Methods for the identification of variant recognition sites for rare-cutting engineered double-strand-break-inducing agents and compositions and uses thereof
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. An in vitro method to identify a Cas endonuclease double strand break site in genomic DNA, said method comprising:
a. contacting the genomic DNA with a Cas endonuclease-guide RNA complex, wherein the Cas endonuclease-guide RNA complex introduces a double-strand break in the genomic DNA, resulting in a blunt end;
b. creating a nucleotide overhang from the blunt end of (a);
c. ligating a first adapter to the nucleotide overhang of (b), resulting in a ligated DNA;
d. obtaining fragments of the ligated DNA of (c) 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;
e. amplifying and sequencing the DNA fragments of (d);
f. aligning nucleotide sequences of the DNA fragments of (e) with a reference DNA sequence; and,
g. identifying the double-strand break site.
2. The method of claim 1 wherein the nucleotide overhang is a 3′ nucleotide overhang.
3. The method of claim 1 wherein the nucleotide overhang is a 5′ nucleotide overhang.
4. The method of claim 1 wherein the genomic DNA is selected from the group consisting of a prokaryotic DNA, eukaryotic DNA, and synthetic DNA.
5. The method of claim 4 wherein the eukaryotic DNA is isolated from a plant, yeast or animal.
6. The method of claim 5 wherein the plant is selected from the group consisting of soybean, sunflower, cotton, alfalfa, canola, tobacco, potato, Arabidopsis , safflower, maize, rice, sorghum, barley, wheat, millet, oats, sugarcane, turfgrass, and switch grass.