COMPOSITIONS AND METHODS OF NUCLEIC ACID-TARGETING NUCLEIC ACIDS
This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.
1 - 26 . (canceled)
27 . A method for quantifying genome-editing events of genomic DNA in a population of cells wherein the genomic DNA comprises a target nucleic acid, the method comprising:
contacting the genomic DNA of cells in the population with a Cas9 protein in a complex with a nucleic acid targeting nucleic acid comprising a crRNA and a tracrRNA wherein (i) the Cas9 protein in the complex with the nucleic acid targeting nucleic acid can cut the target site to create a first end and a second end, (ii) when the Cas9 protein in the complex with the nucleic acid targeting nucleic acid cuts the target site, a cellular repair mechanism rejoins the first end and the second end destroying the target site, and (iii) the contacting produces a number of cells comprising genomic DNA having the target site and a number of cells comprising genomic DNA wherein the target site was destroyed;
isolating the genomic DNA from the population of cells;
performing polymerase chain reaction (PCR) with the isolated genomic DNA using primers that flank the target site to produce an amplified PCR product;
contacting the amplified PCR product with the Cas9 protein in the complex with the nucleic acid targeting nucleic acid; and
quantifying genome-editing events by comparing the amount of amplified PCR product derived from genomic DNA that comprises the target site in the amplified PCR product to the amount of amplified PCR product derived from genomic DNA wherein the target site was destroyed.
28 . The method of claim 27 for quantifying genome-editing events of genomic DNA, wherein the quantifying is carried out using gel electrophoresis.