CRISPR-Cas9 Mechanism and Applications

CRISPR-Cas9 has revolutionized genome editing. (a) Describe the molecular mechanism by which Cas9 generates a double-strand break (DSB). (b) Compare the two DNA repair pathways: non-homologous end joining (NHEJ) vs. homology-directed repair (HDR). (c) What is the role of the PAM sequence and how does it limit targetable sites? (d) How do base editors and prime editors improve upon standard CRISPR-Cas9? (e) Calculate the probability of an off-target site with 3 mismatches in a 20-nt guide RNA being cleaved, if each mismatch reduces cleavage efficiency by 90%.