Fig. P1.

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.