Patent ID: 11866726
Assignee: EDITAS MEDICINE, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 6:
7. A method for determining the outcome of a gene editing event at a cleavage site in a target nucleic acid in a cell using an exogenous oligonucleotide donor template,
wherein the target nucleic acid comprises a first strand comprising: a first homology arm 5′ to a cleavage site, a first priming site either within the first homology arm or 5′ to the first homology arm, a second homology arm 3′ to the cleavage site, and a second priming site either within the second homology arm or 3′ to the second homology arm, and
wherein a first strand of the exogenous oligonucleotide donor template comprises from 5′ to 3′,
a first donor homology arm, a first stuffer, a priming site that is substantially identical to the second priming site, a cargo, a priming site that is substantially identical to the first priming site, a second stuffer, and a second donor homology arm,
wherein the first stuffer and the second stuffer each comprise a random or heterologous sequence having a GC content of approximately 40%,
the method comprising:
i) forming at least one single- or double-strand break at or near the cleavage site in the target nucleic acid;
ii) recombining the exogenous oligonucleotide donor template with the target nucleic acid via homologous recombination to produce an altered nucleic acid; and
iii) amplifying the altered nucleic acid using a first primer which binds to the first priming site and/or the priming site that is substantially identical to the first priming site; and/or a second primer which binds to the second priming site and/or the priming site that is substantially identical to the second priming site;
thereby determining the outcome of the gene editing event in the cell.