A research team used CRISPR-Cas9 to knock out a gene encoding a surface receptor in human cell lines. After editing, some cells were found to contain the intended deletion, while others carried small insertions at the target site. Explain why the presence of insertions at the cut site would be expected, and suggest why these insertions may disrupt gene function even though the original gene sequence has not been deleted.
Written & reviewed by James Millett — Biology (Imperial College London), PGCE Science (University of Cambridge).
CRISPR-Cas9 is a genome-editing tool that uses a guide RNA to direct the Cas9 endonuclease to a specific DNA sequence, where it introduces a double-strand break. The cell then repairs this break using one of two main pathways: non-homologous end joining (NHEJ) or homology-directed repair (HDR).
Model answer (4 marks)
Insertions are a common outcome of the error‑prone, imprecise non‑homologous end joining (NHEJ) repair pathway that repairs the Cas9‑induced double‑strand break.
The small insertions alter the coding sequence, causing a frameshift mutation.
The frameshift changes the reading frame, producing a different, often truncated polypeptide.
A truncated or altered protein cannot fold correctly or bind its ligand, so the surface receptor is non‑functional.
The small insertions alter the coding sequence, causing a frameshift mutation.
The frameshift changes the reading frame, producing a different, often truncated polypeptide.
A truncated or altered protein cannot fold correctly or bind its ligand, so the surface receptor is non‑functional.
Examiner tips
- Use the term NHEJ and explain it is error‑prone; link to insertions. Show how a frameshift changes the reading frame. Mention loss of function (truncated protein, loss of ligand binding).
Common mistakes
- Confusing NHEJ with HDR; not mentioning frameshift. Assuming insertions are always deletions. Failing to link the altered protein to loss of receptor function.
Mark scheme (4 marks)
- Insertions arise because NHEJ is error-prone / imprecise
- Insertions cause a frameshift mutation in the coding sequence
- The altered reading frame produces a different (non-functional) amino acid sequence / truncated polypeptide
- The non-functional receptor means the protein cannot perform its normal role / cannot bind its ligand / correct tertiary structure is lost
Key terms in this question
Related
- All OCR A-Level Biology A (H420) revision notes →
- How to answer a "Explain" question →
- Decode the mark scheme abbreviations →
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