Method for modifying genome sequence to introduce specific mutation to targeted dna sequence by base-removal reaction, and molecular complex used therein

Abstract

The present invention provides a method of modifying a targeted site of a double stranded DNA, including a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a selected double stranded DNA and DNA glycosylase with sufficiently low reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) are bonded, with the double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into the targeted site, without cleaving at least one strand of the double stranded DNA in the targeted site.

1 . A method of modifying a targeted site of a double stranded DNA in a cell, comprising a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a given double stranded DNA and DNA glycosylase with sufficiently low reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) are bonded, with said double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into said targeted site, without cleaving at least one strand of said double stranded DNA in the targeted site. 
     
     
         2 . The method according to  claim 1 , wherein the nucleic acid sequence-recognizing module is selected from the group consisting of a CRISPR-Cas system wherein at least one DNA cleavage ability of Cas is inactivated, a zinc finger motif, a TAL effector and a PPR motif. 
     
     
         3 . The method according to  claim 1 , wherein the nucleic acid sequence-recognizing module is a CRISPR-Cas system wherein at least one DNA cleavage ability of Cas is inactivated. 
     
     
         4 . The method according to  claim 1 , wherein the double stranded DNA is further contacted with a factor that changes a DNA double stranded structure. 
     
     
         5 . The method according to  claim 1 , which uses two or more kinds of nucleic acid sequence-recognizing modules each specifically binding to a different target nucleotide sequence. 
     
     
         6 . The method according to  claim 5 , wherein the different target nucleotide sequences are present in different genes. 
     
     
         7 . The method according to  claim 1 , wherein the DNA glycosylase has cytosine-DNA glycosylase (CDG) activity or thymine-DNA glycosylase (TDG) activity. 
     
     
         8 . The method according to  claim 7 , wherein the DNA glycosylase having CDG activity or TDG activity is a mutant of uracil-DNA glycosylase (UDG). 
     
     
         9 . The method according to  claim 1 , further comprising contacting the double stranded DNA with an AP endonuclease having binding capacity to an abasic site but lacking nuclease activity. 
     
     
         10 . The method according to  claim 1 , wherein the DNA glycosylase has natively low reactivity with a DNA having an unrelaxed double helix structure. 
     
     
         11 . The method according to  claim 10 , wherein the DNA glycosylase is a mutant of UDG derived from a virus belonging to Poxviridae and having CDG activity or TDG activity. 
     
     
         12 . The method according to  claim 11 , wherein the double stranded DNA is further contacted with A20 protein. 
     
     
         13 . The method according to  claim 1 , wherein the DNA glycosylase is a mutant having reduced reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) as compared to a wild-type one. 
     
     
         14 . The method according to  claim 1 , wherein the DNA glycosylase, and an element of the nucleic acid sequence-recognizing module which is directly bonded to the DNA glycosylase are respectively split into two fragments, the fragments of either of the DNA glycosylase and the element are respectively linked to the fragments of the other to provide two partial complexes, and when the partial complexes are refolded with each other, the nucleic acid sequence-recognizing module is capable of specifically binding to the target nucleotide sequence and the specific bond enables the DNA glycosylase to exhibit enzyme activity. 
     
     
         15 . The method according to  claim 14 , wherein the element of the nucleic acid sequence-recognizing module which is directly bonded to the DNA glycosylase is a Cas protein wherein at least one of the DNA cleavage abilities is inactivated. 
     
     
         16 . The method according to  claim 14 , wherein the two partial complexes are provided as separate molecule complexes, and are refolded by association thereof in the cell. 
     
     
         17 . The method according to  claim 1 , wherein the double stranded DNA is contacted with the complex by introducing a nucleic acid encoding the complex into a cell having the double stranded DNA. 
     
     
         18 . The method according to  claim 17 , wherein the cell is a prokaryotic cell. 
     
     
         19 . A nucleic acid-modifying enzyme complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a given double stranded DNA and DNA glycosylase with sufficiently low reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) are bonded, which converts one or more nucleotides in the targeted site to other one or more nucleotides or deletes one or more nucleotides, or inserts one or more nucleotides into said targeted site, without cleaving at least one strand of said double stranded DNA in the targeted site. 
     
     
         20 . A nucleic acid encoding the nucleic acid-modifying enzyme complex according to  claim 19 .