Tracking and manipulating cellular rna via nuclear delivery of crispr/cas9

Abstract

Cas9 polypeptides which target RNA and methods of using them are provided.

1 .- 20 . (canceled) 
     
     
         21 . A nucleic acid molecule encoding a 2-component RNA targeting system comprising:
 (a) nucleic acid sequence encoding a truncated nuclease-inactive RNA-targeted Cas9 (RCas9) polypeptide, wherein the truncated nuclease-inactive RCas9 polypeptide lacks all or part of its HNH domain compared to a wild-type (WT) Cas9 protein; and   (b) a single guide RNA (sgRNA) sequence comprising:
 (i) on its 5′ end, an RNA sequence that hybridizes to or binds to a target RNA sequence comprising a repeat sequence selected from the group consisting of CUG, CCUG, CAG, and GGGGCC; and 
 (ii) on its 3′ end, an RNA sequence capable of binding to or associating with the truncated nuclease-inactive RCas9 polypeptide, wherein the RNA sequence capable of binding to or associating with the truncated nuclease-inactive RCas9 polypeptide comprises or is derived from a wild type guide RNA of an archaeal or a bacterial organism from which the truncated nuclease-inactive RCas9 is derived; and 
   wherein the 2-component RNA targeting system recognizes and alters the target RNA in a cell in the absence of a PAMmer.   
     
     
         22 . The nucleic acid molecule of  claim 21 , wherein the truncated nuclease-inactive RCas9 is missing residues 775-909 from full-length Cas9 protein. 
     
     
         23 . The nucleic acid molecule of  claim 21 , wherein the sequences of a) and b) are in a single vector. 
     
     
         24 . The nucleic acid molecule of  claim 23 , wherein the sequences of a) and b) are a size of less than about 4.5 kb, or a size of less than about 4.7 kb. 
     
     
         25 . The nucleic acid molecule of  claim 21 , wherein the nuclease-inactive RCas9 polypeptide and the RNA sequence capable of binding to or associating with the nuclease-inactive RCas9 polypeptide comprises or is derived from  Haloferax mediteranii, Mycobacterium tuberculosis, Francisella tularensis  subsp.  novicida, Pasteurella multocida, Neisseria meningitidis, Campylobacter jejune, Streptococcus thermophilus, Campylobacter lari, Mycoplasma gallisepticum  str.  F, Nitratifractor salsuginis  str. DSM 16511,  Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria cinerea, Gluconacetobacter diazotrophicus, Azospirillum  B510,  Sphaerochaeta globus  str. Buddy,  Flavobacterium columnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasma mobile, Lactobacillus farciminis, Streptococcus pasteurianus, Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filifactor alocis, Treponema denticola, Legionella pneumophila  str. Paris,  Sutterella wadsworthensis, Corynebacter diphtheriae, Streptococcus aureus, Francisella novicida, Francisella novicida , and an  Natronobacterium gregoryi.    
     
     
         26 . The nucleic acid molecule of  claim 21 , wherein the 5′ end of the sgRNA is between about 15 to 25 nucleotides in length, and wherein the RNA sequence of the RCas9 polypeptide scaffold is between about 85 and 100 nucleotides in length. 
     
     
         27 . The nucleic acid molecule of  claim 21 , wherein the truncated nuclease-inactive RCas9 polypeptide is linked to an effector polypeptide, a targeting agent, an enzyme, a detectable moiety, or a combination thereof. 
     
     
         28 . The nucleic acid molecule of  claim 27 , wherein the effector polypeptide comprises an RNA modifying polypeptide. 
     
     
         29 . nucleic acid molecule of  claim 27 , wherein the targeting agent comprises:
 (a) a cytoplasmic polyadenylation element binding protein (CPEB), a zinc finger binding protein (ZBP), TIA-1, PSF, a DNA-binding domain (DBD) of PSF, fragile X mental retardation protein (FMRP), IGF-II mRNA-binding protein (IMP)-1 (IMP 1), IMP2, IMP3, a cytoskeleton binding protein, a transmembrane protein, or   (b) an engineered protein comprising a combination of domains of the proteins of (a).   
     
     
         30 . The nucleic acid molecule of  claim 28 , wherein the RNA modifying polypeptide comprises a splicing factor or an RNA splicing domain, a RBFOX2 domain-containing protein, a protein known to influence RNA splicing, an RNA cleaving domain or a PTN domain-containing protein. 
     
     
         31 . The nucleic acid molecule of  claim 30 , wherein the RNA cleaving domain comprises an endonuclease. 
     
     
         32 . The nucleic acid molecule of  claim 21 , wherein the truncated nuclease-inactive RCas9 lacks all of its HNH domain. 
     
     
         33 . The nucleic acid molecule of  claim 21 , wherein the sequence of a) comprises a promoter. 
     
     
         34 . The nucleic acid molecule of  claim 21 , wherein the sequence of b) comprises a U6 polymerase III promoter. 
     
     
         35 . the nucleic acid molecule of  claim 21 , wherein the sequence of b) comprises one or more point mutations that remove a transcription termination sequence. 
     
     
         36 . A method of recognizing and altering target RNA in a cell comprising contacting the cell with the nucleic acid molecule of  claim 21 , whereby the nucleic acid molecule encodes an RNA targeting system which recognizes and alters target RNA in the cell in the absence of a PAMmer. 
     
     
         37 . A method of treating a disease, condition, or infection associated with an RNA microsatellite repeat expansion in a patient, comprising administering the nucleic acid molecule of  claim 21  to the patient, whereby the nucleic acid molecule encodes an RNA targeting system which recognizes and alters target RNA corresponding to the RNA microsatellite repeat expansion, and wherein the disease, condition, or infection is selected from the group consisting of myotonic dystrophy, Huntington's disease, familial ALS, cancer, spinal muscular atrophy, spinocerebellar ataxia, Fragile X-associated tremor/ataxia syndrome, Spinal-bulbar muscular dystrophy, Oculopharyngeal muscular dystrophy, Fragile X syndrome, a viral infection, a bacterial infection, a Herpesviridae or herpes simplex virus infection, a human immunodeficiency virus infection, an Epstein Barr virus infection, a hepatitis virus infection, a Hepatitis A infection, a Hepatitis B infection, a Hepatitis C infection, a Hepatitis D infection, a Hepatitis E infection, a Zika virus infection, an enterovirus infection, a Human Papillomavirus (HPV) infection, an influenza virus infection, a Marburg virus infection, an Ebola virus infection, a Mumps virus infection, a cytomegalovirus infection, a rotavirus infection, a Rubella virus infection, a Varicella zoster virus infection, a severe acute respiratory syndrome (SARS) infection, a coronavirus infection, a Paramyxoviridae or measles virus infection, a West Nile virus infection, a Yellow fever virus infection, or a Dengue fever virus infection. 
     
     
         38 . A cell or tissue comprising the nucleic acid molecule of  claim 21 . 
     
     
         39 . A nucleic acid molecule encoding a 2-component RNA targeting system comprising:
 (A) nucleic acid sequence encoding a  Campylobacter jejune  RNA-targeted Cas9 (Rcas9) polypeptide; and   (B) a single guide RNA (sgRNA) sequence comprising:
 (i) on its 5′ end, an RNA sequence that hybridizes to or binds to a target RNA sequence comprising a repeat sequence selected from the group consisting of CUG, CCUG, CAG, and GGGGCC; and 
 (ii) on its 3′ end, an RNA sequence capable of binding to or associating with the  Campylobacter jejune  Rcas9 polypeptide, wherein the RNA sequence capable of binding to or associating with the  Campylobacter jejune  Rcas9 polypeptide comprises or is derived from a wild type guide RNA of a  Campylobacter jejune  organism from which the  Campylobacter jejune  RCas9 is derived; 
   wherein the sequences of (a) and (b) are in a single vector; and wherein the 2-component RNA targeting system recognizes and alters the target RNA in the absence of a PAMmer.   
     
     
         40 . A nucleic acid molecule encoding a 2-component RNA targeting system comprising:
 (a) nucleic acid sequence encoding an RNA-targeted Cas9 (Rcas9) polypeptide; and   (b) a single guide RNA (sgRNA) sequence comprising:
 (1) on its 5′ end, an RNA sequence that hybridizes to or binds to a target RNA sequence comprising a repeat sequence selected from the group consisting of CUG, CCUG, CAG, and GGGGCC; and 
 (2) on its 3′ end, an RNA sequence capable of binding to or associating with the RCas9 polypeptide, wherein the RNA sequence capable of binding to or associating with the RCas9 polypeptide comprises or is derived from a wild type guide RNA of an archaeal or a bacterial organism from which the RCas9 is derived; 
   wherein the sequences of (a) and (b) are in a single vector, wherein the sequences of (a) and (b) are a size of less than about 4.5 kb, or a size of less than about 4.7 kb, and wherein the 2-component RNA targeting system recognizes and alters the target RNA in a cell in the absence of a PAMmer.