In CRISPR-Cas9 gene editing, guide RNA (gRNA) directs Cas9 nuclease to a target DNA sequence. Which additional DNA sequence is an absolute requirement adjacent to the target for Cas9 to cleave?

• A Kozak sequence upstream of the target
• B TATA box promoter element upstream of the target
• C PAM sequence (protospacer adjacent motif, 5'-NGG-3' for SpCas9) immediately 3' of the target ✓
• D Polyadenylation signal downstream of the target

Explanation

For Streptococcus pyogenes Cas9 (SpCas9), the PAM sequence 5'-NGG-3' must be present immediately 3' of the 20-nt protospacer sequence on the non-template strand. Cas9 first recognises the PAM, then unwinds the DNA to check gRNA complementarity. If the 20-nt spacer matches the target, Cas9 cleaves both DNA strands (HNH domain cleaves the strand complementary to gRNA; RuvC domain cleaves the non-complementary strand) to create a blunt-ended double-strand break 3 bp upstream of the PAM. Absence of a PAM prevents Cas9 cleavage even with perfect gRNA complementarity. This PAM requirement also protects the Cas9-encoding genome from self-cleavage. Kozak, TATA box and polyA signals are RNA/protein synthesis elements unrelated to CRISPR targeting.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

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