CRISPR-Cas9 achieves site-specific DNA cleavage by:
- A A guide RNA (gRNA) with a 20-nt spacer complementary to the target DNA plus a PAM sequence recognised by Cas9, directing double-strand DNA cleavage ✓
- B Cas9 protein recognising the target sequence through direct protein-DNA interactions alone
- C Two Cas9 molecules binding opposite strands independently to introduce a nick at the same position
- D Cas9 acting as a restriction endonuclease requiring specific palindromic sequences
Correct answer: A. A guide RNA (gRNA) with a 20-nt spacer complementary to the target DNA plus a PAM sequence recognised by Cas9, directing double-strand DNA cleavage
Explanation
The Cas9 endonuclease is directed by a single guide RNA (sgRNA) containing a 20-nucleotide spacer complementary to the target DNA strand; Cas9 also requires a PAM (protospacer adjacent motif — 5'-NGG-3' for SpCas9) immediately 3' of the target sequence for strand melting and cleavage. The RuvC domain cuts the non-complementary strand and HNH cuts the complementary strand, creating a blunt-ended DSB. Nickase Cas9 uses two nicked sgRNAs but this is a modification, not the standard mechanism.
Reference: Harper's Illustrated Biochemistry, 32nd ed.
High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP
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