Cas9

Cas9 (CRISPR-associated protein 9) is a DNA cutting endonuclease that is part of the CRISPR immune system in bacteria and archaea. It has been adapted for use as a tool. The CRISPR-Cas9 system is widely used in genetic engineering, medicine, agriculture, and biotechnology.

The Cas9 endonuclease introduces site-specific double-stranded breaks in DNA, enabling gene inactivation, removal, or insertion through cellular repair mechanisms such as non-homologous end joining or homologous recombination.

Cas9 was discovered in S. pyogenes and later in S. thermophilus. It functions with two small RNAs—crRNA and tracrRNA—to form a four-component complex. In 2012, Doudna and Charpentier fused the RNAs into a single-guide RNA, enabling Cas9 to cut specific DNA sequences. These developments spurred genome modification efforts, including groups led by Zhang and Church who showed genome editing in human cells.

Variants of Cas9, including Cas9 nickase (Cas9n), that create single-strand breaks, and versions recognizing different PAM sequences, address specific Cas9 limitations.

CRISPR-Cas9 technology has raised ethical and regulatory concerns, particularly regarding human germline editing and genetic enhancement.