Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

The Cas9–crRNA complex generates two distinct DNA nicks on opposing dsDNA strands that match the loaded small, interfering crRNA sequence. (A) After phage DNA entry into the cell, a piece of the invading DNA is inserted as a spacer into the CRISPR locus. (B) The CRISPR repeat-spacer array is transcribed and processed into short crRNAs. (C) The crRNA forms a ribonucleoprotein complex with Cas9, which recognizes invading DNA homologous to the crRNA sequence and mediates interference. (D) Invading DNA cleavage by Cas9–crRNA. In the presence of Mg²⁺ ions, the signature Cas9 protein nicks each DNA strand 3 nt upstream of the PAM sequence to generate blunt DNA ends through RuvC- and HNH-like active sites that act on separate DNA strands.