Hi everybody,
I have a relatively simple question but couldn't find an exact answer. If you want to KO a gene with a CRISPR/Cas9 system, and the gene of interest as a paralogous counterpart gene, and you only want to KO one of the genes (the sequence of the guide RNA is gonna be quite similar since the genes sequences are pretty similar in many parts, but can differ for 2 or 3 nucleotides), is the stringency more important in 5' or 3' of the guide RNA? To explain better what I'm asking, the example of a primer for a PCR is a good one. Indeed, the stringency of your primer in 3' is crucial for a good PCR amplification, so my question is, is there also something similar for sgRNA, and would it influences the action of the Cas9 if the sgRNA sequence differs from the targeted sequence by 2 or nucleotides in the 5' or 3' guide RNA sequence? (I wanna know if I can avoid the off-target effect by targeting only one gene out of the 2 paralogous genes) I appreciate your help