CRISPR-Cas9 genome editing requires two essential components. The Cas9 endonuclease is guided to the target DNA sequence by:
- A A restriction enzyme recognition sequence immediately adjacent to the cut site
- B A single guide RNA (sgRNA) that base-pairs with the target DNA strand upstream of a PAM sequence ✓
- C A donor DNA template with homology arms flanking the desired insertion site
- D A zinc-finger nuclease domain fused to the Cas9 protein
Correct answer: B. A single guide RNA (sgRNA) that base-pairs with the target DNA strand upstream of a PAM sequence
Explanation
Cas9 forms a ribonucleoprotein complex with a single guide RNA (sgRNA), a synthetic fusion of the CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The ~20-nucleotide spacer sequence at the 5' end of the sgRNA base-pairs with the complementary DNA strand at the target locus immediately upstream of a protospacer adjacent motif (PAM, typically 5'-NGG-3' for SpCas9). PAM recognition by the Cas9 PAM-interacting domain is essential for target binding and HNH/RuvC nuclease domain activation, creating a double-strand break.
Reference: Harper's Illustrated Biochemistry, 32nd ed.
High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP
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