Methods and compositions for directed genome editing

Abstract

Provided herein are compositions and methods for increasing editing efficiency of a target nucleic acid. A composition may comprise a guide nucleic acid, a Cas9 nickase, or a reverse transcriptase. The reverse transcriptase may be fused to the Cas9 nickase. The reverse transcriptase may heterodimerize with the Cas9 nickase. The reverse transcriptase may bind to a guide nucleic acid. The reverse transcriptase may be engineered to increase processivity. The guide nucleic acid may be engineered to facilitate synthesis or editing of a sequence. The guide nucleic acid, Cas9 nickase, and reverse transcriptase may be engineered to fit within AAV vectors. The guide nucleic acid may comprise a region that binds to another region on the guide nucleic acid to improve gene editing.

1 - 131 . (canceled) 
     
     
         132 . A method, comprising:
 contacting a target nucleic acid with one or more nucleic acids collectively comprising:
 a spacer reverse complementary to a first region of the target nucleic acid; 
 a scaffold configured to bind to a nuclease; 
 a reverse transcriptase template encoding a sequence to be inserted into the target nucleic acid; 
 a primer binding site reverse complementary to a second region of the target nucleic acid; and 
 at least one of (i) or (ii):
 (i) a guide nucleic acid positioning system (GPS) region and a GPS binding site that hybridizes to the GPS region, wherein the GPS region and the GPS binding site are at least 10 nucleotides in length and are at least 60% reverse complementary to each other, or 
 (ii) a modification in the reverse transcriptase template that disrupts a track of at least 4 consecutive nucleotides of the same base in the target nucleic acid. 
 
   
     
     
         133 . The method of  claim 132 , wherein the one or more nucleic acids collectively comprise (i). 
     
     
         134 . The method of  claim 133 , wherein the one or more nucleic acids comprise a first nucleic acid comprising the spacer, scaffold, reverse transcriptase template, primer binding site, GPS region and GPS binding site. 
     
     
         135 . The method of  claim 133 , wherein the one or more nucleic acids comprise a first nucleic acid and a second nucleic acid, and wherein the first nucleic acid and the second nucleic acid collectively comprise the spacer, scaffold, reverse transcriptase template, primer binding site, GPS region and GPS binding site. 
     
     
         136 . The method of  claim 135 , wherein the first nucleic acid comprises the reverse transcriptase template and the primer binding site, and the first nucleic acid or the second nucleic acid comprises the spacer. 
     
     
         137 . The method of  claim 135 , wherein the first nucleic acid comprises the spacer, and the second nucleic acid comprises a second, different spacer. 
     
     
         138 . The method of  claim 135 , wherein the first nucleic acid comprises the reverse transcriptase template, and the second nucleic acid does not comprise a reverse transcriptase template. 
     
     
         139 . The method of  claim 135 , wherein the first nucleic acid comprises the primer binding site, and the second nucleic acid does not comprise a primer binding site. 
     
     
         140 . The method of  claim 135 , wherein the first nucleic acid comprises the GPS region and the second nucleic acid comprises the GPS binding site. 
     
     
         141 . The method of  claim 135 , wherein the first nucleic acid comprises the GPS region and the GPS binding site. 
     
     
         142 . The method of  claim 133 , wherein the GPS region and GPS binding site do not consist of the primer binding site and the spacer. 
     
     
         143 . The method of  claim 133 , wherein the GPS region is no more than 100 nucleotides in length. 
     
     
         144 . The method of  claim 134 , wherein the GPS region and the GPS binding site are separated by at least part of the reverse transcriptase template. 
     
     
         145 . The method of  claim 133 , wherein hybridization of the GPS region and the GPS binding site positions the primer binding site closer to the second region of the target nucleic acid. 
     
     
         146 . The method of  claim 132 , wherein the one or more nucleic acids collectively comprise (ii). 
     
     
         147 . The method of  claim 146 , wherein the track of at least 4 consecutive nucleotides of the same base comprise a polyA track. 
     
     
         148 . The method of  claim 132 , wherein the nuclease comprises a Cas nickase. 
     
     
         149 . The method of  claim 132 , wherein the one or more nucleic acids comprise RNA. 
     
     
         150 . The method of  claim 132 , wherein contacting the target nucleic acid with the one or more nucleic acids comprises delivering the one or more nucleic acids to a cell via a viral vector, wherein the cell comprises the target nucleic acid. 
     
     
         151 . The method of  claim 132 , further comprising contacting the target nucleic acid with the nuclease and the reverse transcriptase.