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

Abstract

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

What is claimed is: 
     
         1 . An engineered nucleoprotein complex comprising:
 (a) a Cas9 polypeptide, wherein the Cas9 polypeptide:   (i) lacks all or part of an HNH domain, all or part of at least one RuvC nuclease domain, all or part of a Cas9 polypeptide DNase active site, all or part of a ββα-metal fold comprising a Cas9 polypeptide active site, or combinations thereof as compared to a corresponding wild type (WT) Cas9 polypeptide;   (ii) lacks DNase and/or DNA cleaving capability or activity, or nickase activity, wherein the DNase or DNA cleaving capability or activity is removed by mutation or removal of all or part of an HNH domain, all or part of at least one RuvC nuclease domain of Cas9, all or part of a Cas9 polypeptide DNase active site or a ββα-metal fold comprising a Cas9 polypeptide active site, or combinations thereof; and   (iii) has a reduced polypeptide size that permits packaging of the Cas9-coding nucleotide in a viral or other delivery vector;   and   (b) a recombinant or synthetic single guide RNA (sgRNA) which is engineered or designed to comprise:
 (1) on its 5′ end, an RNA sequence that hybridizes to or binds to a target RNA; and 
 (2) on its 3′ end:
 (i) an RNA sequence capable of binding to or associating with the Cas9 polypeptide and/or a Cas9 polypeptide binding scaffold sequence, or 
 (ii) a linker that binds or covalently or non-covalently links the 5′ RNA-hybridizing or binding end of the sgRNA with the Cas9 polypeptide, 
 
   wherein the sgRNA 3′ end or “scaffold sequence” comprises all or part of, or is derived from, the wild type (WT) cognate guide nucleic acid of any one of the archaeal or bacterial Cas9 polypeptide of (a) (iv);   wherein the nucleoprotein complex does not comprise a PAMmer oligonucleotide and does not cleave or nick DNA or genomic loci.   
     
     
         2 . The engineered nucleoprotein complex of  claim 1 , wherein said Cas9 polypeptide is noncovalently associated with said effector polypeptide, detectable moiety, or RNA modifying polypeptide. 
     
     
         3 . The engineered nucleoprotein complex of  claim 1 , wherein said Cas9 polypeptide is fused to or covalently linked to said effector polypeptide, detectable moiety, or RNA modifying polypeptide. 
     
     
         4 . The engineered nucleoprotein complex of  claim 1 , wherein said RNA modifying polypeptide comprises a splicing factor or an RNA splicing domain, RBFOX2 domain-containing protein, a protein known to influence RNA splicing, an RNA cleaving domain (endonuclease), or a PIN domain-containing protein. 
     
     
         5 . The engineered nucleoprotein complex of  claim 1 , wherein said single guide RNA (sgRNA) carries extensions of, or comprises, secondary RNA structures in the 3′ end scaffold sequence, wherein said single guide RNA (sgRNA) comprises one or more point mutations that improve expression levels of the single guide RNAs via removal of partial or full transcription termination sequences or sequences that destabilize sgRNAs after transcription via action of trans-acting nucleases by at least about 5%, 10%, 15% or more, or
 wherein said single guide RNA comprises an alteration at the 5′ end which stabilizes said single guide RNA against degradation, or wherein said single guide RNA comprises an alteration at the 5′ end which improves RNA targeting, or 
 wherein said single guide RNA comprises one or more methylphosphonate, thiophosponoaceteate, or phosphorothioate linkages that reduce nuclease activity on the target RNA. 
 
     
     
         6 . The engineered nucleoprotein complex of  claim 5 , wherein said single guide RNA comprises sufficient sequence antisense to the target RNA to allow it to hybridize to at least a portion of the target RNA. 
     
     
         7 . The engineered nucleoprotein complex of  claim 5 , wherein said portion is at least about 5%, 10%, 15% or more of the target RNA. 
     
     
         8 . The engineered nucleoprotein complex of  claim 1 , further comprising a CRISPR-targeting RNA (crRNA) and a trans-activating cRNA (tracrRNA), wherein said Cas9 polypeptide is complexed with or linked to, or covalently or non-covalently associated with, the CRISPR-targeting RNA (crRNA) in combination with the trans-activating cRNA (tracrRNA). 
     
     
         9 . The engineered nucleoprotein complex of  claim 1 , wherein said RNA modifying polypeptide is further complexed with, or is linked to covalently or non-covalently, an antisense oligonucleotide. 
     
     
         10 . The engineered nucleoprotein complex of any one of  claim 9 , wherein said antisense oligonucleotide further comprises a PAMmer oligonucleotide. 
     
     
         11 . The engineered nucleoprotein complex of  claim 1 , wherein said Cas9 polypeptide comprises a nuclear localization signal or a linker peptide. 
     
     
         12 . The engineered nucleoprotein complex of  claim 1 , wherein said Cas9 polypeptide is, comprises or is derived from an archaeal or bacterial Cas9 polypeptide selected from the group consisting of:  Haloferax mediteranii, Mycobacterium tuberculosis, Francisella tularensis  subsp.  novicida, Pasteurella multocida, Neisseria meningitidis, Campylobacter jejune, Streptococcus thermophilus  LMD-9 CRISPR 3,  Campylobacter lari  CF89-12,  Mycoplasma gallisepticum  str. F,  Nitratifractor salsuginis  str. DSM 16511 , Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria cinerea , a  Gluconacetobacter diazotrophicus , an  Azospirillum  B510, a  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 diphtherias, Streptococcus aureus, Francisella novicida, Francisella novicida  Cpf1, and an  Natronobacterium gregoryi  Argonaute polypeptide modified or repurposed to target RNA. 
     
     
         13 . The engineered nucleoprotein complex of  claim 1 , wherein said target RNA comprises a repeat sequence, and the 5′ end RNA sequence that recognizes by hybridization (that hybridizes to or binds to) the target RNA comprises a sequence capable of hybridizing to, or is complementary to, the repeat sequence. 
     
     
         14 . The engineered nucleoprotein complex of  claim 13 , wherein said target RNA is associated with a disease, condition, or infection. 
     
     
         15 . A method of treating or ameliorating a disease, condition, or infection in a mammalian or a human subject comprising administering to said mammalian or human subject:
 (a) engineered nucleoprotein complex of  claim 1 , wherein said Cas9 polypeptide is adapted to be associated with, or that binds to or is covalently or non-covalently linked to, an effector polypeptide; or   (b) a nucleic acid, nucleic acids, vector or vectors, encoding the engineered nucleoprotein complex of  claim 1 , wherein said Cas9 polypeptide is adapted to be associated with, or that binds to or is covalently or non-covalently linked to, an effector polypeptide, and wherein the nucleic acid is, or nucleic acids are, expressed intracellularly and express the engineered nucleoprotein complex,   thereby modifying an RNA in a cell and treating or ameliorating the disease, condition, or infection.   
     
     
         16 . The method of  claim 15 , wherein the nucleic acid or nucleic acids encoding one or all of the components of the engineered nucleoprotein complex is/are carried by or is/are contained in a single vector, or each component (the Cas9 polypeptide, the sgRNA, the effector polypeptide, or a combination of components), is carried by or is contained in a separate vector,
 and the vector or vectors are adenovirus vectors, or one or more adeno-associated virus (AAV) vectors, a retrovirus, a herpes simplex virus, a human immunodeficiency virus (HIV), or a synthetic vector.   
     
     
         17 . The method of  claim 15 , wherein the disease or condition is caused by a repeat sequence selected from the group consisting of CTG, CCTG, CAG, GGGGCC, and any combination thereof. 
     
     
         18 . The method of  claim 15 , wherein:
 (a) said disease or condition is caused by or is associated with a RNA microsatellite repeat expansion, or 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, and Fragile X syndrome,   (b) said infection is a viral or bacterial infection, or   (c) said infection is a Herpesviridae or herpes simplex virus, a human immunodeficiency virus, Epstein Barr virus, hepatitis virus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Zika virus, enteroviruses, Human Papillomavirus (HPV), influenza virus, Marburg virus, Ebola virus, Mumps virus, cytomegalovirus, rotavirus, Rubella virus, Varicella zoster virus, severe acute respiratory syndrome (SARS) coronavirus, a Paramyxoviridae or measles virus, West Nile virus, Yellow fever virus, or Dengue fever virus infection.   
     
     
         19 . A pharmaceutical composition comprising
 (a) the engineered nucleoprotein complex of  claim 1 ; or   (b) a nucleic acid, or nucleic acids, vector or vectors, encoding the engineered nucleoprotein complex of  claim 1 ,   and an excipient.   
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein the engineered nucleoprotein complex or the nucleic acid or vector encoding the nucleoprotein complex is carried or contained in a nanoparticle, a particle, a micelle or a liposome or lipoplex, a polymersome, a polyplex or a dendrimer, which optionally can further comprise or express a cell penetrating moiety or peptide.