I noticed in one of my recent CRISPR experiments that my HDR template, which is inserted at low frequency, was being further edited AFTER current insertion into the genome. This was particularly surprising since I had taken care to mutate the gAGG PAM sequence into aCCC to avoid targeting my repair template (and I used the eCas9). I will now introduce additional modifications in the HDR template to avoid additional edits. While I do so, please share your thoughts on this unanticipated outcome.
Thanks.
Hi Kristian,
I think you're taking the right approach by introducing further mutations in the repair template. I understand that a single mutation in the PAM may not be sufficient to prevent post-insertion editing. I use a further 2-3 silent mutations in the template.
Be interested to know how you got on.
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