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 . A method of increasing gene editing efficiency in a cell having a low deoxynucleoside triphosphate (dNTP) concentration and comprising a DNA polymerase for the gene editing, the method comprising:
 increasing the dNTP concentration in the cell, relative to a baseline dNTP concentration.   
     
     
         2 . The method of  claim 1 , wherein increasing the dNTP concentration in the cell comprises inhibiting a deoxynucleotide triphosphate triphosphohydrolase in the cell. 
     
     
         3 . The method of  claim 2 , wherein the deoxynucleotide triphosphate triphosphohydrolase comprises SAM domain and HD domain-containing protein 1 (SAMHD1). 
     
     
         4 . The method of  claim 3 , wherein inhibiting SAMHD1 comprises contacting the SAMHD1 with a Vpx protein, or expressing the Vpx protein in the cell, or contacting the SAMHD1 with a BGLF4 protein, or expressing the BGLF4 protein in the cell. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 3 , wherein inhibiting SAMHD1 comprises contacting an mRNA encoding the SAMHD1 with a microRNA or siRNA that hybridizes to the mRNA, or expressing the microRNA or siRNA in the cell, or contacting SAMHD1 with a small molecule SAMHD1 inhibitor. 
     
     
         7 - 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the DNA polymerase comprises a reverse transcriptase. 
     
     
         15 . The method of  claim 1 , wherein the cell further comprises a Cas9 programmable nuclease, a guide nucleic acid, or a combination thereof. 
     
     
         16 - 20 . (canceled) 
     
     
         21 . A composition comprising a Cas nickase and a reverse transcriptase, wherein at least part of the Cas nickase and the reverse transcriptase are included in separate polypeptide chains, and wherein the Cas nickase and the reverse transcriptase form a Cas-reverse transcriptase heterodimer. 
     
     
         22 . The composition of  claim 21 , wherein the Cas-reverse transcriptase heterodimer comprises a first heterodimer domain fused to the Cas nickase and a second heterodimer domain fused to the reverse transcriptase, wherein the first heterodimer domain binds the second heterodimer domain to form the Cas-reverse transcriptase heterodimer. 
     
     
         23 . The composition of  claim 22 , wherein the first heterodimer domain is a leucine zipper and the second heterodimer domain is a leucine zipper. 
     
     
         24 . The composition of  claim 21 , wherein the reverse transcriptase comprises a sequence having at least 80% sequence identity to of any one of SEQ ID NO: 3-SEQ ID NO: 22 or SEQ ID NO: 40-SEQ ID NO: 80, or a fragment thereof. 
     
     
         25 . The composition of  claim 21 , wherein the reverse transcriptase comprises a domain from a non-long terminal repeat retrotransposable element fused to part of the Cas nickase, a sequence from a bacterial group II intron fused to part of the Cas nickase, or a domain from a retroviral gag-pol polyprotein fused to part of the Cas nickase. 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . A composition comprising a Cas nickase, a reverse transcriptase, and a guide nucleic acid, wherein a first polypeptide comprises the Cas nickase and a second polypeptide comprises the reverse transcriptase and the guide nucleic acid binds to the Cas nickase and the reverse transcriptase. 
     
     
         29 . The composition of  claim 21 , wherein the reverse transcriptase comprises an mcp peptide. 
     
     
         30 . The composition of  claim 21  wherein the reverse transcriptase comprises a loop region. 
     
     
         31 . (canceled) 
     
     
         32 . The composition of  claim 28 , wherein the guide nucleic acid comprises a MS2 hairpin. 
     
     
         33 . A composition comprising a reverse transcriptase with a sequence having at least 80% sequence identity to of any one of SEQ ID NO: 3-SEQ ID NO: 22 or SEQ ID NO: 40-SEQ ID NO: 80, or a fragment thereof fused to a Cas nickase. 
     
     
         34 . A composition comprising a reverse transcriptase comprising a domain from a non-long terminal repeat retrotransposable element fused to a Cas nickase, a bacterial group II intron fused to a Cas nickase, or a domain from a retroviral gag-pol polyprotein fused to a Cas nickase. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . A composition comprising a Cas nickase and a reverse transcriptase, wherein the Cas nickase and the reverse transcriptase comprise separate polypeptide chains, and wherein the Cas nickase and reverse transcriptase are not engineered to heterodimerize. 
     
     
         38 . The composition of  claim 21 , further comprising a guide nucleic acid that forms a complex with the Cas nickase. 
     
     
         39 - 47 . (canceled) 
     
     
         48 . A guide nucleic acid comprising:
 a spacer reverse complementary to a first region of a target nucleic acid,   a scaffold configured to bind to a Cas nickase,   a reverse transcriptase template encoding a sequence to be inserted into the target nucleic acid, and   a first strand primer binding site reverse complementary to a second region of the target nucleic acid.   
     
     
         49 . The guide nucleic acid of  claim 48 , further comprising a second strand primer comprising a sequence of a region of the reverse transcriptase template. 
     
     
         50 . The guide nucleic acid of  claim 48 , wherein the first region of the target nucleic acid is on a first strand of the target nucleic acid and the second region of the target nucleic acid is on a second strand of the target nucleic acid. 
     
     
         51 - 71 . (canceled) 
     
     
         72 . A method of increasing genome editing efficiency comprising delivering an Orflp to a cell expressing the composition of  claim 21 . 
     
     
         73 . One or more nucleic acids encoding the composition of  claim 21 . 
     
     
         74 . A viral vector comprising the nucleic acid of  claim 73 . 
     
     
         75 . A cell comprising the composition of  claim 21 . 
     
     
         76 . (canceled) 
     
     
         77 . (canceled) 
     
     
         78 . A method of increasing genome editing efficiency comprising expressing a Vpx protein in a cell. 
     
     
         79 . (canceled) 
     
     
         80 . A method of increasing genome editing efficiency by increasing the dNTP concentration in a cell, for example a method of increasing genome editing efficiency comprising inhibiting SAMHD1 in a cell. 
     
     
         81 - 83 . (canceled) 
     
     
         84 . A composition comprising a Cas9 programmable nuclease comprising one or more point mutations or insertion mutations that enable or improve intein catalysis. 
     
     
         85 - 106 . (canceled) 
     
     
         107 . The composition of  claim 21 , wherein the reverse transcriptase comprises a point mutation at position P51, S67, Q84, L139, Q221, V223, T197, D653, T664, L671, L435, H204, or D524. 
     
     
         108 - 110 . (canceled) 
     
     
         111 . The composition of  claim 21 , wherein the Cas nickase and RT are encoded by polynucleotides. 
     
     
         112 . AAVs comprising the polynucleotides of  claim 111 . 
     
     
         113 . (canceled) 
     
     
         114 . Adeno-associated viruses (AAVs) comprising: a first AAV comprising a first polynucleotide encoding at least part of a Cas nickase, and a second AAV comprising a second polynucleotide encoding a reverse transcriptase. 
     
     
         115 - 120 . (canceled) 
     
     
         121 . A method of genome editing, comprising administering a composition comprising the first or second AAV of  claim 114  to a subject or cell. 
     
     
         122 . (canceled) 
     
     
         123 . (canceled) 
     
     
         124 . A method of increasing gene editing efficiency in a cell having a low deoxynucleoside triphosphate (dNTP) concentration, comprising:
 contacting the cell with a gene editing enzyme modified for efficient catalysis in the low dNTP concentration, or expressing the gene editing enzyme in the cell.   
     
     
         125 . (canceled) 
     
     
         126 . (canceled) 
     
     
         127 . A system comprising an RNA or polynucleotide comprising a spacer, a reverse transcriptase template comprising a desired edit, and a primer binding site, in which the primer binding site binds to a nucleic acid that does not comprise any part of the region of the nucleic acid targeted or bound by the spacer or the nucleic acid reverse complementary to the nucleic acid targeted or bound by the spacer. 
     
     
         128 - 131 . (canceled)