Explain how the CRISPR‑Cas9 system introduces a site‑specific double‑strand break in a target DNA sequence.
Answer
CRISPR‑Cas9 uses a single‑guide RNA to direct the Cas9 endonuclease to a complementary DNA sequence, where Cas9 binds a PAM site and cleaves both strands, creating a double‑strand break.
Step-by-step solution
- The guide RNA (sgRNA) contains a 20‑nt sequence that is complementary to the target DNA.
- The Cas9 protein binds the sgRNA forming a ribonucleoprotein complex.
- The complex scans the genome for a PAM sequence (e.g., NGG) adjacent to the target region.
- Upon finding the PAM and complementary base‑pairing, Cas9 undergoes a conformational change that activates its nuclease domains.
- The HNH domain cuts the strand complementary to the guide, and the RuvC domain cuts the non‑complementary strand, generating a double‑strand break.
Common mistake: Assuming Cas9 can recognize DNA targets without the guide RNA
Concept tested: CRISPR‑Cas9 mechanism
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