Rna targeting methods and compositions

Abstract

Provided herein are CRISPR/Cas methods and compositions for targeting RNA molecules, which can be used to detect, edit, or modify a target RNA.

We claim: 
     
         1 . A method of splicing or perturbing the splicing of one or more target RNA molecules, comprising:
 contacting one or more target RNA molecules with a non-naturally occurring or engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system comprising:   at least one isolated protein, or a nucleic acid molecule encoding the at least one isolated protein, wherein the at least one isolated protein comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 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, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 138, 147, 149, 153, 155, 158, 160, 162, 164, 166, 168, 170, 175, 177, 179, 181, 183, 185, 187, 189, 194, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 310, 311, 312, or 313; and   at least one gRNA that hybridizes with the one or more target RNA molecules, or at least one nucleic acid molecule encoding the gRNA,   whereby the isolated protein forms a complex with the gRNA, wherein the gRNA directs the complex to the one or more target RNA molecules thereby splicing or perturbing the splicing of the one or more target RNA molecules.   
     
     
         2 . The method of  claim 1 , wherein the at least one gRNA comprises one or more direct repeat (DR) sequences comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, 148, 150, 151, 152, 154, 156, 157, 159, 161, 163, 165, 167, 169, 176, 178, 180, 182, 184, 186, 188, 190, 191, 192, 193, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, or 254, or a truncated version thereof. 
     
     
         3 . The method of  claim 1 , wherein the one or more target RNA molecules is a non-coding RNA. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid molecule encoding the at least one isolated protein comprises:
 at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 124, 125, 126, 127, 128, 139, 140 or 141;   at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 142, 143, 144, or 145; or   encodes a protein sequence comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 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, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 138, 147, 149, 153, 155, 158, 160, 162, 164, 166, 168, 170, 175, 177, 179, 181, 183, 185, 187, 189, 194, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 310, 311, 312, or 313.   
     
     
         5 . The method of  claim 1 , wherein the method treats a disease, and wherein the one or more target RNA molecules is associated with a disease. 
     
     
         6 . The method of  claim 1 , wherein the at least one isolated protein further comprises:
 a subcellular localization signal;   a mutation in at least one native HEPN domain; or   both a subcellular localization signal and a mutation in at least one native HEPN domain.   
     
     
         7 . The method of  claim 6 , wherein the at least one isolated protein further comprises a mutated HEPN1 domain or a mutated HEPN2 domain. 
     
     
         8 . The method of  claim 6 , wherein the mutation in the at least one native HEPN domain comprises a mutation in a sequence comprising RXXXXH, wherein the R of the sequence is mutated, the H of the sequence is mutated, or both the R and the H of the sequence are mutated. 
     
     
         9 . The method of  claim 1 , wherein the nucleic acid molecule encoding the at least one isolated protein is part of a recombinant vector. 
     
     
         10 . The method of  claim 9 , wherein:
 the recombinant vector comprises a plasmid or viral vector;   the isolated nucleic acid molecule is operably linked to a promoter; and/or   the recombinant vector further comprises at least one gRNA.   
     
     
         11 . The method of  claim 1 , wherein contacting the one or more target RNA molecules with the non-naturally occurring or engineered CRISPR-Cas system comprises introducing into a cell containing the one or more target RNA molecules the non-naturally occurring or engineered CRISPR-Cas system. 
     
     
         12 . The method of  claim 11  wherein the CRISPR-Cas system is introduced into the cell using endocytosis, a liposome, a particle, an exosome, a microvesicle, a gene gun, electroporation, a virus, or combinations thereof. 
     
     
         13 . The method of  claim 11 , wherein the cell is a eukaryotic cell. 
     
     
         14 . The method of  claim 11 , wherein the cell is a non-bacterial cell. 
     
     
         15 . The method of  claim 1 , wherein the method is performed ex vivo, in vitro, or in a cell-free system. 
     
     
         16 . The method of  claim 1 , wherein the gRNA further comprises one or more spacer sequences specific for the one or more target RNA molecules, a nucleic acid aptamer, or both. 
     
     
         17 . The method of  claim 1 , wherein the at least one isolated protein and the one or more target RNA molecules are part of a ribonucleoprotein (RNP) complex. 
     
     
         18 . The method of  claim 1 , wherein the at least one isolated protein is catalytically inactive for ribonuclease activity.