Methods for the identification of variant recognition sites for rare-cutting engineered double-strand-break-inducing agents and compositions and uses thereof

Abstract

Methods for the identification of variant recognition sites for rare cutting engineered double strand break inducing agents and compositions thereof are provided. Further provided are nucleic acid constructs, yeast, plants, plant cells, explants, seeds and grain having the of variant recognition sites. Various methods of identifying variant recognition sites with increased substrate activity for a rare cutting engineered double strand break inducing agents are provided.

1 ) A method to identify a double-strand break site for a double-strand-break-inducing agent in genomic DNA, said method comprising:
 a. contacting genomic DNA with a double-strand-break-inducing agent capable of introducing a double-strand break into said genomic DNA, wherein the double-strand break results in a nucleotide overhang;   b. ligating a first adapter to said nucleotide overhang of (a), resulting in ligated DNA;   c. obtaining fragments of the ligated DNA obtained in step (b) and ligating a second adapter to the DNA fragments to allow for the amplification and sequencing of the DNA fragments that each comprise the first adapter on one end and the second adapter on the other end;   d. amplifying and sequencing the DNA fragments of (c);   e. aligning nucleotide sequences of the DNA fragments obtained in (d) with a reference genome DNA sequence; and,   f. identifying a variant recognition site comprising at least one nucleotide base alteration when compared to the intended recognition site of said engineered double-strand break-inducing agent.   
     
     
         2 ) The method of  claim 1  wherein the double-strand-break-inducing agent is selected from the group consisting of: a meganuclease, a zinc finger nuclease, a TAL effector nuclease, a transposase, a Cas endonuclease, and a site-specific recombinase. 
     
     
         3 ) The method of  claim 1  wherein the nucleotide overhang is a 3′ nucleotide overhang. 
     
     
         4 ) The method of  claim 1  wherein the nucleotide overhang is a 5′ nucleotide overhang. 
     
     
         5 ) The method of  claim 1  wherein the first adapter ligated to the nucleotide overhang is a non-5′ phosphorylated adapter. 
     
     
         6 ) The method of  claim 1  wherein the genomic DNA is selected from the group consisting of a prokaryotic DNA, eukaryotic DNA and synthetic DNA. 
     
     
         7 ) The method of  claim 6  wherein the eukaryotic DNA is isolated from a plant, yeast, or animal. 
     
     
         8 ) The method of  claim 7  wherein the plant is selected from the group consisting of soybean, sunflower, cotton, alfalfa, canola, cotton, tobacco, potato,  Arabidopsis , safflower, maize, rice, sorghum, barley, wheat, millet, oats, sugarcane, turfgrass, and switch grass. 
     
     
         9 ) (canceled) 
     
     
         10 ) A method to identify a variant double-strand break site with improved cleavage activity for a double-strand-break-inducing agent capable of introducing a double strand break in an intended recognition site, said method comprising:
 a. contacting genomic DNA with double-strand-break-inducing agent capable of introducing a double strand break into said genomic DNA, wherein the double strand break results in a nucleotide overhang;   b. ligating a first adapter to said nucleotide overhang;   c. shearing the ligated DNA obtained in step (b) and ligating a second adapter to the sheared nucleotide end to allow for the amplification and sequencing of genomic DNA fragments surrounding the double strand break;   d. amplifying and sequencing the DNA fragments of (c);   e. aligning nucleotide sequences of the DNA fragments obtained in (d) with a reference genome DNA sequence; and,   f. identifying a variant double-strand break site comprising at least one nucleotide base alteration when compared to the intended double-strand break site of said rare-cutting engineered double-strand-break-inducing agent;   g. analyzing the a rare-cutting engineered double-strand-break-inducing agent activity at the variant double-strand break site of (e); and,   g. identifying a variant double-strand break site that results in an increased activity of the double-strand-break-inducing agent when compared to the activity at the intended double-strand break site.   
     
     
         11 ) The method of  claim 10  wherein the increased activity of the double-strand-break-inducing agent is evidenced by
 a. a higher percent (%) cleavage of the variant double-strand break site when compared to the percent (%) cleavage of intended double-strand break site, wherein the double-strand break sites are located on genomic DNA; 
 b. a higher percent (%) cleavage of the variant double-strand break site when compared to the percent (%) cleavage of intended double-strand break site, wherein the double-strand break sites are located on plasmid DNA; 
 c. a higher yeast assay score for the variant double-strand break site when compared to the intended double-strand break site; or, 
 d. any combination of (a), (b) and (c) 
 
     
     
         12 ) A method for introducing into the genome of a cell a variant double-strand break site for a double-strand-break-inducing agent capable of introducing a double strand break in an intended double-strand break site, said method comprising:
 a. providing a donor DNA comprising a variant double-strand break site for a double-strand-break-inducing agent capable of introducing a double-strand break in an intended double-strand break site, wherein said a double-strand-break-inducing agent is also capable of introducing a double-strand break in said variant double-strand break site;   b. providing a plant cell;   c. contacting the plant cell with the donor DNA; and,   d. identifying at least one plant cell from (c) comprising in its genome said variant double-strand break site.   
     
     
         13 ) The method of  claim 12  wherein the double-strand-break-inducing agent is selected from the group consisting of: a meganuclease, a zinc finger nuclease, a TAL effector nuclease, a transposase, a Cas endonuclease, and a site-specific recombinase. 
     
     
         14 )- 42 ) (canceled)