For my Edit-R experiments, my DNA mismatch assay shows a low percentage of editing. Why is that?
Mutation analysis assays which utilize mismatch-specific DNA endonucleases such as Surveyor (IDT) or T7EI (NEB) rely on PCR amplification of a genomic DNA target site and subsequent observation of cleavage commonly by gel electrophoresis. While these assays are a straightforward approach for detecting insertions and deletions (indels) introduced by CRISPR-Cas9 gene editing, sensitivity varies between endonucleases and generally underestimates editing efficiency for several reasons:
1. Cas9 cleavage followed by DNA repair through non-homologous end-joining (NHEJ) results in deletions, insertions, and mutations of various sizes. Mismatch DNA endonuclease cleavage can produce smeared bands on a gel which are not easily visualized or quantified.
2. Mismatch DNA endonuclease digestion can lead to non-specific cuts that degrade the PCR product and reduce the intensity of the desired bands, especially at longer incubation times.
3. If the CRISPR-Cas9 gene editing generates large inserts or deletions, primer binding sites can be impacted and the mutation thus will not be detected by the DNA mismatch assay.