batch of 293T cells was used for the transfection procedure), and the average results of the triplicates were obtained as shown in Table 5 and Figure 5.

[0394] Table 4. PTBP1 RNA knockdown test results

[0395] [Table 4]

[0396] Table 5. AQp1 RNA knockdown test results

[0397] [Table 5]

[0398] Note: "-" means not detected.

[0399] C13-2-BsaI and CasRx-blank were controls that did not target either AQp1 or PTBP1. The experimental results showed that, when combined with the gRNAs in this example, C13-2 exhibited significant editing activity, with higher editing activity targeting PTBP1 and higher editing activity targeting AQp1 than CasRx.

[0400] Experimental Example 5: Comparison of editing efficiency with the disclosed Cas13 protein 1. Construction of an editing vector targeting the endogenous gene PTBP1 Patent publication number US10476825B2 discloses the Cas13 protein from BMZ-11B_GL0037771, referred to in this example as C13-113 (the amino acid sequence of this protein is set forth in SEQ ID NO: 32 of the sequences herein). The direct repeat sequence corresponding to C13-113 used in this example is set forth in SEQ ID NO: 33.

[0401] GenBank discloses the Cas13 protein MBR0191107.1, referred to in this application as C13-114 (the amino acid sequence of this protein is set forth in SEQ ID NO: 34 of the Sequences herein). The direct repeat sequence corresponding to C13-114 used in this example is set forth in SEQ ID NO: 35.

[0402] The expression vectors C13-113-BsaI (SEQ ID NO: 36) and C13-114-BsaI (SEQ ID NO: 37) for C13-113 and C13-114, respectively, were synthesized by Reagent Co., Ltd.

[0403] Following the method of Example 4, a fragment targeting the target site was obtained using primer annealing, and the primers are shown below:

[0404] Targeting PTBP1 C13-113: Upstream primer: 5'-CAACGTGGTTGGAGAACTGGATGTAGATGGGCTG-3' (SEQ ID NO: 38) Downstream primer: 5'-AAAACAGCCCATCTACATCCAGTTCTCCAACCAC-3' (SEQ ID NO: 23) C13-114: Upstream primer: 5'-atctGTGGTTGGAGAACTGGATGTAGATGGGCTG-3' (SEQ ID NO: 39) Downstream primer: 5'-AAAACAGCCCATCTACATCCAGTTCTCCAACCAC-3' (SEQ ID NO: 23)

[0405] Following the method of Example 4, the synthesized C13-113-BsaI and C13-114-BsaI plasmids were digested with BsaI endonuclease, followed by T4 ligation with the annealed product. After transforming E. coli, positive clones were selected and the plasmids were extracted. Detection was performed 72 hours after transfection of 293T cells (a different batch from that used in Example 4). Blank controls were transfected with C13-2-BsaI from Example 4.

[0406] RNA was extracted from the cells 72 hours after transfection, reverse transcribed, and qPCR was performed according to the method of Experimental Example 4 (the primers were the same as those of Experimental Example 4).

[0407] Assemble the reaction according to the instructions for the SYBR® Green Premix Pro Taq HS qPCR Kit and run it on the QuantStudio TM 5 Detection was performed using a Real-Time PCR System.

[0408] The amount of PTBP1 RNA was 2 -△△Ct The calculation was performed using the method described below: △Ct = Ct(PTBP1)-Ct(GAPDH) △△Ct = △Ct (sample to be tested) - △Ct (C13-2-BsaI) 2 -△△Ct =2^(-△△Ct)

[0409] The experimental results are shown in Table 6 and FIG.

[0410] Table 6. Comparison of PTBP1 RNA knockdown test results

[0411] [Table 6]

[0412] The data in the table show that when the gRNAs in this experiment were combined to target PTBP1, higher editing efficiency was observed in C13-2, which was superior to C13-113 and C13-114. No significant editing was observed in the C13-113 group.

[0413] Experimental Example 6: Off-target testing of C13-2 Prediction was performed on the whole genome and whole cDNA sequences of the target species (Homo sapiens) using the EMBOSS-water program and the NCBI-Blast program, and the sense and antisense strands of the gRNA guide sequence were used for comparison. The prediction results were filtered according to the following: the difference in length between the predicted target and the gRNA guide sequence does not exceed 4 bases, and the mismatch + gap does not exceed 4 bases. The potential off-target information is shown in Table 7:

[0414] Table 7. Number of predicted potential off-target genes

[0415] [Table 7]

[0416] The vector plasmid C13-2 targeting PTBP1 of Experimental Example 4, the shRNA-PTBP1 plasmid (the expressed shRNA is referred to as "shRNA2" in this Experimental Example), and the CasRx-blank plasmid were used.

[0417] The sequence of the CasRx-PTBP1 plasmid (a positive control vector for CasRx targeting PTBP1) prepared using conventional methods is shown in SEQ ID NO: 18, and the plasmid structure is CMV-CasRx-U6-gRNA.

[0418] A plasmid expressing shRNA1 was constructed according to the method of Example 4 (the difference from the shRNA-PTBP1 plasmid is only the guide sequence of the encoded shRNA, which also targets PTBP1, but does not have an additional g after the U6 promoter).

[0419] The above plasmids were transfected into 293T cells according to the method described in Experimental Example 4. 24-well plates were transfected according to the Lipofectamine 2000 (Thermo) instructions, and RNA was extracted 72 hours later using the SteadyPure Universal RNA Extraction Kit AG21017.

[0420] The extracted RNA samples were subjected to PE150bp RNA-Seq sequencing, and the resulting fastq files were compared with the reference genome of the target species using HISAT or STAR software to generate a BAM file. Transcripts and the expression levels of each gene were detected using kallisto, RSEM, or HTSeq.

[0421] Using DESeq2, limma-voom, and edger, differential expression analysis of each group (difference from the CasRx-blank group) was performed, and genes with p.adj<0.05, |log2FoldChange|>=0.5, and basemean>2.5 were defined as differentially expressed genes (DEGs). The total DEG information obtained is as follows:

[0422] Table 8. Number of differentially expressed genes and predicted intersections with potential off-target genes

[0423] [Table 8]

[0424] NOTE: Up means that expression is upregulated, and Down means that expression is downregulated.

[0425] Sig indicates significant differences in gene expression from the control group (CasRx-blank group).

[0426] Isec represents the number of intersections of DEGs with “potential off-target genes” predicted by the program.

[0427] As can be seen from the data in the table above, the number of off-target sites after transcriptome sequencing and predicted off-target cross-reactions of CasRx and C13-2 were all zero. C13-2 had almost no off-target sites, while shRNA1 and shRNA2 had a large number of off-target sites. In terms of off-target safety, C13-2 was superior to shRNA1 and shRNA2 and comparable to CasRx. Furthermore, C13-2 is only 893 aa in size, much smaller than the 967 aa of CasRx, making it easier to package and deliver with gRNA into AAV.

[0428] Experimental Example 7: Targeted editing of ANGPTL3 This experiment was carried out in accordance with the method of Experimental Example 4. A vector Lv-ANGPTL3-T2a-GFP (sequence number 52) was constructed to overexpress the ANGPTL3 gene and the EGFP gene, with ANGPTL3 and EGFP separated by a 2A peptide. The Lv-ANGPTL3-T2a-GFP plasmid-packaged lentivirus was transduced into 293T cells to obtain a 293T cell line (referred to as 293T-ANGPTL3 cells) that stably overexpresses the ANGPTL3 gene. Primer annealing was used to obtain fragments targeting the target site. The C13-2-BsaI vector (SEQ ID NO: 15) was digested with BsaI endonuclease, and the annealed product and the purified scaffold recovered after digestion were ligated with T4 ligation. After transforming E. coli, positive clones were selected and the C13-2 plasmid targeting ANGPTL3 was extracted (CMV drives C13-2 protein expression, the U6 promoter drives gRNA expression, the DR sequence of gRNA is SEQ ID NO: 3, and the gRNA guide sequence is shown in Table 9). The plasmid was then transfected into 293T-ANGPTL3 cells. The negative control group was transfected with the C13-2-BsaI plasmid.

[0429] Table 9. gRNA guide sequences targeting ANGPTL3 RNA

[0430] [Table 9]

[0431] 72 hours after transfection, RNA was extracted and reverse transcribed, and the reverse transcription products were detected by qPCR.

[0432] The primers used for qPCR are as follows: Detection of ANGPTL3: Upstream primer: 5'-CCAGAACACCCAGAAGTAACT-3' (SEQ ID NO: 50) Downstream primer: 5'-TCTGTGGGTTCTTGAATACTAGTC-3' (SEQ ID NO: 51) Detection of internal reference GAPDH: Upstream primer: 5'-CCATGGGGAAGGTGAAGGTC-3' (SEQ ID NO: 30) Downstream primer: 5'-GAAGGGGTCATTGATGGCAAC-3' (SEQ ID NO: 31)

[0433] Relative quantification method 2 -△△CtThe target RNA content was calculated using the method described above. Cells transfected with the recombinant plasmid, RNA extraction, reverse transcription, and qPCR were all performed in three independent biological replicates, and the average results of the triplicates were obtained. Table 10 and Figure 7 show the results.

[0434] Table 10. ANGPTL3 RNA knockdown test results

[0435] [Table 10]

[0436] Experimental data showed that C13-2 could effectively knock down ANGPTL3 RNA, among which the editing effects of gRNA2, gRNA4, gRNA5, and gRNA6 were significant.

[0437] Example 8: Editing using a C13-2 inactivation mutant (dC13-2) A C13-2-VEGFA vector (SEQ ID NO: 72) capable of expressing the C13-2 protein and a gRNA targeting VEGFA was constructed, and the gRNA guide sequence is TGGGTGCAGCCTGGGACCACTTGGCATGG (SEQ ID NO: 73).

[0438] Next, a C13-2 R4xH mutant validation vector was constructed from the C13-2-VEGFA vector using conventional homologous recombination methods, as shown in Table 11.

[0439] The mutant vector introduced the following mutations into the C13-2 coding sequence of the C13-2-VEGFA vector: R210A+H215A: AGAAACGCCACCGCCCAC (SEQ ID NO: 74) → GCAAACGCCACCGCCGCC (SEQ ID NO: 75), R750A+H755A: AGAAAGACCAAGAGACAC (SEQ ID NO: 76) → GCAAAGACCAAGAGAGCC (SEQ ID NO: 77), and / or R785A+H790A: AGAAACGACGTGGAGCAC (sequence number 78) → GCAAACGACGTGGAGGCC (sequence number 79).

[0440] Table 11. Vectors used to detect C13-2 inactivating mutations

[0441] [Table 11]

[0442] The vector was transfected into 293T cell line. Transfection was performed according to the Lipofectamine 2000 (Thermo) instructions. After 72 hours, RNA was extracted using the SteadyPure Universal RNA Extraction Kit. The RNA extracted from three batches of experiments was sent to a sequencing company for RNAseq sequencing to detect the amount of VEGFA RNA, as shown in Table 12 below:

[0443] Table 12. VEGFA RNA abundance determined by RNAseq sequencing

[0444] [Table 12]

[0445] The experimental data in Table 12 showed that editing activity remained high after introducing the R750A+H755A mutation, weak editing activity was retained after introducing the R210A+H215A and / or R785A+H790A mutations, and editing activity was almost completely lost after simultaneously introducing the R210A+H215A, R750A+H755A, and R785A+H790A mutations.

[0446] Experimental Example 9: Testing of C13-2 cleavage products Construction of a validation vector for C13-2 truncations targeting the endogenous gene VEGFA Using the three-fragment homologous recombination method, validation vectors for each truncation shown in Table 13 were constructed from the C13-2-VEGFA plasmid (SEQ ID NO: 72) that can express each truncation protein and a gRNA targeting VEGFA (the only difference from the C13-2-VEGFA vector is that the C13-2 coding sequence has been truncated).

[0447] Table 13. Constructed C13-2 truncation validation vectors

[0448] [Table 13]

[0449] The validation and control vectors were transfected into 293T cells according to the Lipofectamine 2000 (Thermo) instructions. After 72 hours, RNA was extracted using the SteadyPure Universal RNA Extraction Kit and reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR Reverse Transcription Kit. The reaction system was set up according to the instructions for the SYBR® Green Premix Pro Taq HS qPCR Kit, and the PCR was performed using the QuantStudio TM 5 Detection was performed using a Real-Time PCR System.

[0450] The primers used for qPCR are as follows: Detection of VEGFA: ACCTCCACCATGCCAAGTGG (SEQ ID NO: 88) CAGGGTCTCGATTGGATGGC (SEQ ID NO: 89) Detection of internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 30) GAAGGGGTCATTGATGGCAAC (SEQ ID NO: 31)

[0451] Relative quantification method 2 -△△CtThe target RNA was calculated using the method. Multiple batches were tested and the results were averaged. Table 14 shows the results.

[0452] Table 14. Relative amounts of VEGFA RNA after truncation editing

[0453] [Table 14]

[0454] The C13-2 truncation in this example retains specific RNA editing activity.

[0455] Experimental Example 10: Testing different DR (direct repeat) sequences Validation vectors (shown in Table 16) encoding different DR sequences (shown in Table 15) and targeting the endogenous genes VEGFA and PTBP1 were constructed.

[0456] Table 15. Different designed DR sequences

[0457] [Table 15]

[0458] Table 16. Construction of vector plasmids for expressing various gRNAs

[0459] [Table 16]

[0460] Validation vectors expressing various different crRNA sequences (5'-guide-DR-3') in Table 16 were constructed by conventional methods from the C13-2-VEGFA vector in Experimental Example 8 and the C13-2 vector plasmid targeting PTBP1 in Experimental Example 4 (only the crRNA expression cassette sequence was replaced).

[0461] According to the experimental method of the above experimental example, 293T cells were transfected with the test vector and the control vector. After 72 hours, RNA was extracted, reverse transcribed, and detected using a qPCR kit. The primers used for qPCR were as follows: Detection of VEGFA: ACCTCCACCATGCCAAGTGG (SEQ ID NO: 88) CAGGGTCTCGATTGGATGGC (SEQ ID NO: 89) Detection of PTBP1: ATTGTCCCAGATATAGCCGTTG (SEQ ID NO: 26) GCTGTCATTTCCGTTTGCTG (SEQ ID NO: 27) Detection of internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 30) GAAGGGGTCATTGATGGCAAC (SEQ ID NO: 31)

[0462] 2 -△△Ct The change in target RNA was calculated using the method. Multiple batches were tested and the results were averaged. Table 17, Table 18, Figure 9, and Figure 10 show the results.

[0463] Table 17. Relative amounts of VEGFA RNA after different gRNA edits

[0464] [Table 17]

[0465] Table 18. Relative amounts of PTBP1 RNA after different gRNA editing

[0466] [Table 18]

[0467] The experimental results in Tables 17 and 18 show that the editing efficiency was highest when DRrc or DR-hf2 was used, and was superior to other DR sequences (P<0.05), and that relatively high editing efficiency could also be achieved when DR2rc, DR3, or DR4 was used, and was superior to DR2, DR3rc, DR4rc, and DR-hf1.

[0468] The results of sequence comparison of the direct repeat sequences DRrc, DR-hf2, and DR2rc are shown in FIG.

[0469] The RNA secondary structure of the direct repeat sequence DR-hf2 predicted by RNAfold is shown in Figure 19.

[0470] Experimental Example 11: Comparison of C13-2 with mainstream Cas13 tools The validation and control vectors in this example (Table 19) all use the same scaffold sequence, C13-2-BsaI (SEQ ID NO: 15), with only the Cas13 and crRNA coding sequences differing. The NLS sequence and its linking sequence are identical for each vector. All vectors have the structure CMV-NLS-Cas13-2xNLS-U6-crRNA, and all Cas13s have one NLS at the N-terminus and two NLS at the C-terminus, for a total of 3xNLS.

[0471] Because 293T cells do not contain the EGFP sequence, a vector targeting GFP was used as a negative control.

[0472] The guide sequence targeting GFP is tgccgttcttctgcttgtcggccatgatat (SEQ ID NO: 90). The guide sequence targeting PTBP1 is GTGGTTGGAGAACTGGATGTAGATGGGCTG (SEQ ID NO: 6). The guide sequence targeting VEGFA is TGGGTGCAGCCTGGGACCACTTGGCATGG (SEQ ID NO: 73).

[0473] Table 19. Validation and control vectors constructed for comparison with mainstream Cas13 tools.

[0474] [Table 19]

[0475] To save space, the sequences of four vectors in Table 19 are shown as examples (SEQ ID NOs: 91-94).

[0476] The validation vector and control vector were transfected into 293T cells. 293T cells not transfected with any plasmid were used as blank controls.

[0477] Transfection was performed according to the Lipofectamine 2000 (Thermo) instructions. After 48 hours, RNA was extracted using the SteadyPure Universal RNA Extraction Kit, and the RNA concentration was detected using an ultra-microvolume spectrophotometer. The RNA product was reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR reverse transcription kit. The reaction was set up according to the SYBR® Green Premix Pro Taq HS qPCR Kit instructions, and the PCR product was analyzed using the QuantStudio™ PCR Kit. TM 5 Detection was performed using a Real-Time PCR System.

[0478] The primers used for qPCR are as follows: Detection of VEGFA: ACCTCCACCATGCCAAGTGG (SEQ ID NO: 88) CAGGGTCTCGATTGGATGGC (SEQ ID NO: 89) Detection of PTBP1: ATTGTCCCAGATATAGCCGTTG (SEQ ID NO: 26) GCTGTCATTTCCGTTTGCTG (SEQ ID NO: 27) Detection of internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 30) GAAGGGGTCATTGATGGCAAC (SEQ ID NO: 31)

[0479] 2 -△△Ct The amount of target RNA was calculated using the qRT-PCR method, and each Cas13 protein used its respective target GFP group as a negative control. Multiple batches were tested and the results were averaged. Table 20, Table 21, Figure 11, and Figure 12 show the results.

[0480] Table 20. C13-2 and known Cas13 tools targeting VEGFA RNA

[0481] [Table 20]

[0482] Comparing the editing efficiency of VEGFA targets, the editing efficiency of C13-2 was significantly superior, even surpassing that of the currently mainstream Cas13 editing tool, with the editing efficiency ranking C13-2 > PspCas13b > Cas13X.1 > Cas13Y.1.

[0483] Table 21. C13-2 and known Cas13 tools targeting PTBP1 RNA

[0484] [Table 21]

[0485] Comparing the editing efficiency of PTBP1 targets, C13-2 showed superior editing efficiency, with the editing efficiency ranking being C13-2 > CasRx > PspCas13b > Cas13X.1 > Cas13Y.1. The editing efficiency of C13-2 was significantly superior to that of PspCas13b, Cas13X.1, and Cas13Y.1 (P<0.05).

[0486] Example 12: Construction of a single nucleotide editor from dC13-2 In this example experiment, the target used to verify the single-base editing effect is EGFP, and the guide sequence used to target this EGFP is tgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccagcttgtgccc (sequence number 95).

[0487] The vectors in this example, related to dC13-2, express C13-2 inactivating mutants containing the R210A+H215A, R750A+H755A, and R785A+H790A mutations.

[0488] Table 22. Plasmid vectors and descriptions relevant to single base editing

[0489] [Table 22]

[0490] The dC13-2-BsaI and dC13-2-EGFP vectors were constructed using conventional methods.

[0491] The single base editing validation vectors dC13-2-ADAR-EGFP, dC13-2-A(EAAAK)3A-ADAR-EGFP, and dC13-2-(GGGGS)3-ADAR-EGFP were obtained by homologous recombination from the dC13-2-BsaI plasmid, dC13-2-EGFP plasmid, and pC0055-CMV-dPspCas13b-GS-ADAR2DD plasmid.

[0492] pC0055-CMV-dPspCas13b-GS-ADAR2DD was used as a positive control. Because this plasmid does not contain a gRNA expression cassette, the gRNA expression vector PLKO-PURO-PspGRNA-EGFP was synthesized by a contractor.

[0493] Transfection was performed according to the Lipofectamine 2000 (Thermo) instructions, with the vector to be tested and the EGFP reporter vector pAAV-CMV-EGFP transfected at a 4:1 ratio. The transfection protocol is shown in Table 23 below. pC0055-CMV-dPspCas13b-GS-ADAR2DD and PLKO-PURO-PspGRNA-EGFP-AD express the PspCas13b-ADAR protein and gRNA, respectively, and co-transfection was performed at 200 ng each.

[0494] Table 23. Transfection protocols for single base editing validation vectors and reporter vectors

[0495] [Table 23]

[0496] Forty-eight hours after transfection, RNA was extracted using the SteadyPure Universal RNA Extraction Kit, and RNA concentrations were detected using an ultra-microvolume spectrophotometer. The RNA products were reverse-transcribed using the Evo M-MLV RT-PCR Universal Reverse Transcriptase Kit, and the reverse-transcribed products were subjected to PCR using identified primers. The PCR products were then sent to a sequencing company for sequencing.

[0497] The sequences of the identifying primers are as follows (product length 704 bp): agggcgaggagctgtt (SEQ ID NO: 103), gtacagctcgtccatgccg (SEQ ID NO: 104).

[0498] The sequencing results are shown in Figure 13 and indicate that the dC13-2 editor constructed by the present inventors successfully induced single-base editing, resulting in an A→G conversion at the corresponding position on the target RNA at base 48 of the guide sequence.

[0499] The negative control dC13-2-EGFP group did not induce base conversion, while the positive control dPspCas13b-ADAR group induced base conversion. Base conversion was induced when no linker peptide was used, when the rigid linker peptide A(EAAAK)3A was used, or when the flexible linker peptide (GGGGS)3 was used between dC13-2 and ADAR.

[0500] Example 13: First round mutation of C13-2 Principle of first-round mutation: The structure of C13-2 was predicted using AlphaFold v2.1, and N→A and R→A mutations were performed on aa 1-89 and aa 263-417 belonging to the REC lobe.

[0501] The designed mutants are shown in Table 24 below.

[0502] Table 24: First round mutations

[0503] [Table 24]

[0504] Construction of validation vectors The first round of mutations was validated using the VEGFA target. The C13-2-VEGFA vector from the above example was used as a wild-type C13-2-encoding vector, which was then modified using the VazymeMut Site-Directed Mutagenesis Kit Express II Fast Mutagenesis Kit V2 to generate C13-2 mutants and expression constructs (validation vectors) for each mutant to express gRNAs targeting VEGFA. The primers used are listed in Table 25 below.

[0505] Table 25. Primer sequences used for vector validation

[0506] [Table 25-1]

[0507] [Table 25-2]

[0508] [Table 25-3]

[0509] Transfection 293T cells were transfected with the test and control vectors according to the Lipofectamine 2000 (Thermo) instructions. The C13-2-BsaI control group was transfected with the C13-2-BsaI vector from the above experimental example, and the WT control group was transfected with the C13-2-VEGFA vector. Both groups expressed wild-type C13-2. A separate control group of 293T cells was also set up without transfection of the plasmid.

[0510] qPCR detection of target RNA levels RNA was extracted from cells 48 hours posttransfection using the SteadyPure Universal RNA Extraction Kit, and the RNA concentration was detected using an ultra-microvolume spectrophotometer. The RNA products were reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR reverse transcription kit, and the reverse transcription products were detected using the SYBR Green Premix Pro Taq HS qPCR Kit (Low Rox Plus).

[0511] The primers used for qPCR were SEQ ID NOs: 88, 89, 30, and 31.

[0512] Assemble the reaction system according to the instructions for the SYBR® Green Premix Pro Taq HS qPCR Kit (Rox Plus) and run it on the QuantStudio. TM5 Detection was performed using a Real-Time PCR System.

[0513] 2 -△△Ct The edited target RNA levels were calculated using the method. Three experiments were repeated and the results were averaged and shown in Table 26 and Figure 14.

[0514] Table 26. VEGFA RNA levels after targeted editing of mutants by qPCR detection

[0515] [Table 26]

[0516] As can be seen from the qPCR detection results, all point mutants still retained high editing activity.

[0517] RNA sequencing The total RNA samples extracted after editing were subjected to RNA sequencing, the library construction type was lncRNA strand-specific library, the sequencing data amount was 16G, and the sequencing strategy was PE150.

[0518] RNAseq analysis principle: Data quality was controlled using fastqc and multiqc, and low-quality reads were removed using fastp. The sequences were filtered against human rRNA sequences and compared to the hg38 reference genome using Hisat2 comparison software. After comparison, the expression levels of genes were quantified using Kallisto software, and then differential expression analysis was performed using sleuth software. Genes with |b|>0.5, qval<0.05, and mean_obs>2 were considered as differentially expressed genes, and the 293T cell control group was used as the benchmark. Using EMBOSS water software, guide sequences were compared to a reference cDNA. Transcripts with a minimum of 18 base matches, 6 mismatches, and a minimum of 8 consecutive paired bases were considered as predicted off-target transcripts, and the corresponding genes were considered as predicted potential off-target genes. The significantly downregulated differentially expressed genes and predicted potential off-target genes were intersected, and after removing the on-target VEGFA gene, an off-target gene set was obtained.

[0519] RNASeq result analysis Using the 293T cell control group as a benchmark, the VEGFA gene expression levels of each group were analyzed, and the results are shown in Table 27 and FIG.

[0520] Table 27. VEGFA editing efficiency measured by RNAseq

[0521] [Table 27]

[0522] The RNAseq data were essentially consistent with the qPCR results.

[0523] When combined with the gRNAs of this example, the editing activity of the M02, M07, M08, M10, M12, M14, M15, and M18 mutants was slightly improved compared to wild-type C13-2.

[0524] The number of differentially expressed genes and the number of off-target genes are shown in Table 28.

[0525] The number of differentially expressed genes whose intracellular expression was downregulated after editing was lower in the M04, M09, M17, M22, M25, M27, and M28 groups than in the WT group.

[0526] Regarding the number of off-target genes determined after crossover, the number was 0 in all of the M01 to M28 groups, that is, there were no off-target genes.

[0527] Table 28. Differentially expressed genes and off-target genes of different mutants targeting VEGFA

[0528] [Table 28]

[0529] Example 14: Second round mutation of C13-2 Mutant design Based on the results of Experimental Example 1, five low off-target mutation sites (M09 N87A, M17 R308A, M28 N394A, M04 R47A, and M13 R290A) were obtained and either internally linked around the five sites or linked to other mutation sites. In addition, conservative mutations at the conserved sites were also designed. These are shown in Table 29.

[0530] Table 29. Mutants designed in the second round

[0531] [Table 29]

[0532] Construction of validation vectors In the second round of mutations, the human AR (androgen receptor) target was chosen for testing, and the C13-2-AR-h3 plasmid vector (SEQ ID NO: 161, capable of expressing wild-type C13-2 and h3 gRNA targeting AR, the guide sequence of which is SEQ ID NO: 162, i.e., ATAACATTTCCGAAGACGACAAGAT) was synthesized.

[0533] The C13-2-AR-h3 plasmid vector was modified using the VazymeMut Site-Directed Mutagenesis Kit Mut Express MultiS Fast Mutagenesis Kit V2 to obtain expression constructs (validation vectors) for each mutant to express the C13-2 mutant and h3 gRNA. The primers used are listed in Table 30 below.

[0534] Table 30. Primers used for construction of mutant expression vectors

[0535] [Table 30-1]

[0536] [Table 30-2]

[0537] [Table 30-3]

[0538] [Table 30-4]

[0539] [Table 30-5]

[0540] [Table 30-6]

[0541] Transfection 293T cells were transfected with the test and control vectors according to the Lipofectamine 2000 (Thermo) instructions. The C13-2-BsaI control group was transfected with the C13-2-BsaI vector from the above experiment, and the WT control group was transfected with the C13-2-AR-h3 vector. Both groups expressed wild-type C13-2. A separate control group of 293T cells was also set up without transfection of the plasmid.

[0542] qPCR detection of target RNA levels RNA was extracted from cells 48 hours posttransfection using the SteadyPure Universal RNA Extraction Kit, and the RNA concentration was detected using an ultra-microvolume spectrophotometer. The RNA products were reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR reverse transcription kit, and the reverse transcription products were detected using the SYBR Green Premix Pro Taq HS qPCR Kit (Low Rox Plus).

[0543] The primers used for qPCR are as follows: Detection of AR: CCAGGGACCATGTTTTGCC (SEQ ID NO: 275) CGAAGACGACAAGATGGACAA (SEQ ID NO: 276) Detection of internal reference GAPDH: SEQ ID NO: 30, 31.

[0544] Assemble the reaction system according to the instructions for the SYBR® Green Premix Pro Taq HS qPCR Kit (Rox Plus) and run it on the QuantStudio. TM 5 Detection was performed using a Real-Time PCR System.

[0545] 2 -△△Ct The edited target RNA levels were calculated using the method. Three experiments were repeated and the results were averaged and shown in Table 31, Figure 16.

[0546] Table 31. AR RNA levels after target editing of mutants detected by qPCR

[0547] [Table 31]

[0548] RNA sequencing The total RNA samples extracted after editing were subjected to RNA sequencing, the library construction type was lncRNA strand-specific library, the sequencing data amount was 16G, and the sequencing strategy was PE150.

[0549] RNAseq analysis principle: Data quality was controlled using fastqc and multiqc, and low-quality reads were removed using fastp. The sequences were filtered against the human rRNA sequence and compared to the hg38 reference genome using Hisat2. After comparison, the expression levels of genes were quantified using Kallisto software, and then differential expression analysis was performed using sleuth software, and genes with |b|>0.5, qval<0.05, and mean_obs>2 were considered as differentially expressed genes. Using EMBOSS water software, the guide sequences were compared to the reference cDNA. Transcripts with a minimum of 18 base matches, 6 mismatches, and a minimum of 8 consecutive paired bases were considered as predicted off-target transcripts, and the corresponding genes were considered as predicted off-target genes. Significantly downregulated differentially expressed genes and predicted off-target genes were intersected, and after removing on-target AR genes, an off-target gene set was obtained.

[0550] RNASeq result analysis Using the 293T cell control group as a benchmark, the AR gene expression level and editing efficiency (average value) of each group were analyzed, and the results are shown in Table 32 and Figure 17.

[0551] Table 32. AR editing efficiency measured by RNAseq

[0552] [Table 32]

[0553] The RNAseq data were essentially consistent with the qPCR results.

[0554] When combined with the gRNAs of this example, the editing activity of the M2-1, M2-2, M2-3, M2-4, M2-9, M2-10, M2-16, M2-17, M2-18, M2-19, M2-23, M2-24, M2-25, M2-26, M2-32, M2-33, M2-34, M2-35, M2-39, M2-40, and M2-41 mutants was higher than that of wild-type C13-2.

[0555] The number of differentially expressed genes and the number of off-target genes are shown in Table 33.

[0556] The number of differentially expressed genes whose intracellular expression was downregulated after editing was lower in the M2-1, M2-2, M2-3, M2-4, M2-5, M2-6, M2-7, M2-8, M2-10, M2-13, M2-14, M2-15, M2-16, M2-21, M2-22, M2-23, M2-24, M2-25, M2-26, M2-27, M2-28, M2-29, M2-30, M2-31, M2-32, M2-33, M2-34, M2-35, M2-36, M2-37, M2-38, M2-39, M2-40, and M2-41 groups than in the WT group.

[0557] Regarding the number of off-target genes determined after crossover, the M2-1, M2-2, M2-3, M2-4, M2-5, M2-6, M2-7, M2-8, M2-10, M2-13, M2-14, M2-15, M2-16, M2-21, M2-22, M2-23, M2-24, M2-25, M2-26, M2-27, M2-28, M2-29, M2-30, M2-31, M2-32, M2-33, M2-35, M2-36, M2-37, M2-38, M2-39, M2-40, and M2-41 groups were lower than the WT group. Of these, M2-1, M2-6, M2-7, M2-14, M2-15, M2-22, M2-31, M2-38, and M2-39 had no off-target activity.

[0558] Table 33. Altered expression and off-target genes of mutants when targeting AR

[0559] [Table 33-1]

[0560] [Table 33-2]

[0561] Example 15: Testing target protein levels

[0562] The C13-2-VEGFA vector and the negative control vector C13-2-BsaI were transfected into 293T cells (Lipofectamine 2000, Thermo Fisher Scientific) and cultured at 37°C for 72 hours. The supernatant was then collected. VEGFA protein levels were detected using the Elascience Human VEGF-A (Vascular Endothelial Cell Growth Factor A) ELISA Kit, which showed that VEGFA protein expression was downregulated by 97.4% compared to the negative control.

[0563] In 293T cells, after editing using a vector to express CasRx and a gRNA containing the same guide sequence, VEGFA protein expression was downregulated by 75.7% compared to the CasRx negative control.

[0564] array:

[0565] SEQ ID NO: 1 (wild type C13-2 protein sequence, 893 aa):

[0566] MSKDKKTKAKRMGVKALLAHGEDKLTMTTFGKGNRSKIEFTEGYHGRALETPKHFGIRGFEVRRIDENVDLCGDLEEGKTIEALLVNPSEKVGEDYLKLKGTLEKRFFGRE FPHDNIRIQLIYNILDIYKILGMNVADILYALGNMQDTELDIDMFGQSLNNEDNLKECLKRMRPYMGYFGDIFKISPKGENIADREHNKKVLRCISVLRNATAHDKQDEYPW FKSSDIYETKIFKADMWKIIKDQYREKIKKVNKDFLSKNAVNMAILFDLLNARDVEQKKQITDEFYRFTIRKDGKNLGMNLVKIREIIIDRYASGLRDKKHDPHRQKINVI ADFLIFRALSQNQGIIDKTVSSLRLTKDEEEKDHVYQNAAELVWGMVSNCLTPYFNDPKNKYILKYKDAKTPGDFEDWITSKISEDDGEPFVKVLSFLCNFLEGKEINELLT AYIHKFECIQDFLNVISSLGENVQFQPRFALFNNASFAQNVAVQLRILASIGKMKPDLTEAKRPLYKAAIRMLCPPEKWEKYTSDEWLEKNMLLNSEDRKNDKKKKQVNPF RNFIAGNVIESRRFMYLVRYSKPKAVRAIMQNRSIVNYVLHRLPSEQVHRYASVFPENFADLEQEIDFLTKKLFEFSFEELLHEKDVILNNSRSHKPSLEIERLKAITGLYL SVAYIAIKNIVKANARYYIAFAVFERDKELVKAKDARIQTKIPETDFPDYFCLTQYYLDRDEEKKFPGDPRDKEAFFEHLRKTKRHFSKQWREWLNEKIADAKSSQATGLLL REARNDVEHLNVLRAIPDYIQDFRHGEKGETAMNSYFELYHYLMQRLMLKNTELDLSHWSGWIMRSGRPDRDLIQIAFVSLAYNLPRYRNLTKEHHFDDTVLQKIREKESLD

[0567] SEQ ID NO: 2 (amino acid sequence of CasRx expressed by CasRx-AQp1 plasmid, 966 aa):

[0568] IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFSKEGRNYINYIGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDL LTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEFTRQINREKAKTALNAYLRNTKWNVIIREDLRIDNKTCTLFRNKAVHLEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS

[0569] SEQ ID NO: 3 (C13-2 gRNA direct repeat / scaffold sequence):

[0570] 5'-GGAAGAUAACUCUACAAACCUGUAGGGUUCUGAGAC-3'

[0571] SEQ ID NO: 4 (Guide sequence targeting EGFP):

[0572] ugccguucuucugcuugucggccaugauau

[0573] SEQ ID NO: 5 (Guide sequence targeting AQp1):

[0574] AGGGCAGAACCGAUGCUGAUGAAGAC

[0575] SEQ ID NO: 6 (Guide sequence targeting PTBP1):

[0576] GUGGUUGGAGAACUGGAUGUAGAUGGGCUG

[0577] SEQ ID NO: 7 (shRNA targeting AQp1):

[0578] CCACGACCCUCUUUGUCUUCACUCGAGUGAAGACAAAGAGGGUCGUGG

[0579] SEQ ID NO: 8 (shRNA targeting PTBP1):

[0580] CAGCCCAUCUACAUCCAGUUCCUCGAGGAACUGGAUGUAGAUGGGCUG

[0581] SEQ ID NO:9 (wild-type C13-2 DNA coding sequence):

[0582]

[0583] SEQ ID NO: 10 (C13-2-pET28 recombinant vector):

[0584]

[0585] SEQ ID NO: 11 (recombinant C13-2 protein):

[0586] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSPAAKKKKLDGSVDMSKDKKTKAKRMGVKALLAGEDKLTMTTFGKGNRSKIEFTEGYHGRALETPKHFGIRGFEVRRIDENVDLCGDLEEGKTIEALLVNPSEKVGEDYLKLKGTLEKRFFGREFPHDNIRIQLIYNILDIYKILGMNVADILYALGNMQDTELDIDMFGQSLNNEDNLKECLKRMRPYMGYFGDIFKISPKGENIADREHNKKVLRCISVLRNATAHDKQDEYPWFKSSDIYETKIFKADMWKIIKDQYREKIKKVNKDFLSKNAVNMAILFDLLNARDVEQKKQITDEFYRFTIRKDGKNLGMNLVKIREIIIDRYASGLRDKKHDPHRQKINVIADFLIFRALSQNQGIIDKTVSSLRLTKDEEEKDHVYQNAAELVWGMVSNCLTPYFNDPKNKYILKYKDAKTPGDFEDWITSKISEDDGEPFVKVLSFLCNFLEGKEINEL LTAYIHKFECIQDFLNVISSLGENVQFQPRFALFNNASFAQNVAVQLRILASIGKMKPDLTEAKRPLYKAAIRMLCPPEKWEKYTSDEWLEKNMLLNSEDRKNDKKKKQVNPFRNFIAGNVIESRRFMYLVRYSKPKAVRAIMQNRSIVNYVLHRLPSEQVHRYASVFPENFADLEQEIDFLTKKLFEFSFEELLHEKDVILNNSRSHKPSLEIERLKAITGLYLSVAYIAIKNIVKANARYYIAFAVFERDKELVKAKDARIQTKIPETDFPDYFCLTQYYLDRDEEKKFPGDPRDKEAFFEHLRKTKRHFSKQWREWLNEKIADAKSSQATGLLLREARNDVEHLNVLRAIPDYIQDFRHGEKGETAMNSYFELYHYMQRLMLKNTELDLSHWSGWIMRSGRPDRDLIQIAFVSLAYNLPRYRNLTKEHHFDDTVLQKIREKESLDTGGGPGGGAAAGSGSPKKRKVGSGSKRPAATKKAGQAKKKK

[0587] SEQ ID NO: 12 (exogenous EGFP reporter gene sequence):

[0588] atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgaggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc ctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctacccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgcc gaccactaccagcagaacaccccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa

[0589] SEQ ID NO: 13 (Plasmid sequence of exogenous EGFP reporter gene expression vector):

[0590]

[0591] SEQ ID NO: 14 (Plasmid sequence of C13-2 expression vector targeting EGFP):

[0592]

[0593] SEQ ID NO: 15 (C13-2-BsaI vector):

[0594]

[0595] SEQ ID NO: 16 (Lv-AQp1-T2a-GFP vector):

[0596]

[0597] SEQ ID NO: 17 (CasRx-AQp1 plasmid, positive control vector of CasRx targeting AQp1):

[0598]

[0599] SEQ ID NO: 18 (CasRx-PTBP1 plasmid, positive control vector of CasRx targeting PTBP1):

[0600]

[0601] SEQ ID NO: 19 (shRNA-AQp1 plasmid, positive control vector for shRNA targeting AQp1):

[0602]

[0603] SEQ ID NO: 20 (shRNA-PTBP1 plasmid, positive control vector for shRNA targeting PTBP1):

[0604]

[0605] SEQ ID NO: 21 (CasRx-blank plasmid, blank control vector expressing CasRx but not containing gRNA targeting AQp1 or PTBP1):

[0606]

[0607] SEQ ID NO: 22 (PTBP1 upstream primer):

[0608] AGACGTGGTTGGAGAACTGGATGTAGATGGGCTG

[0609] SEQ ID NO: 23 (PTBP1 downstream primer):

[0610] AAAACAGCCCATCTACATCCAGTTCTCCAACCAC

[0611] SEQ ID NO: 24 (AQp1 upstream primer):

[0612] AGACAGGGCAGAACCGATGCTGATGAAGAC

[0613] SEQ ID NO: 25 (AQp1 downstream primer):

[0614] AAAAGTCTTCATCAGCATCGGTTCTGCCCT

[0615] SEQ ID NO: 26 (PTBP1 upstream primer):

[0616] ATTGTCCCAGATATAGCCGTTG

[0617] SEQ ID NO: 27 (PTBP1 downstream primer):

[0618] GCTGTCATTTCCGTTTGCTG

[0619] SEQ ID NO: 28 (AQp1 upstream primer):

[0620] GCTCTTCTGGAGGGCAGTGG

[0621] SEQ ID NO: 29 (AQp1 downstream primer):

[0622] CAGTGTGACAGCCGGGTTGAG

[0623] SEQ ID NO: 30 (GAPDH upstream primer):

[0624] CCATGGGGAAGGTGAAGGTC

[0625] SEQ ID NO: 31 (GAPDH downstream primer):

[0626] GAAGGGGTCATTGATGGCAAC

[0627] SEQ ID NO: 32 (C13-113 protein sequence, 928 aa):

[0628] MSKDKKTKAKRMGVKALLAHGEDKLTMTTFGKGNRSKIEFTEGYHGRALETPKHFGKRGFEVRKIDENVDLYGDLEEGKTIEALLINPSEKVGEDYLKLKGTLEKRFFGREFPHDNIRIQLIYNILDIYKILGMNVADILYALGNMQDTELDIDMFGQSLNNPEKAKDVVQRMKPYMGFFGGIFRTQKKEDRKKDSNLSEEEKEEKKKKQEELLQKDLQHNMDVARCISALRHATAHNKPATAHDKQDEYPWFKSSDIYETKIFKAGMWSVIEADYKKKIQDVNKQFFSKNKVNLAILFDLLDVRDVKQKKRISDEFYRFTIRKDGKNLGMNLVKIREIIIDRYASGLRDKKHDPHRQKINVIADFLIFRALSQNQGIIDKTVSSLRLTKDEEEKDHVYQNAAELVWGMVSNCLTPYFNDPKNKYILKYKDAKTPGDFEDWITSKISEDDGEPFVKVLSFLCNFLEGKEINELLTAYIHKFECIQDFLNVISSLGENVQFQPRFALFNNASFAQNVAVQLRILASIGKMKPDLTEAKRPLYKAAIRMLCPPEKWEKYTSDEWLEKNMLLNSEDRKNDKKKKQVNPFRNFIAGNVIESRRFMYLVRYSKPKAVRAIMQNRSIVNYVLHRLPSEQIKTYSRVFPEDFSDTEAEIDFLVNKLSEFSFETFISNQQTILNNSKRGFSPNRRETAEEIERLKAITGLYLSVAYIAIKNIVKANARYYIAFAVFERDKELVKAKDARIQTTIPNTKFTNYFCLTQYYLDRDEEKKFPGDPRDKEAFFEHLRKTKRHFSKQWREWLNEKIADAKSSQATGLLLREARNDVEHLNVLRAIPDYIQDFRHGEKGETAMNSYFELYHYLMQRLMLKNTELDLSHWSGWIMRSGRPDRDLIQIAFVSLAYNLPRYRNLTKEHHFDDTVLQKIREKESLD

[0629] Sequence number 33 (C13-113 direct repeat sequence):

[0630] GGUCAACAGUGUGGGGAAUUACCCGCACUCUACAAC

[0631] SEQ ID NO: 34 (C13-114 protein sequence):

[0632] MSKDKKTKAKRMGVKALLAHGEDKLTMTTFGKGNRSQIEFTEGYHGRALETPKRFGKRGFEVRKIDENVDLYGDLDEGKTIEALLVNPSENVGEDYLKLKSTLENYFFGREFPHDNIRIQLIYNILDIYKILGMNVADILYTLGNLQDAEGDIDLFGKSLNNEDNIKESLNRMRPYMGYFGEVFKKDNREHNKKVLRCISALRNATAHGKQDEYPWFKSSDIYEKTIFKADKWRIIEDQYREKIRKVNNEFFSKNKVNLAILFDLLHARDVEPKKQIADEFYRFTIRKDGKNLGMNLVKIREKIIDRFARDLRDKKHDPHRQKIHVIADFLIFRALSQNQEVIDKTVSRLRLTKDEEEKDRVYQNAAELVWGMVSNCLSPYFKDPKKYIIQYKSGGKIKIFDDWITSKISAKDGEPFVKVLSFLCNFLEGKEINELLTAYIHKFECIQDFLKVISSLGERAQFQPRFDLFNKPDFAQNVAVQLRILASIGKMKPDLTEAKRPLYKAAIQMLCPPEKWEKYTSDEWLEENMLLNSEDRQNKEKREKVNPFRNFIAGNVIESRRFMYLVRYSKPKAVRALMKNRSVVNYVLHRLPPEQIKTYSRVFPDDFNDTEAEIDFLVNKLSEFSFETFITSRQTILANSKRGFSPNRRETAEEIERLKAITGLYLSVAYIAIKNIVKANARYYIAFAVFERDKELVKAKDARIQTTIPNTTYTNYFCLTQYYLDRDEEKKFQGDPRDKEAFFEHLRKKKTHFSKQWREWLNEKIADAKSAQETGLLLTEARNDVEHLNVLRAIPDYIQDFRHGDKGETPMNSYFELYHYLLQRLMLKNRLLDLSSWRSWIERSGRPDRDLIQIAFVSLAYNLPRYRNLTKEHHFDDTVLQKIRERKSLD

[0633] SEQ ID NO: 35 (C13-114 direct repeat sequence):

[0634] agUcUcagaacccUacaggUUUgUagagUUaUcU

[0635] SEQ ID NO: 36 (C13-113-BsaI expression vector):

[0636]

[0637] SEQ ID NO: 37 (C13-114-BsaI expression vector):

[0638]

[0639] SEQ ID NO: 38 (PTBP1 upstream primer):

[0640] CAACGTGGTTGGAGAACTGGATGTAGATGGGCTG

[0641] SEQ ID NO: 39 (PTBP1 upstream primer):

[0642] ATCTGTGGTTGGAGAACTGGATGTAGATGGGCTG

[0643] SEQ ID NO: 40 (Guide sequence of shRNA targeting PTBP1):

[0644] GCCCAUCUACAUCCAGUUCUC

[0645] SEQ ID NO: 41 (Guide sequence of shRNA targeting PTBP1):

[0646] CAGCCCAUCUACAUCCAGUUC

[0647] SEQ ID NO: 42 (ANGPTL3-gRNA1 guide sequence)

[0648] CAUGAAAAAACUUGAGAGUUGCUGGGUCUGA

[0649] SEQ ID NO: 43 (ANGPTL3-gRNA2 guide sequence)

[0650] GAAUUAGUUAGUUAGUUGCUCUUCUAAAU

[0651] SEQ ID NO: 44 (ANGPTL3-gRNA3 guide sequence)

[0652] CGAUGUUGAAUUAAUUGUCCAUGGACUACCU

[0653] SEQ ID NO: 45 (ANGPTL3-gRNA4 guide sequence)

[0654] GAUAGAGAAAUUUCUGUGGGUUCUUGAAUA

[0655] SEQ ID NO: 46 (ANGPTL3-gRNA5 guide sequence)

[0656] CUGGAGAAGGUCUUUGAUGCUAUUAUCUUG

[0657] SEQ ID NO: 47 (ANGPTL3-gRNA6 guide sequence)

[0658] CACUAUGGAGUAUAUCUUCUCUAGGCCCAA

[0659] SEQ ID NO: 48 (ANGPTL3-gRNA7 guide sequence)

[0660] CCACACUCAUCAUGCCCACCACCAGCCUCCU

[0661] SEQ ID NO: 49 (ANGPTL3-gRNA8 guide sequence)

[0662] GACCAUCUAAAAUUGAUUCCCACAUCACAA

[0663] SEQ ID NO: 50 (ANGPTL3 upstream primer):

[0664] CCAGAACACCCAGAAGTAACT

[0665] SEQ ID NO: 51 (ANGPTL3 downstream primer):

[0666] TCTGTGGGTTCTTGAATACTAGTC

[0667] SEQ ID NO: 52 (Lv-ANGPTL3-T2a-GFP vector):

[0668]

[0669] NLS:

[0670] NLS1:SPKKKRKVEAS (SEQ ID NO: 53)

[0671] NLS2: GPKKKRKVAAA (SEQ ID NO: 54)

[0672] NLS3: PKKKRKV (SEQ ID NO: 55)

[0673] NLS4: KRPAATKKA GQA KKKK (SEQ ID NO: 56)

[0674] NLS5:PAAKRVKLD (SEQ ID NO: 57)

[0675] NLS6: RQRRNELKRSP (SEQ ID NO: 58)

[0676] NLS7: NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 59)

[0677] NLS8:RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 60)

[0678] NLS9:VSRKRPRP (SEQ ID NO: 61)

[0679] NLS10:PPKKARED (SEQ ID NO: 62)

[0680] NLS11:POPKKKPL (SEQ ID NO: 63)

[0681] NLS12: SALIKKKKKMAP (SEQ ID NO: 64)

[0682] NLS13:DRLRR (SEQ ID NO: 65)

[0683] NLS14:PKQKKRK (SEQ ID NO: 66)

[0684] NLS15:RKLKKKIKKL (SEQ ID NO: 67)

[0685] NLS16:REKKKFLKRR (SEQ ID NO: 68)

[0686] NLS17:KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 69)

[0687] NLS18:RKCLQAGMNLEARKTKK (SEQ ID NO: 70)

[0688] NLS19:PAAKKKKLD (SEQ ID NO: 71)

[0689] SEQ ID NO: 72 (C13-2-VEGFA vector of Example 8):

[0690]

[0691] SEQ ID NO: 73 (Guide sequence of gRNA targeting VEGFA):

[0692] TGGGTGCAGCCTGGGACCACTTGGCATGG

[0693] SEQ ID NO: 74 (Sequence before R210A+H215A mutation):

[0694] AGAAACGCCACCGCCCAC (SEQ ID NO: 74)

[0695] SEQ ID NO: 75 (sequence after R210A+H215A mutation):

[0696] GCAAACGCCACCGCCGCC (SEQ ID NO: 75)

[0697] SEQ ID NO: 76 (sequence before R750A+H755A mutation):

[0698] AGAAAGACCAAGAGACAC

[0699] SEQ ID NO: 77 (sequence after R750A+H755A mutation):

[0700] GCAAAGACCAAGAGAGCC

[0701] SEQ ID NO: 78 (sequence before R785A+H790A mutation):

[0702] AGAAACGACGTGGAGCAC

[0703] SEQ ID NO: 79 (sequence after R785A+H790A mutation):

[0704] GCAAACGACGTGGAGGCC

[0705] SEQ ID NO: 80 (designed DR sequence, i.e., DR2):

[0706] CCGCACAGTCCCTACAGGTTTGTAGAGTCATCTTCC

[0707] SEQ ID NO: 81 (designed DR sequence, i.e., DR2rc):

[0708] GGAAGATGACTCTACAAACCTGTAGGGACTGTGCGG

[0709] SEQ ID NO: 82 (designed DR sequence, i.e., DR3):

[0710] GGTGTACAGGGTGCCTGGATTTGACAGGGTTACAGC

[0711] SEQ ID NO: 83 (designed DR sequence, i.e., DR3rc):

[0712] GCTGTAACCCTGTCAAATCCAGGCACCCTGTACACC

[0713] SEQ ID NO: 84 (designed DR sequence, i.e., DR4):

[0714] GGTGTACAGGGTGCCTAGATTTGACAGGGTTACAGC

[0715] SEQ ID NO: 85 (designed DR sequence, i.e., DR4rc):

[0716] GCTGTAACCCTGTCAAATCTAGGCACCCTGTACACC

[0717] SEQ ID NO: 86 (designed DR sequence, i.e., DR-hf1):

[0718] GGAAGAACTCTACAAACCTGTAGGGTTCTGAGAC

[0719] SEQ ID NO: 87 (designed DR sequence, i.e., DR-hf2):

[0720] GGAAGATAACTCTACAAACCTGTAGAGTTCTGAGAC

[0721] SEQ ID NO: 88 (qPCR primer used for detection of VEGFA):

[0722] ACCTCCACCATGCCAAGTGG

[0723] SEQ ID NO: 89 (qPCR primer used for detection of VEGFA):

[0724] CAGGGTCTCGATTGGATGGC (SEQ ID NO: 89)

[0725] SEQ ID NO: 90 (Guide sequence targeting GFP):

[0726] tgccgttcttctgcttgtcggccatgatat

[0727] SEQ ID NO: 91 (Plasmid vector CasRx-3NLS-PTBP1 targeting PTBP1):

[0728]

[0729] SEQ ID NO: 92 (Plasmid vector PspCas13b-3NLS-PTBP1 targeting PTBP1):

[0730]

[0731] SEQ ID NO: 93 (Plasmid vector Cas13X.1-3NLS-PTBP1 targeting PTBP1):

[0732]

[0733] SEQ ID NO: 94 (Plasmid vector Cas13Y.1-3NLS-PTBP1 targeting PTBP1):

[0734]

[0735] SEQ ID NO: 95 (Guide sequence targeting EGFP in single base editing experiments):

[0736] tgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccagcttgtgccc

[0737] SEQ ID NO: 96 (dC13-2-BsaI vector sequence in single base editing experiment):

[0738]

[0739] SEQ ID NO: 97 (dC13-2-EGFP vector sequence in single base editing experiment):

[0740]

[0741] SEQ ID NO: 98 (dC13-2-ADAR-EGFP vector sequence in single base editing experiment):

[0742]

[0743] SEQ ID NO: 99 (dC13-2-A(EAAAK)3A-ADAR-EGFP vector sequence in single base editing experiment):

[0744]

[0745] SEQ ID NO: 100 (dC13-2-(GGGGS)3-ADAR-EGFP vector sequence in single base editing experiments):

[0746]

[0747] SEQ ID NO: 101 (pAAV-CMV-EGFP vector sequence in single base editing experiments):

[0748]

[0749] SEQ ID NO: 102 (PLKO-PURO-PspGRNA-EGFP vector sequence in single base editing experiment):

[0750]

[0751] SEQ ID NO: 103 (identifying primer sequence in single base editing experiment):

[0752] agggcgaggagctgtt (SEQ ID NO: 103),

[0753] SEQ ID NO: 104 (identifying primer sequence in single base editing experiment):

[0754] gtacagctcgtccatgccg (SEQ ID NO: 104).

Claims

1. A Cas13 protein, characterized in that the amino acid sequence has at least 90% sequence identity compared to SEQ ID NO:

1.

2. The Cas13 protein of claim 1, characterized in that the Cas13 protein can form a CRISPR complex with a guide polynucleotide that includes a direct repeat sequence linked to a guide sequence engineered to induce sequence-specific binding of the CRISPR complex to a target RNA.

3. The Cas13 protein of claim 1, characterized in that the Cas13 protein contains one or more mutations in the catalytic domain and has reduced RNA cleavage activity.

4. The Cas13 protein of claim 1, characterized in that the Cas13 protein contains one or more mutations in one or two HEPN domains and substantially lacks RNA cleavage activity.

5. The Cas13 protein of claim 1, characterized in that it has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to SEQ ID NO:

1.

6. The Cas13 protein comprises at least one mutation at a position corresponding to amino acid residues 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 a reference protein whose amino acid sequence is set forth in SEQ ID NO:1; and / or The Cas13 protein is a protein having an amino acid sequence corresponding to the following amino acid residues of a comparative protein 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+N6 8, any one or more mutations at 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; Preferably, the mutations are 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, R2 90A+R314A, R47A+R290A+R314A, R308A+N68A, N394A+N68A, N87A+N68A, R308A+N 265A, N394A+N265A, N87A+N265A, R308A+N68A+N265A, N87A+N68A+N265A, T7S, A16S, S260E, A263K, M266I, 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.

7. The Cas13 protein includes any one or more mutations at positions corresponding to the following amino acid residues of a comparative protein whose amino acid sequence is set forth in SEQ ID NO:1: N34, R64, N68, N265, R276, R294, N299, R314, R47+R290, R47+R314, R290+R314, R47+R290+R314, N394+N265, N87+N265, A263, M266, N274, F288, V305, I311, D313, H324, T360, E365, A373, M380, D402, and D411; 2. The Cas13 protein of claim 1, wherein the mutations are N34A, R64A, N68A, N265A, R276A, R294A, N299A, R314A, R47A+R290A, R47A+R314A, R290A+R314A, R47A+R290A+R314A, N394A+N265A, N87A+N265A, A263K, M266I, N274K, F288Y, V305K, I311M, D313E, H324Y, T360S, E365D, A373E, M380K, D402L and D411E.

8. The Cas13 protein according to any one of claims 1 to 7, characterized in that the Cas13 protein is derived from a species comprising a genome having an average nucleotide identity (ANI) of ≥ 95% with the genome shown in the CNGB database (China National Genome Bank) under the number CNA0009596.

9. A fusion protein comprising a Cas13 protein or a functional fragment thereof according to any one of claims 1 to 8 fused to a protein domain and / or a polypeptide tag, which is selectable and does not alter the original function of the Cas13 protein and / or functional fragment after being fused.

10. The fusion protein of claim 9, wherein the Cas13 protein or a functional fragment thereof is fused to a nuclear localization signal (NLS).

11. The fusion protein according to claim 9, characterized in that the Cas13 protein or a functional fragment thereof is fused to a nuclear export signal (NES).

12. The fusion protein of claim 9, wherein the Cas13 protein or a functional fragment thereof is covalently linked to a protein domain.

13. The fusion protein according to claim 9, characterized in that the Cas13 protein or a functional fragment thereof is fused to any one or more protein domains and / or polypeptide tags selected from a cytosine deaminase domain, an adenosine deaminase domain, a translation activation domain, a translation 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.

14. The fusion protein according to any one of claims 9 to 13, characterized in that the structure of the fusion protein is NLS-Cas13 protein-SV40 NLS-nucleoplasmin NLS.

15. (i) comprising a direct repeat sequence having at least 50% sequence identity to any one of SEQ ID NO: 3 and SEQ ID NO: 80 to 87, the direct repeat sequence being linked to a guide sequence (ii) engineered to hybridize to a target RNA, the guide polynucleotide forming a CRISPR complex with a Cas13 protein and capable of inducing sequence-specific binding of the CRISPR complex to the target RNA, preferably the Cas13 protein is Cas13a, Cas13b, Cas13c or Cas13d, more preferably the Cas13 protein has at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; Alternatively, the base at position 26 of the direct repeat corresponding to SEQ ID NO:3 is A; Optionally, the direct repeat sequence is １ ACTCTACAAAAACCTGTAGN ２ G.N. ３ N ４ N ５ N ６ N ７ N ８ N ９ N １０ N １１ where N １ and N ３ ~N １１ is arbitrarily selected from A, C, G, and T; N ２ is arbitrarily selected from A and G; In a further alternative, the direct repeat sequence is １２ ACTCTACAAAAACCTGTAGN １３ G.N. １４ N １５ N １６ N １７ N １８ N １９ N ２０ N ２１ N ２２ where N １２ , N １３ , N １９ and N ２１ is arbitrarily selected from A and G; N １４ is arbitrarily selected from A and T; N １５ and N １６ is arbitrarily selected from C and T; N １７ and N １８ is arbitrarily selected from G and T; N ２０ and N ２２ is arbitrarily selected from C and G.

16. 16. The guide polynucleotide of claim 15, wherein the direct repeat sequence has at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 3, 81, 82, 84, and 87.

17. 16. The guide polynucleotide of claim 15, wherein the guide sequence is located at the 3' end of the direct repeat sequence.

18. The guide polynucleotide of claim 15, wherein the guide sequence comprises 15 to 35 nucleotides.

19. 16. The guide polynucleotide of claim 15, wherein the guide sequence hybridizes to the target RNA with no more than one nucleotide mismatch.

20. The guide polynucleotide of claim 15, wherein the direct repeat sequence comprises 25 to 40 nucleotides.

21. The guide polynucleotide of claim 15, further comprising an aptamer sequence.

22. 22. The guide polynucleotide of claim 21, wherein the aptamer sequence is inserted into a loop of the guide polynucleotide.

23. 22. The guide polynucleotide of claim 21, wherein the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

24. 16. The guide polynucleotide of claim 15, wherein the guide polynucleotide comprises modified nucleotides.

25. 25. The guide polynucleotide of claim 24, wherein the modification comprises a 2'-O-methyl, a 2'-O-methyl-3'-thiophosphate, or a 2'-O-methyl-3'-thio PACE modification.

26. The guide polynucleotide according to any one of claims 15 to 25, characterized in that the target RNA is located in the nucleus of a eukaryotic cell.

27. The guide polynucleotide according to any one of claims 15 to 26, characterized in that the target RNA is arbitrarily selected from TTR RNA, SOD1 RNA, PCSK9 RNA, VEGFA RNA, VEGFR1 RNA, PTBP1 RNA, AQp1 RNA or ANGPTL3 RNA, preferably the target RNA is arbitrarily selected from PTBP1 RNA, AQp1 RNA or ANGPTL3 RNA.

28. The guide sequence is arbitrarily selected from the sequences shown in SEQ ID NOs: 5-6, 42-49, preferably the guide sequence is arbitrarily selected from the sequences shown in SEQ ID NOs: 5-6, 43, 45-47. The guide polynucleotide according to claim 27.

29. A Cas13 protein according to any one of claims 1 to 8 or a fusion protein according to any one of claims 9 to 14, or a nucleic acid encoding said Cas13 protein or said fusion protein, and A guide polynucleotide or a nucleic acid encoding said guide polynucleotide, said guide polynucleotide comprising a direct repeat sequence linked to a guide sequence engineered to hybridize to a target RNA; The guide polynucleotide forms a CRISPR complex with the Cas13 protein or fusion protein and is capable of inducing sequence-specific binding of the CRISPR complex to a target RNA. A CRISPR-Cas13 system.

30. The target RNA is optionally selected from TTR RNA, SOD1 RNA, PCSK9 RNA, VEGFA RNA, VEGFR1 RNA, PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA, and is selectable, and the target RNA is optionally selected from PTBP1 RNA, AQp1 RNA, or ANGPTL3 RNA. The CRISPR-Cas13 system of claim 29.

31. The guide sequence is arbitrarily selected from the sequences shown in SEQ ID NOs: 5 to 6, 42 to 49, and preferably, the guide sequence is arbitrarily selected from the sequences shown in SEQ ID NOs: 5 to 6, 43, and 45 to 47. The CRISPR-Cas13 system according to claim 30.

32. The CRISPR-Cas13 system according to any one of claims 29 to 31, characterized in that the direct repeat sequence has at least 70% sequence identity compared to any one of SEQ ID NO: 3 and SEQ ID NO: 80 to 87.

33. A CRISPR-Cas13 system comprising a guide polynucleotide according to any one of claims 15 to 28 or a nucleic acid encoding the same, and a Cas13 protein or a nucleic acid encoding the same.

34. A vector system comprising the CRISPR-Cas13 system of any one of claims 29 to 33, comprising one or more vectors comprising a polynucleotide sequence encoding the Cas13 protein or fusion protein and a polynucleotide sequence encoding the guide polynucleotide.

35. An adeno-associated virus vector comprising a CRISPR-Cas13 system according to any one of claims 29 to 33, comprising DNA encoding the Cas13 protein or fusion protein and a guide polynucleotide.

36. A lipid nanoparticle comprising the CRISPR-Cas13 system of any one of claims 29 to 33, comprising the guide polynucleotide and an mRNA encoding the Cas13 protein or fusion protein.

37. 34. A lentiviral vector comprising the CRISPR-Cas13 system of any one of claims 29 to 33, comprising the guide polynucleotide and an mRNA encoding the Cas13 protein or fusion protein, preferably pseudotyped with an envelope protein and selectable, wherein the mRNA encoding the Cas13 protein or fusion protein is linked to an aptamer sequence.

38. A ribonucleoprotein complex formed from the guide polynucleotide and a Cas13 protein or fusion protein, comprising the CRISPR-Cas13 system of any one of claims 29 to 33.

39. A virus-like particle comprising a ribonucleoprotein complex formed from the guide polynucleotide and a Cas13 protein or fusion protein, optionally wherein the Cas13 protein or fusion protein is fused to a gag protein, comprising the CRISPR-Cas13 system of any one of claims 29 to 33.

40. 34. A eukaryotic cell comprising a Cas13 protein according to any one of claims 1 to 8, a fusion protein according to any one of claims 9 to 14, a guide polynucleotide according to any one of claims 15 to 28, or a CRISPR-Cas13 system according to any one of claims 29 to 33, and which is selectable and is a mammalian cell.

41. A pharmaceutical composition comprising a Cas13 protein according to any one of claims 1 to 8, a fusion protein according to any one of claims 9 to 14, a guide polynucleotide according to any one of claims 15 to 28, or a CRISPR-Cas13 system according to any one of claims 29 to 33.

42. An in vitro composition comprising the CRISPR-Cas13 system according to any one of claims 29 to 32 and a labeled detector RNA that cannot hybridize to the guide polynucleotide.

43. An isolated nucleic acid, characterized in that it encodes a Cas13 protein according to any one of claims 1 to 8 or a fusion protein according to any one of claims 9 to 14.

44. An isolated nucleic acid, characterized in that it encodes a guide polynucleotide according to any one of claims 15 to 28.

45. Use of a Cas13 protein according to any one of claims 1 to 8, a fusion protein according to any one of claims 9 to 14, a guide polynucleotide according to any one of claims 15 to 28, a CRISPR-Cas13 system according to any one of claims 29 to 33, or an isolated nucleic acid according to claim 42 or claim 43 in the detection of a target RNA in a nucleic acid sample suspected of containing the target RNA, or in the preparation of a reagent for detecting a target RNA in a nucleic acid sample suspected of containing the target RNA.

46. 23. Use of the Cas13 protein of any one of claims 1 to 8, the fusion protein of any one of claims 9 to 14, the guide polynucleotide of any one of claims 15 to 28, the CRISPR-Cas13 system of any one of claims 29 to 33, or the isolated nucleic acid of claim 42 or claim 43 in any one of the following schemes: cleaving one or more target RNA molecules or nicking one or more target RNA molecules, activating or upregulating one or more target RNA molecules, activating or inhibiting translation of one or more target RNA molecules, inactivating one or more target RNA molecules, visualising, labelling or detecting one or more target RNA molecules, binding to one or more target RNA molecules, transporting one or more target RNA molecules and masking one or more target RNA molecules, or in the preparation of a reagent for achieving any one of the above schemes.

47. A method for diagnosing, treating or preventing a disease or condition associated with a target RNA, comprising administering to a sample from or to a subject in need thereof a Cas13 protein according to any one of claims 1 to 8, a fusion protein according to any one of claims 9 to 14, a guide polynucleotide according to any one of claims 15 to 28, a CRISPR-Cas13 system according to any one of claims 29 to 33, or an isolated nucleic acid according to claim 42 or claim 43.

48. Use of a Cas13 protein according to any one of claims 1 to 8, a fusion protein according to any one of claims 9 to 14, a guide polynucleotide according to any one of claims 15 to 28, a CRISPR-Cas13 system according to any one of claims 29 to 33, or an isolated nucleic acid according to claim 42 or claim 43 in the preparation of a medicament for diagnosing, treating or preventing a disease or condition associated with a target RNA.

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