Guide RNA targeting, PAM recognition, R-loop formation, DSB and repair
Mechanism Steps
Details
Cas9 + sgRNA: SpCas9 (from S. pyogenes) is a 1368 aa protein with two nuclease domains — RuvC (cuts non-target strand) and HNH (cuts target strand).
The single guide RNA (sgRNA) = crRNA + tracrRNA fusion. The 20 nt spacer sequence is programmable — change spacer, change target. Cas9 searches genome by 3D diffusion + 1D sliding.
Repair Pathways
NHEJ (Non-Homologous End Joining): default in most cells. Error-prone — inserts or deletes bases (indels). Used to knock out genes.
HDR (Homology-Directed Repair): requires a DNA template. ~1% efficiency in most cells, higher in S-phase. Used for precise corrections and insertions.