Molecular Mechanism - Edubirdie

University:
University of California, San Diego
Course:
BIMM 100 | Molecular Biology
Academic year:

2024
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241

Pages:

1
Author:

customer-8930767

molecular mechanism of CRISPR-Cas9 gene editing, including the roles of tracrRNA, crRNA, and PAM sequences. CRISPR-Cas9 is an adaptive immune system in Streptococcus pyogenes repurposed for gene editing. The system requires two RNA molecules: CRISPR RNA (crRNA) and trans-activating crRNA ( tracrRNA), often fused into a single guide RNA (sgRNA). The crRNA contains a 20-nucleotide spacer complementary to the target DNA; the tracrRNA forms a scaffold that binds Cas9. Cas9 is an endonuclease with two catalytic domains: RuvC (cleaves non-complementary strand) and HNH (cleaves complementary strand). The mechanism begins with Cas9 binding to the sgRNA. Target recognition requires a protospacer adjacent motif (PAM)—a short sequence (5’-NGG-3’ for SpCas9) immediately downstream of the target site in the DNA. Cas9 scans DNA by PAM interrogation; upon PAM binding, the target DNA is melted, allowing crRNA–DNA base pairing. If complementarity exists, Cas9 undergoes conformational activation, positioning the HNH and RuvC domains to cleave each DNA strand 3 bp upstream of the PAM, creating a double-strand break (DSB). DSB repair occurs via non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ introduces insertions/deletions (indels), causing gene disruption. HDR, using a donor template, enables precise sequence replacement or insertion. Off-target effects arise from partial complementarity tolerance. High-fidelity variants (e.g., SpCas9-HF1) reduce off-target activity. Thus, CRISPR-Cas9 enables targeted mutagenesis, gene knockouts, and corrections.

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