Use of exonucleases to improve crispr/cas-mediated genome editing

Abstract

The present disclosure is directed to methods of producing a modified nucleic acid comprising a precise deletion in a target nucleic acid in a cell comprising generating, within the cell, a first single strand break on a first strand of the target nucleic acid and a second single strand break on a second strand of the target nucleic acid, thereby forming a double strand break in the target nucleic acid having a first 3′ overhang and a second 3′ overhang; processing the first 3′ overhang and the second 3′ overhang with an exonuclease molecule, thereby deleting the segment of the target nucleic acid that was located between the first single strand break and the second single strand break, and forming a processed double strand break; and allowing the processed double strand break to be repaired by at least one DNA repair pathway, thereby producing the modified nucleic acid comprising the precise deletion in the target nucleic acid in the cell. Gene editing systems, vectors, polynucleotides, and methods of treatment are also disclosed herein.

1 . A gene editing system comprising:
 a first gRNA molecule and a second gRNA molecule;   at least one nickase molecule, or fragment thereof; and   a 3′ to 5′ exonuclease molecule;   wherein the first gRNA molecule, the second gRNA molecule, and the at least one nickase molecule are configured to associate with a target nucleic acid and form a DNA double strand break having a 3′ overhang.   
     
     
         2 . The gene editing system of  claim 1 ,
 wherein the first gRNA molecule and the at least one nickase molecule can associate with a target nucleic acid and generate a first single strand break on a first strand of the target nucleic acid;   wherein the second gRNA molecule and the at least one nickase molecule can associate with the target nucleic acid and generate a second single strand break on a second strand of the target nucleic acid, thereby forming a double strand break in the target nucleic acid having a first 3′ overhang and a second 3′ overhang;   wherein the exonuclease molecule can process the first 3′ overhang and the second 3′ overhang, forming a processed double strand break; and   wherein the processed double strand break can be repaired by at least one DNA repair pathway.   
     
     
         3 .- 17 . (canceled) 
     
     
         18 . An isolated polynucleotide encoding the gene editing system of  claim 1 . 
     
     
         19 . A vector or plurality of vectors encoding the gene editing system of  claim 1 . 
     
     
         20 . A lipid particle comprising the gene editing system of  claim 1 . 
     
     
         21 . A pharmaceutical composition comprising the gene editing system of  claim 1 . 
     
     
         22 .- 26 . (canceled) 
     
     
         27 . A method of deleting a segment of a target nucleic acid in a cell, the method comprising:
 contacting the cell with a first gRNA molecule, a second gRNA molecule, and at least one enzymatically active Cas9 (eaCas9) nickase molecule; and   contacting the cell with a 3′ to 5′ exonuclease;   wherein the first gRNA molecule, the second gRNA molecule, and the at least one eaCas9 nickase molecule are configured to associate with the target nucleic acid and form a DNA double strand break having a first 3′ overhang and a second 3′ overhang.   
     
     
         28 . A method of deleting a segment of a target nucleic acid in a cell, the method comprising:
 generating, within the cell, a first single strand break on a first strand of the target nucleic acid and a second single strand break on a second strand of the target nucleic acid, wherein the first single strand break is located at least 25 base pairs away from the second single strand break, thereby forming a double strand break in the target nucleic acid having a first 3′ overhang and a second 3′ overhang; and   processing the first 3′ overhang and the second 3′ overhang using a 3′ to 5′ exonuclease molecule, thereby forming a processed double strand break;   wherein the processed double strand break is repaired by at least one DNA repair pathway, thereby deleting the segment of the target nucleic acid that is located between the first single strand break or within 5 base pairs thereof, and the second single strand break or within 5 base pairs thereof.   
     
     
         29 . The method of  claim 27 , wherein the first gRNA molecule and the at least one eaCas9 nickase molecule associate with the target nucleic acid and generate the first single strand break, and wherein the second gRNA molecule and the at least one eaCas9 nickase molecule associate with the target nucleic acid and generate the second single strand break. 
     
     
         30 . The method of  claim 27 , wherein the segment of the target nucleic acid is at least about 15, 20, 25, 30, 40, 50, 75, or 100 base pairs in length. 
     
     
         31 . The method of  claim 28 , wherein the step of generating the first single strand break and the second single strand break comprises contacting the cell with a first gRNA molecule, at least one enzymatically active Cas9 (eaCas9) nickase molecule, and a second gRNA molecule. 
     
     
         32 . The method of  claim 27 , wherein the target nucleic acid is a promoter region of a gene, a coding region of a gene, a non-coding region of a gene, an intron of a gene, or an exon of a gene. 
     
     
         33 .- 35 . (canceled) 
     
     
         36 . The method of  claim 27 , wherein the 3′ to 5′ exonuclease is a Trex2 molecule. 
     
     
         37 .- 41 . (canceled) 
     
     
         42 . The method of  claim 27 , wherein the cell is a mammalian cell. 
     
     
         43 .- 44 . (canceled) 
     
     
         45 . The method of  claim 27 , wherein the cell is a population of cells, and wherein at least 20% of the cells in the population of cells comprise a deletion of the segment of the target nucleic acid. 
     
     
         46 . (canceled) 
     
     
         47 . The method of  claim 29 , wherein the segment of the target nucleic acid is located between the first single strand break or within 5 base pairs thereof, and the second single strand break or within 5 base pairs thereof. 
     
     
         48 . The method of  claim 28 , wherein at least a portion of the segment of the target nucleic acid corresponds to the first 3′ overhang, or a fragment of the first 3′ overhang, and/or wherein at least a portion of the segment of the target nucleic acid corresponds to the second 3′ overhang, or a fragment of the second 3′ overhang. 
     
     
         49 .- 52 . (canceled) 
     
     
         53 . An isolated population of cells modified by the method of  claim 28 , wherein the population of cells comprises a distribution of lengths of the segment of the target nucleic acid
 a) having a mean length and/or a median length within 5 base pairs of the number of base pairs between the first single strand break and the second single strand break; and   b) having a median absolute deviation that is lower than a corresponding median absolute deviation in the distribution of lengths of the segment of the target nucleic acid in a second isolated population of cells modified by contacting the second population of cells with the first gRNA molecule, the second gRNA molecule, and the at least one enzymatically active Cas9 (eaCas9) nickase molecule, without contacting the second population of cells with the 3′ to 5′ exonuclease.   
     
     
         54 . The isolated population of cells of  claim 53 , wherein a difference between the mean length and the median length of the distribution of lengths of the segment of the target nucleic acid in the isolated population of cells is smaller than a corresponding difference between a mean length and a median length of a distribution of lengths observed in the second isolated population of cells. 
     
     
         55 . The isolated population of cells of  claim 53 , wherein a difference between the mean length and the median length of the distribution of lengths of the segment of the target nucleic acid in the isolated population of cells is less than 5 base pairs.