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 sequence encoding an RNA-guided target RNA-binding fusion protein comprising (a) a first RNA-binding polypeptide or portion thereof, and (b) a second RNA-binding polypeptide, wherein the first RNA-binding polypeptide binds a target RNA when guided by a gRNA sequence, and wherein the second RNA-binding polypeptide comprises RNA-nuclease activity. 
     
     
         2 . The composition of  claim 1 , wherein the first RNA-binding polypeptide or portion thereof is a CRISPR/Cas polypeptide or portion thereof. 
     
     
         3 . The composition of  claim 2 , wherein the CRISPR/Cas polypeptide or portion thereof is selected from the group consisting of Cas9, Cpf1, Cas13a, Cas13b, Cas13c and CasRX/Cas13d, wherein the CRISPR/Cas polypeptide has native, reduced or null activity. 
     
     
         4 . The composition of  claim 1 , wherein the second RNA-binding polypeptide binds RNA in a manner in which it associates with RNA. 
     
     
         5 . The composition of  claim 4 , wherein the second RNA-binding polypeptide associates with RNA in a manner in which it cleaves RNA. 
     
     
         6 . The composition of  claim 1 , wherein the nucleic acid sequence comprises a promoter. 
     
     
         7 . The composition of  claim 6 , wherein the promoter is a constitutive promoter or a tissue-specific promoter. 
     
     
         8 . The composition of  claim 1 , wherein the nucleic acid sequence further comprises a gRNA sequence, wherein the gRNA sequence comprises a spacer sequence that specifically binds a target sequence within an RNA molecule and a scaffold sequence that specifically binds to the first RNA-binding polypeptide. 
     
     
         9 . The composition of  claim 8 , 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. 
     
     
         10 . The composition of  claim 8 , wherein the nucleic acid sequence comprises a promoter which drives expression of the gRNA sequence. 
     
     
         11 . The composition of  claim 9 , wherein the promoter is a polymerase III promoter. 
     
     
         12 . The composition of  claim 10 , wherein the polymerase III promoter is a U6 promoter. 
     
     
         13 . The composition of  claim 1 , wherein the promoter is a tRNA promoter. 
     
     
         14 . The composition of  claim 1 , wherein the fusion protein comprises an NLS, NES or tag. 
     
     
         15 . A vector comprising the composition of  claim 1 . 
     
     
         16 . The vector of  claim 15 , wherein the vector is selected from the group consisting of: adeno-associated virus, retrovirus, lentivirus, adenovirus, nanoparticle, micelle, liposome, lipoplex, polymersome, polyplex, and dendrimer. 
     
     
         17 . A cell comprising the vector of  claim 15 . 
     
     
         18 . The composition of  claim 1 , wherein the second RNA-binding polypeptide is selected from the group consisting of: RNAse1, RNAse4, RNAse6, RNAse7, RNAse8, RNAse2, RNAse6PL, RNAseL, RNAseT2, RNAse11, RNAseT2-like, NOB 1, ENDOV, ENDOG, ENDOD1, hFEN1, hSLFN14, hLACTB2, APEX2, ANG, HRSP12, ZC3H12A, RIDA, PDL6, NTHL, KIAA0391, APEX1, AGO2, EXOG, ZC3H12D, ERN2, PELO, YBEY, CPSF4L, hCG_2002731, ERCC1, RAC1, RAA1, RAB1, DNA2, FLJ35220, FLJ13173, ERCC4, Rnase1(K41R), Rnase1(K41R, D121E), Rnase1(K41R, D121E, H119N), Rnase1(H119N), Rnase1(R39D, N67D, N88A, G89D, R91D, H119N), Rnase1(R39D, N67D, N88A, G89D, R91D, H119N, K41R, D121E), Rnase1(R39D, N67D, N88A, G89D, R91D), TENM1, TENM2, RNAseK, TALEN, and ZNF638. 
     
     
         19 . A composition comprising:
 (a) a guide RNA (gRNA) sequence comprising a spacer sequence that specifically binds a target sequence within an RNA molecule and a scaffold sequence that specifically binds to the first RNA-binding polypeptide;   (b) a nucleic acid sequence encoding a fusion protein, the fusion protein comprising 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.   
     
     
         20 . A method for modifying the level of expression of a target RNA molecule or a protein encoded by the RNA molecule, the method comprising contacting the composition of  claim 19  and the RNA molecule under conditions suitable for binding of the fusion protein or a portion thereof to the RNA molecule.