CRISPR-Cas9 gene editing requires a guide RNA (gRNA) and Cas9 endonuclease. The gRNA directs Cas9 to the target by Watson-Crick base pairing with the DNA strand adjacent to which essential sequence that Cas9 recognises?

• A TATA box in the promoter region
• B Kozak sequence upstream of the start codon
• C Poly-A signal at the 3' end of the gene
• D Protospacer adjacent motif (PAM) — typically NGG for SpCas9 — immediately 3' of the target sequence ✓

Explanation

SpCas9 (from Streptococcus pyogenes) requires a PAM sequence (5'-NGG-3') on the non-template strand immediately 3' to the protospacer (the ~20 nt target). The gRNA (sgRNA) base-pairs with the complementary DNA strand; Cas9 unwinds DNA and the PAM-adjacent strand is cleaved by the HNH domain while the other strand is cleaved by the RuvC domain, creating a blunt double-strand break 3 bp upstream of the PAM. Without a PAM, Cas9 cannot bind productively. This restricts targetable sites to NGG-flanked sequences (~1 per 8 bp on average). TATA boxes, Kozak sequences, and poly-A signals are eukaryotic gene regulatory elements unrelated to Cas9 recognition.

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

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

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