Cas9 is a 160 kilodalton protein that is a vital component of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system in certain bacteria. It is a RNA-guided DNA endonuclease enzyme.

Mechanism of Action

Cas9 interrogates DNA by unwinding foreign DNA and checking for sites complementary to the 20 nucleotide spacer region of the guide RNA (gRNA). If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. Once Cas-9 has found a target site with the appropriate PAM, it triggers local DNA melting followed by the formation of RNA-DNA hybrid, but the mechanism of how Cas-9 enzyme melts target DNA sequence was not clearly understood yet. Then, the Cas-9 protein is activated for DNA cleavage. The Cas9:crRNA:tracrRNA complex recognizes the foreign DNA by using PAM sequence and DNA:RNA complementarity and triggers Cas9-catalyzed DNA. The Cas-9 nuclease makes double-stranded breaks (DSBs) at a site 3 base pair upstream to PAM.

Structure

Cas9 features a bi-lobed architecture with the guide RNA nestled between the alpha-helical lobe (blue) and the nuclease lobe (cyan, orange, and gray). These two lobes are connected through a single bridge helix. There are two nuclease domains located in the multi-domain nuclease lobe, the RuvC (gray) which cleaves the non-target DNA strand, and the HNH nuclease domain (cyan) that cleaves the target strand of DNA. A key feature of the target DNA is that it must contain a protospacer adjacent motif (PAM) consisting of the three-nucleotide sequence- NGG.

dCas9

The two crucial catalytic residues of the RuvC and HNH domain can be mutated to alanine abolishing all endonuclease activity of Cas9. The resulting protein coined 'dead' Cas9 or 'dCas9' for short, can still tightly bind to dsDNA. This catalytically inactive Cas9 variant has been used for both mechanistic studies into Cas9 DNA interrogative binding and as a general programmable DNA binding RNA-Protein complex.