Methods and compositions for editing rnas

Abstract

Provided are methods for editing RNA by introducing a deaminase-recruiting RNA in a host cell for deamination of an adenosine in a target RNA. Further provided are deaminase-recruiting RNAs used in the RNA editing methods and compositions comprising the same.

1 . A deaminase-recruiting RNA (dRNA) of 60-200 nucleotides, wherein:
 a) the dRNA comprises a complementary RNA sequence capable of hybridizing to a target RNA;   b) the dRNA is capable of recruiting a deaminase or a construct comprising a deaminase or a construct comprising a catalytic domain of a deaminase to deaminate a target adenosine in the target RNA; and   c) the dRNA comprises one or more chemical modifications.   
     
     
         2 . The dRNA of  claim 1 , wherein the dRNA is longer than about any of 60 nt, 65 nt, 70 nt, 80 nt, 90 nt, 100 nt, or 110 nt. 
     
     
         3 . The dRNA of  claim 1 , comprising one or more mismatches, wobbles and/or bulges with the complementary target RNA region. 
     
     
         4 . The dRNA of  claim 1 , wherein the complementary RNA sequence comprises a cytidine, adenosine or uridine directly opposite to a target adenosine in the target RNA. 
     
     
         5 . The dRNA of  claim 4 , wherein the cytidine, adenosine or uridine directly opposite to the target adenosine locates at least about 7 nucleotides away from the 3′ end. 
     
     
         6 . The dRNA of  claim 4 , wherein the cytidine, adenosine or uridine directly opposite to the target adenosine locates at least about 25 nucleotides away from the 5′ end. 
     
     
         7 . The dRNA of  claim 4 , wherein the lengths of the 5′ and 3′ sequences flanking the cytidine, adenosine or uridine directly opposite to the target adenosine are unequal. 
     
     
         8 . The dRNA of  claim 4 , wherein the length of the 5′ sequence flanking the cytidine, adenosine or uridine directly opposite to the target adenosine is longer than the 3′ sequence. 
     
     
         9 . The dRNA of  claim 1 , comprising a cytidine directly opposite to the target adenosine in the target RNA. 
     
     
         10 . The dRNA of  claim 1 , wherein the complementary RNA sequence comprises one or more guanosines each opposite to a non-target adenosine in the target RNA. 
     
     
         11 . The dRNA of  claim 1 , wherein the complementary sequence comprises two or more consecutive mismatch nucleotides opposite to a non-target adenosine in the target RNA. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The dRNA of  claim 1 , wherein the three-base motif is UAG, and wherein the dRNA comprises an A directly opposite to the uridine in the three-base motif, a cytidine directly opposite to the target adenosine, and a cytidine, guanosine or uridine directly opposite the guanosine in the three-base motif. 
     
     
         15 . The dRNA of  claim 14 , comprising a 5′-CCA-3′ directly opposite to the three-base motif of UAG. 
     
     
         16 . The dRNA of  claim 1 , wherein the chemical modification comprises methylation and/or phosphorothioation. 
     
     
         17 . The dRNA of  claim 16 , wherein the chemical modification comprises 2′-O-methylation and/or internucleotide phosphorothioate linkage. 
     
     
         18 . The dRNA of  claim 16 , wherein the chemical modification comprises a 2′-O-methylationin the first and last 1-5, 2-5, 3-5, 4-5 nucleotides and/or phosphorothioations in the first and last 1-5, 2-5, 3-5, 4-5 internucleotide linkages. 
     
     
         19 . The dRNA of  claim 16 , wherein the chemical modification comprises a 2′-O-methylation and/or a 3′-phosphorothioation in the nucleotide opposite to the target adenosine and/or its 5′ and/or 3′ most adjacent nucleotides. 
     
     
         20 . The dRNA of  claim 1 , wherein the chemical modification is selected from a group consisting of:
 1) 2′-O-methylations in the first and last 3 nucleotides and/or phosphorothiations in the first and last 3 internucleotide linkages;   2) 2′-O-methylations in the first and last 3 nucleotides and/or phosphorothiations in the first and last 3 internucleotide linkages, and 2′-O-methylations in a single or multiple or all uridines;   3) 2′-O-methylations in the first and last 3 nucleotides, phosphorothiations in the first and last 3 internucleotide linkages, 2′-O-methylations in a single or multiple or all uridines, and a modification in the nucleotide opposite to the target adenosine, and/or its 5′ and/or 3′ most adjacent nucleotides;   4) 2′-O-methylations in the first and last 3 nucleotides, phosphorothiations in the first and last 3 internucleotide linkages, 2′-O-methylations in a single or multiple or all uridines, and 2′-O-methylation in the nucleotide most adjacent to the 3′ terminus and/or 5′ terminus of the nucleotide opposite to the target adenosine;   5) 2′-O-methylations in the first and last 3 nucleotides, phosphorothiations in the first and last 3 internucleotide linkages, 2′-O-methylations in a single or multiple or all uridines, and phosphorothiation linkage in the nucleotide opposite to the target adenosine and/or its 5′ and/or 3′ most adjacent nucleotides; and   6) 2′-O-methylations in the first and last 1-5 nucleotides and/or phosphorothiations in the first and last 1-5 internucleotide linkages.   
     
     
         21 . The dRNA of  claim 20 , wherein the modification in the nucleotide opposite to the target adenosine, and/or one or two nucleotides most adjacent to the nucleotide opposite to the target adenosine is 2′-O-methylation and/or phosphorothiation linkage. 
     
     
         22 . The dRNA of  claim 1 , which does not comprise an ADAR-recruiting domain capable of forming an intramolecular stem loop structure for binding an ADAR enzyme. 
     
     
         23 . A construct comprising or encoding a dRNA of  claim 1 . 
     
     
         24 . A method for editing a target RNA in a host cell, comprising introducing a dRNA of  claim 1  into host cells. 
     
     
         25 . The method of  claim 24 , further comprises introducing an inhibitor of ADAR3 to the host cell. 
     
     
         26 . The method of  claim 24 , further comprises introducing a stimulator of interferon to the host cell. 
     
     
         27 . The method of  claim 24 , comprising introducing a plurality of the dRNAs each targeting a different target RNA. 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 24 , further comprises introducing an exogenous ADAR to the host cell. 
     
     
         30 . The method of  claim 29 , wherein the ADAR is an ADAR1 comprising an E1008 mutation. 
     
     
         31 . A construction, composition, cell, library or kit comprising the dRNAs of  claim 1 .