Compositions And Methods Of Nucleic Acid-Targeting Nucleic Acids
AssigneeCaribou Biosciences, Inc.
InventorsAndrew Paul May, Rachel E. Haurwitz, Jennifer A. Doudna, James M. Berger, Matthew Merrill Carter, Paul Donohoue
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Abstract
This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof. Genome engineering can refer to altering the genome by deleting, inserting, mutating, or substituting specific nucleic acid sequences. The altering can be gene or location specific. Genome engineering can use nucleases to cut a nucleic acid thereby generating a site for the alteration. Engineering of non-genomic nucleic acid is also contemplated.
Claims21
1-26. (canceled)
A method for quantifying genome-editing events in genomic DNA, wherein the genomic DNA comprises a target nucleic acid, the method comprising:
- contacting genomic DNA in a number of cells with a complex comprising a site-directed nuclease and a nucleic acid targeting nucleic acid wherein the complex is capable of cleaving the target nucleic acid to create a first end and a second end, and further the contacting results in a population of cells comprising (i) cells comprising genomic DNA having the target nucleic acid wherein cleavage of the target nucleic acid does not occur, and (ii) cells comprising genomic DNA wherein cleavage of the target nucleic acid does occur;
- performing amplification using genomic DNA isolated from the population of cells and primers that flank the target nucleic acid to produce an amplified double-stranded DNA product;
- contacting the amplified double-stranded DNA product with the complex comprising the site-directed nuclease and the nucleic acid targeting nucleic acid;
- and
- quantifying genome-editing events by comparing (i) the amount of amplified double-stranded DNA product derived from genomic DNA comprising the target nucleic acid wherein cleavage of the target nucleic acid did not occur, with (ii) the amount of amplified double-stranded DNA product derived from genomic DNA wherein cleavage of the target nucleic acid did occur.
A method for quantifying genome-editing events in genomic DNA, wherein the genomic DNA comprises a target nucleic acid, the method comprising:
- contacting genomic DNA in a number of cells with a complex comprising a Cas9 protein and a nucleic acid targeting nucleic acid comprising RNA, wherein the complex is capable of cleaving the target nucleic acid to create a first end and a second end, and further the contacting results in a population of cells comprising (i) cells comprising genomic DNA having the target nucleic acid wherein cleavage of the target nucleic acid does not occur, and (ii) cells comprising genomic DNA wherein cleavage of the target nucleic acid does occur;
- performing polymerase chain reaction amplification using genomic DNA isolated from the population of cells and primers that flank the target nucleic acid to produce a polymerase chain reaction amplified double-stranded DNA product;
- contacting the amplified double-stranded DNA product with the complex comprising the Cas9 protein and the nucleic acid targeting nucleic acid comprising RNA;
- and
- quantifying genome-editing events by comparing (i) the amount of amplified double-stranded DNA product derived from genomic DNA comprising the target nucleic acid wherein cleavage of the target nucleic acid did not occur, with (ii) the amount of amplified double-stranded DNA product derived from genomic DNA wherein cleavage of the target nucleic acid did occur.
Dependencies are inferred from the claim text.