CRISPR-Cas9 is a programmable nuclease that uses a guide RNA to direct the Cas9 enzyme to a specific genomic sequence, where it introduces a precise double-strand break. The cell's native repair pathways can then be co-opted to delete the targeted region or splice in a new sequence via homology-directed repair. Applications now span therapeutic gene editing for sickle-cell disease, agricultural trait modification, and high-throughput functional genomics. Off-target effects, delivery to specific tissues, and germline-editing ethics remain active areas of research and regulation.

Schematic of the Cas9-guide-RNA complex binding DNA
Schematic of the Cas9-guide-RNA complex binding DNA

MECHANISM

The guide RNA (sgRNA) is a ~20 nt complementary sequence that Watson-Crick base-pairs with the target DNA strand. The Cas9 protein scans the genome for sequences adjacent to a Protospacer Adjacent Motif (PAM, typically 5'-NGG-3' for SpCas9) and forms an R-loop when the sgRNA finds its match. The HNH and RuvC nuclease domains then cleave the target and non-target strands respectively, producing a blunt double-strand break three nucleotides upstream of the PAM.