The CRISPR-Cas9 bacterial immune system and design of a CRISPR-Cas9 target sequence. (A) The CRISPR-Cas system acts as an adaptive immune system in bacteria and archaea. Clustered regularly interspaced short palindromic repeats (CRISPR) regions are stretches of repetitive genomic bacterial or archaea DNA interspersed by segments of foreign DNA sequences captured from bacterial phages and plasmids. A cluster of Cas (CRISPR associated) genes are located near the CRISPR region. The Cas9 gene, which is unique to type II CRISPR systems, codes for an RNA-guided endonuclease. Following foreign DNA infection in type II CRISPR systems, the CRISPR region is transcribed as a single RNA transcript called a pre-crRNA, and in type II systems, the pre-crRNAs are bound by tracrRNAs, processed into individual crRNA:tracrRNA duplexes by RNase III and form a complex with Cas9. The crRNA sequences are complementary to the foreign DNA and direct the Cas9 nuclease to form a complex with the foreign DNA and introduce a double-stranded break. (B) CRISPR-Cas9 target sequences are 20-nt long and are flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG.