Cas9

Cas9 is a DNA endonuclease that forms part of the CRISPR-Cas9 adaptive immune system in bacteria and is widely used for genome editing. In the lab, Cas9 is guided to a target DNA sequence by a programmable RNA molecule, enabling targeted double-strand breaks. The most commonly used variant is Streptococcus pyogenes Cas9 (SpCas9).

Cas9 requires a PAM sequence; with SpCas9 this is NGG, located next to the target site. The enzyme’s HNH and RuvC nuclease domains cleave both DNA strands, producing a blunt break three base pairs upstream of the PAM. Cells repair the break by non-homologous end joining or, with a donor template, by homology-directed repair.

Derived tools include catalytically inactive dCas9, which can recruit transcriptional regulators or fluorescent tags, enabling gene regulation and imaging. Further refinements include high-fidelity Cas9 variants to reduce off-target activity, as well as base editors and prime editors that enable more precise edits without creating a double-strand break in some contexts.

Applications span gene knockout, precise edits, and regulation in research, medicine, and agriculture. Delivery methods include plasmids, mRNA, ribonucleoprotein complexes, and viral vectors; each has trade-offs in efficiency and safety. Off-target effects, immune responses, and ethical considerations are active areas of discussion.

Cas9 was characterized in Streptococcus pyogenes and other bacteria as part of the CRISPR defense system. In 2012–2013, Jennifer Doudna and Emmanuelle Charpentier demonstrated programmable DNA cleavage by Cas9 guided by RNA, catalyzing rapid adoption of CRISPR-Cas9 for genome editing. They received the Nobel Prize in Chemistry in 2020.