Outline how the CRISPR‑Cas9 system can be used to correct a point mutation in a patient’s diseased cell. Include the roles of guide RNA, Cas9, a repair template, and the cellular repair pathway involved.
Answer
Guide RNA directs Cas9 to the mutant site, Cas9 creates a double‑strand break, a donor template with the correct sequence is supplied, and homology‑directed repair replaces the mutant base.
Step-by-step solution
- Design a guide RNA that is complementary to the DNA sequence adjacent to the mutation.
- Deliver Cas9 protein and the guide RNA into the patient’s cells.
- Allow Cas9 to bind the guide RNA and cleave the DNA at the target site, creating a double‑strand break.
- Introduce a repair template (single‑stranded DNA or plasmid) that contains the correct base and homology arms flanking the break.
- Rely on the cell’s homology‑directed repair (HDR) mechanism to use the template and replace the mutant sequence.
- Confirm that the repaired allele now matches the normal sequence, restoring gene function.
Common mistake: Thinking HDR occurs in all cells regardless of cell cycle phase
Concept tested: CRISPR gene editing mechanism
Published · How this solution was prepared and checked
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