Site directed mutagenesis using Cas9 and HDR

I have read papers using the HDR pathway to edit the genome and if I understood correctly, Cas9 system only allows us to make a DSB before the PAM sequence, which then can be repaired by the host using the donor template if it is co-transfected. This means that it is not some kind of homology recombination that the donor DNA replaces the original sequence, rather that the original genomic sequence always stays downstream of the cut site right? My question would be, if one wants to make a only Serine to Alanine mutation in a gene; then besides the Alanine codon; your donor template should also include the rest of the gene sequence until the end of the gene (so that original Serine codon is not transcribed at all) or I am missing something?


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Pink Golemabout 4 years ago

Your donor template should include homology up and downstream of the expected cut site (3bp upstream of the pam). The homology arms can be around 100 nt on either side of the cut. The donor template only needs to include the codon change as well as any silent mutations that help disrupt guideRNA from re-annealing after the recombination has happened. Generally you can do this by disrupting the pam site.

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