A researcher wants to use CRISPR to correct a point mutation that causes a genetic disease in patient cells. What components are required to achieve a precise correction via homology-directed repair?
AOnly a guide RNA matching the target sequence — Cas9 will correct the mutation automatically once it finds the site
BA guide RNA, Cas9 protein, and a DNA repair template containing the corrected sequence flanked by homologous regions
CA guide RNA and activation of NHEJ, which will insert the correct sequence at the cut site
DOnly Cas9 — the guide RNA is needed only for knockout experiments, not for precise corrections
Precise correction requires HDR, which copies from a template. Without a provided template, cells default to NHEJ, which joins broken ends imprecisely and typically introduces indels. HDR requires the guide RNA (for targeting), Cas9 (for cutting), and a template with the desired sequence flanked by sequences homologous to the genomic cut site.
Question 2 Multiple Choice
Why does NHEJ editing with CRISPR typically result in gene disruption rather than a predictable specific change?
AThe Cas9 enzyme degrades nucleotides at the cut site before ligating the ends
BNHEJ ligates broken ends without using a template, often introducing small insertions or deletions that shift the reading frame
CThe guide RNA degrades the target strand before the break is repaired, leaving a permanent gap
DThe indels are introduced by the PAM sequence adjacent to the cut site during repair
NHEJ is a fast but imprecise pathway — it rejoins broken ends without a template, and the ligation is error-prone. The resulting insertions or deletions (indels) are unpredictable in their exact sequence, but if they fall in a coding exon, they frequently disrupt the reading frame and produce a truncated, non-functional protein — a knockout.
Question 3 True / False
CRISPR-Cas9 edits DNA by directly replacing the target sequence with a new sequence specified by the researcher.
TTrue
FFalse
Answer: False
Cas9 only cuts both strands of DNA at the target site; it does not insert or replace any sequence. All sequence changes result from the cell's own DNA repair machinery. NHEJ repairs the break imprecisely (producing indels), while HDR can incorporate a provided template precisely — but in both cases, Cas9's role ends at the cut.
Question 4 True / False
The PAM sequence (e.g., NGG for SpCas9) is required for Cas9 to initiate DNA unwinding and check for guide RNA complementarity at a potential target site.
TTrue
FFalse
Answer: True
Cas9 first scans the genome for PAM sequences — its docking signal. Upon finding a PAM, it unwinds the adjacent DNA and checks whether the guide RNA matches. Without a PAM, Cas9 does not engage the DNA, even if the guide RNA matches the nearby sequence. This two-step recognition (PAM first, then base pairing) provides the specificity of targeting.
Question 5 Short Answer
Explain why the repair pathway used after a CRISPR cut determines the nature of the edit, and why researchers cannot fully control which pathway the cell uses.
Think about your answer, then reveal below.
Model answer: Cas9 creates a double-strand break, but the outcome depends on which of the cell's repair pathways closes that break. NHEJ — the default, faster pathway — rejoins ends without a template, producing imprecise indels that usually disrupt gene function. HDR — a slower, template-dependent pathway — can incorporate a provided sequence precisely, but it requires the cell to be in S or G2 phase and competes with NHEJ. Because NHEJ is more active in most cell types and most of the cell cycle, it wins the competition most of the time. Researchers can bias toward HDR by supplying a template and sometimes by inhibiting NHEJ, but cannot guarantee which pathway the cell will use.
CRISPR's dependency on cellular repair machinery means its outcomes are probabilistic, not fully deterministic. This is why CRISPR is described as creating a break that repair machinery then 'mends — often imperfectly.'