CRISPR-Cas9 uses a short guide RNA (gRNA) to find a complementary 20-base target sequence in the genome next to a PAM motif. Once bound, the Cas9 protein's two nuclease domains cut both DNA strands, creating a double-strand break. The cell then repairs the break — either sloppily via Non-Homologous End Joining (NHEJ, which often introduces small insertions/deletions and disables the gene) or precisely via Homology-Directed Repair (HDR, which uses a donor template to insert an exact edit, but works less often).
Cutting efficiency ≈ f(specificity score, chromatin accessibility)
Off-target risk ↑ as guide-RNA sequence mismatch tolerance ↑
NHEJ: fast, error-prone, indel formation (most common repair)
HDR: slow, precise, requires donor template + active cell division
- Guide-RNA target — selects which base-pair position along the helix Cas9 is programmed to bind and cut.
- Cut efficiency — probability per attempt that Cas9 successfully cleaves the target once bound; higher values mean faster, more frequent cuts.
- Repair pathway toggle — NHEJ reconnects the strand roughly (visualized as a jagged, discolored splice); HDR reconnects it cleanly with an inserted edit segment (visualized as a clean glowing splice).
- Step / Auto-play — advances the scan-bind-cut-repair cycle manually or continuously.
This exact scan-bind-cut-repair cycle is the basis of real CRISPR therapies, such as the FDA-approved sickle-cell treatment that edits a patient's own hematopoietic stem cells.