My DNA mismatch assay shows a low percentage of editing. Why is that?
DNA mismatch assays (e.g., SURVEYOR™, T7EI) rely on PCR amplification of a genomic DNA target site and observation of cleavage by a mismatch endonuclease, usually on a gel. While these assays are a straightforward approach for testing for editing, they likely underestimate editing for the following reasons:
1) Longer periods of mismatch enzyme digestion lead to off-target cuts that degrade the PCR product and reduce the intensity of the specific cut bands.
2) Cas9 cleavage and NHEJ can result in deletions, insertions, and mutations of various sizes; mismatch endonuclease cleavage may result in smeared bands that are not easily visualized on a gel.
3) If editing impacts primer binding (large inserts or deletions), the editing will not be detected.
Observation of any cut bands in the DNA mismatch assay indicate successful editing, usually equal or greater than 1-10%.