In CRISPR-Cas9 gene editing, the guide RNA (sgRNA) directs Cas9 to the target DNA. Cas9 requires a protospacer adjacent motif (PAM) sequence (5'-NGG-3' for SpCas9) immediately 3' of the target for cleavage. What is the biochemical rationale for this PAM requirement?
- A The PAM is recognized by the PAM-interacting (PI) domain of Cas9, triggering conformational activation of the HNH and RuvC nuclease domains to generate a double-strand break ✓
- B The PAM sequence is required for sgRNA transcription by RNA polymerase III in the host cell
- C The NGG PAM recruits the Rad51 homologous recombination machinery to the cleavage site
- D The PAM prevents Cas9 from cutting its own CRISPR array in the bacterial host genome (self-protection)
Explanation
Cas9 remains in an inactive conformation until it encounters a PAM sequence. The PAM-interacting (PI) domain of SpCas9 recognizes 5'-NGG-3' on the non-template strand, triggering local DNA unwinding and RNA-DNA heteroduplex (R-loop) formation between the sgRNA spacer and the target strand. PAM recognition induces allosteric conformational changes that activate both the HNH domain (cleaves the strand complementary to sgRNA) and the RuvC domain (cleaves the opposite strand), generating a blunt-ended double-strand break 3 bp upstream of the PAM. This PAM requirement evolved in bacteria to prevent Cas9 from cleaving its own CRISPR array (which lacks PAM flanking of its spacers).
Reference: Harper's Illustrated Biochemistry, 32nd ed.
High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP
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