In CRISPR-Cas9 gene editing, the guide RNA (gRNA) directs Cas9 to cut double-stranded DNA at a specific site. The absolute prerequisite sequence element present in the target DNA (not in the gRNA) is:
- A Protospacer adjacent motif (PAM): 5'-NGG-3' for SpCas9 ✓
- B TATA box upstream of the target site
- C Kozak consensus sequence for translation initiation
- D CpG island at the promoter of the target gene
Correct answer: A. Protospacer adjacent motif (PAM): 5'-NGG-3' for SpCas9
Explanation
SpCas9 (from Streptococcus pyogenes, the most widely used Cas9) requires a protospacer adjacent motif (PAM) sequence of 5'-NGG-3' immediately 3' of the target site in genomic DNA (the protospacer). The gRNA's 20-nt spacer sequence must match the protospacer; Cas9 then interrogates for the PAM, and upon PAM binding unwinds the DNA and checks gRNA-DNA complementarity, triggering a blunt DSB 3 bp upstream of the PAM. Without the PAM, Cas9 cannot cleave even perfectly matched targets. This PAM requirement provides a safety mechanism limiting off-target cutting.
Reference: Harper's Illustrated Biochemistry, 32nd ed.
High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP
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