I am currently trying to select the best gRNAs for a CrispR-CAS9 mediated deletion. NEB suggests to use "their" T7 endonuclease assay that should generate fragments from PCR products when they form wt/mut heteroduplexes.

Befor doing it for the target CispR_CAS9 locus, I tried to test it on some positive controls (PCR products with 50% of the alleles carrying 10-20 bp insertions). The results look anything else than clear-cut, although one would expect that 50% of the bands are wt/mut heteroduplexes that should be cut by the T7 EN.

I remember that there are other endonucleases that are also specific for sites of small mismatches in double-stranded DNA, such as celery endonuclease (surveyor assay).

Has anybody experience which of the two methods is better in terms of sensitivity and specifity ?

regards, Michael