In CRISPR-Cas9 gene editing, the guide RNA (gRNA) must match a target DNA sequence adjacent to which motif for Cas9 cleavage to occur?

• A TATA box (upstream regulatory element used by Cas9 to find transcriptionally active regions)
• B CpG island (present near gene promoters, which Cas9 recognises for epigenetic editing only)
• C Splice acceptor site (AG dinucleotide) that Cas9 uses to verify intronic versus exonic targeting
• D PAM sequence (Protospacer Adjacent Motif, typically 5'-NGG-3' for SpCas9), located immediately downstream of the target protospacer ✓

Explanation

Cas9 protein requires a PAM (Protospacer Adjacent Motif) sequence — for the most commonly used SpCas9 from Streptococcus pyogenes, the PAM is 5'-NGG-3' on the non-target strand, located immediately 3' of the 20-nt protospacer sequence. The gRNA base-pairs with the protospacer, and Cas9 unwinds the DNA only if the PAM is present adjacent to the target; without PAM recognition, Cas9 does not cleave. This prevents Cas9 from cutting its own genome (where guide sequences exist within CRISPR array loci without adjacent PAM). Different Cas orthologues use different PAM sequences (e.g., Cas12a uses 5'-TTTV-3').

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

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

Written and medically reviewed by the StethoPrep medical team.