Cas9 finds its target through the 20 nucleotides carried on the guide RNA, but it only cuts when a short PAM sequence sits right beside that target; without a PAM even a perfect match goes untouched. After a double-strand break the two repair routes diverge completely: NHEJ rejoins the ends sloppily and usually wrecks the gene, while HDR copies a template and can insert exactly the intended sequence. The biggest risk is the off-target cut, where the guide binds a near-match somewhere else in the genome.
Cas9 finds its target through the 20 nucleotides carried on the guide RNA, but it only cuts when a short PAM sequence sits right beside that target; without a PAM even a perfect match goes untouched. After a double-strand break the two repair routes diverge completely: NHEJ rejoins the ends sloppily and usually wrecks the gene, while HDR copies a template and can insert exactly the intended sequence. The biggest risk is the off-target cut, where the guide binds a near-match somewhere else in the genome.
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