Rna-targeting fusion protein compositions and methods for use

Abstract

Disclosed are compositions comprising: (a) a sequence comprising a guide RNA (gRNA) that specifically binds a target sequence within an RNA molecule and (b) a sequence encoding a fusion protein, the sequence comprising a sequence encoding a first RNA-binding polypeptide and a sequence encoding a second RNA-binding polypeptide, wherein neither the first RNA-binding polypeptide nor the second RNA-binding polypeptide comprises a significant DNA-nuclease activity, wherein the first RNA-binding polypeptide and the second RNA-binding polypeptide are not identical, and wherein the second RNA-binding polypeptide comprises an RNA-nuclease activity. Methods of making and methods of using compositions of the disclosure are also provided. For example, compositions of the disclosure may be used in the treatment of a disease or disorder in a subject. Exemplary disease or disorders of the disclosure include genetic and epigenetic diseases or disorders.

1 . A composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising a first RNA-binding polypeptide and a second RNA-binding polypeptide, wherein the first RNA-binding polypeptide is a CRISPR/Cas polypeptide or RNA binding domain thereof, wherein the second RNA-binding polypeptide comprises RNA-nuclease activity, wherein the second RNA-binding polypeptide comprises a Zinc Finger CCCH-Type Containing 12A (ZC3H12A) polypeptide, and wherein the ZC3H12A polypeptide comprises SEQ ID NO: 42. 
     
     
         2 . The composition of  claim 1 , wherein the ZC3H12A polypeptide comprises SEQ ID NO: 43. 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The composition of  claim 1 , wherein the CRISPR/Cas polypeptide or RNA binding domain thereof is selected from the group consisting of Cas9, Cpf1, Cas13a, Cas13b, Cas13c and Cas13d, and wherein the CRISPR/Cas polypeptide or portion thereof has native, reduced or null activity. 
     
     
         7 . The composition of  claim 1 , wherein the ZC3H12A polypeptide is capable of binding RNA. 
     
     
         8 . The composition of  claim 7 , wherein the ZC3H12A polypeptide is capable of binding and cleaving RNA. 
     
     
         9 . The composition of  claim 1 , wherein the nucleic acid comprises a promoter. 
     
     
         10 . The composition of  claim 9 , wherein the promoter is a constitutive promoter or a tissue-specific promoter. 
     
     
         11 . The composition of  claim 1 , wherein the nucleic acid further comprises a guide RNA (gRNA) sequence, wherein the gRNA sequence comprises a) a spacer sequence that specifically binds a target sequence within an RNA molecule, and b) a scaffold sequence that specifically binds to the first RNA-binding polypeptide. 
     
     
         12 . The composition of  claim 11 , wherein the spacer sequence comprises a sequence comprising at least 1, 2, 3, 4, 5, 6, or 7 repeats of a sequence selected from the group consisting of: CUG (SEQ ID NO: 18), CCUG (SEQ ID NO: 19), CAG (SEQ ID NO: 80), GGGGCC (SEQ ID NO: 81), and a combination thereof. 
     
     
         13 . The composition of  claim 11 , wherein the nucleic acid comprises a promoter which drives expression of the gRNA sequence. 
     
     
         14 . The composition of  claim 13 , wherein the promoter is a polymerase III promoter. 
     
     
         15 . The composition of  claim 14 , wherein the polymerase III promoter is a U6 promoter or a tRNA promoter. 
     
     
         16 . The composition of  claim 1 , wherein the fusion protein comprises an NLS, NES or tag. 
     
     
         17 . A vector comprising the composition of  claim 1 . 
     
     
         18 . The vector of  claim 17 , wherein the vector is selected from the group consisting of: adeno-associated virus, retrovirus, lentivirus, adenovirus, nanoparticle, micelle, liposome, lipoplex, polymersome, polyplex, and dendrimer. 
     
     
         19 . A cell comprising the vector of  claim 17 . 
     
     
         20 . The composition of  claim 11 , wherein the scaffold sequence comprises a direct repeat sequence.