βοΈ CRISPR-Cas9
Step through the most powerful gene-editing tool ever devised: a guide RNA directs the Cas9 protein to a precise location in the genome, the double helix unwinds, 20 base pairs are interrogated one by one, both DNA strands are cut β then the cell repairs the break by error-prone NHEJ or precise HDR.
CRISPR-Cas9 Mechanism (Jinek et al., 2012)
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was adapted from a bacterial immune system into a programmable genome-editing tool. The two key components are:
- Cas9 protein β a molecular scissor with two nuclease domains (RuvC and HNH), each cutting one DNA strand.
- Guide RNA (gRNA) β a ~100 nt RNA consisting of a 20-nt spacer (complementary to the target) fused to a scaffold that binds Cas9.
Cas9 first scans the DNA for a PAM sequence (5β²-NGG-3β²) on the non-template strand. Once found, it locally unwinds the helix and checks base-pair complementarity between the spacer and the target. A near-perfect match triggers cleavage 3 bp upstream of the PAM, creating a blunt-ended double-strand break (DSB).
The cell repairs the DSB by one of two pathways: NHEJ (Non-Homologous End Joining) β fast but error-prone, generating small indels that disrupt gene function β or HDR (Homology-Directed Repair) β precise correction using a donor template, but only active in dividing cells.