The CRISPR/Cas9 system for genomic engineering. (a) A complex consisting of the CRISPR sgRNA and the Cas9 protein binds to a target site in the DNA. Two nuclease active sites in the Cas9 protein are indicated by red asterisks. The cleavage produces a double-strand break or, if one active site is inactivated, a single-strand break (nick). (b) If both nuclease active sites in Cas9 are inactivated, a complex is produced that simply binds tightly to its target sequence. This bound complex can be situated so as to block transcription of a particular gene by RNA polymerase. (c) If both nuclease active sites are inactivated, the Cas9 protein can be fused to a gene activator protein (see Chapter 19). If targeted to a site near an appropriate gene, the complex can be used to trigger enhanced transcription.