CRISPR-Cas9 genome editing uses guide RNA (gRNA) to direct the Cas9 nuclease to a specific genomic locus. After creating a double-strand break (DSB), the cell repairs the break via two major pathways. For therapeutic gene correction in sickle cell disease, which repair pathway would be deliberately utilized to achieve precise correction of the HBB beta-6 codon?

• A Non-homologous end joining (NHEJ) to introduce targeted insertions at the beta-globin locus
• B Base excision repair to convert the mutant valine codon back to glutamic acid
• C Mismatch repair to detect and correct the single nucleotide substitution
• D Homology-directed repair (HDR) using a donor DNA template containing the corrected sequence, exploiting the homologous recombination machinery ✓

Explanation

Cas9-induced DSBs are repaired predominantly by NHEJ in post-mitotic cells, which is error-prone and creates insertions/deletions (indels) — useful for gene disruption. For precise correction (such as changing the HBB codon 6 GTG back to GAG), homology-directed repair (HDR) is exploited by co-delivering a donor template containing the corrected sequence flanked by homology arms. HDR only occurs efficiently in dividing cells (S/G2 phase) since it requires the homologous recombination machinery. Current clinical trials (e.g., CTX001/exagamglogene autotemcel for SCD) use CRISPR to reactivate fetal hemoglobin via BCL11A disruption rather than direct HBB correction, circumventing HDR efficiency limitations.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

Written and medically reviewed by the StethoPrep medical team.