I'm trying to insert few point mutations close to each other in a gene of interest and I want to use CRISPR/Cas9. Clearly, I am aiming at HDR rather than NHEJ, so I will provide a template DNA molecule that HDR can use to repair. There is a good number of PAM sites in my gene of interest, but since I need to target a single gene in a family of 3, I need to select the most specific region I can find. So, my question is: how close to the site I want to edit should the gRNA anneal? Is it important that the double strand break is inserted very close to the site I'm editing or it can be distant. If yes, how distant?

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