The 20‑nt guide RNA directs Cas9 to a complementary genomic locus immediately upstream of an NGG PAM. Cas9 unwinds DNA at that site and cleaves both strands. If no repair template is provided, error‑prone NHEJ rejoins ends—often introducing indels that disrupt the gene. When a donor template is supplied, HDR leverages homology arms to insert or correct sequences precisely. Strategies like truncated guides, high‑fidelity Cas9 variants, and careful PAM selection reduce off‑target cuts
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How might guide RNA and PAM recognition drive CRISPR’s pinpoint accuracy?
asked 6 days ago
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In CRISPR‑Cas9 editing, I want to explore how the sequence and design of guide RNA plus the protospacer adjacent motif work together to ensure on‑target binding, and compare how cells employ NHEJ versus HDR to knock out or precisely insert DNA sequences.
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