A cystic fibrosis patient has a G→A nonsense mutation in the CFTR gene that creates a premature stop codon at position 508. Design a CRISPR‑Cas9 strategy to correct this point mutation, specifying the roles of guide RNA, Cas9, repair template, and the cellular repair pathway involved.
Answer
Use a gRNA that targets the mutant sequence, Cas9 induces a double‑strand break, provide a single‑strand DNA template with the correct G, and rely on homology‑directed repair to restore the wild‑type CFTR allele.
Step-by-step solution
- Identify the mutant base (A) at position 508 in the CFTR coding sequence.
- Design a 20‑nt guide RNA complementary to the sequence immediately upstream of the mutant site, ensuring the presence of an NGG PAM for SpCas9.
- Form a Cas9–gRNA ribonucleoprotein complex and deliver it into patient cells (e.g., via lipid nanoparticles).
- Cas9 recognizes the gRNA target and generates a 3’–phosphate–ended double‑strand break 3 bp upstream of the PAM.
- Supply a single‑stranded oligodeoxynucleotide (ssODN) repair template containing the wild‑type G base, flanked by 60‑80 nt homology arms on each side of the break.
- The cell’s homology‑directed repair (HDR) machinery uses the ssODN to repair the break, inserting the correct G and restoring the full‑length CFTR transcript.
- After HDR, the mutant allele is replaced with the wild‑type allele, allowing production of functional CFTR protein.
Common mistake: Omitting the NGG PAM requirement in the guide RNA design.
Concept tested: CRISPR‑Cas9 gene editing
Published · How this solution was prepared and checked
← All Molecular Basis of Inheritance questions