pairing of its first 20 nt with the corresponding DNA sequence in the genome (Jinek et al. 2013). The only limitation to the targeting is a requirement for a protospacer adjacent motif (PAM) sequence adjacent to the target site, which is not present in the guide RNA, but is recognised by the Cas9 protein. In the case of the most widely used Streptococcus pyogenes Cas9 protein, this is NGG, which in a genome with an even base distribution and composition should occur every 8 bp. However, should this be a limitation, orthologues of Cas9 in other species have been discovered with alternative PAM requirements (Hou et al. 2013; Ran et al. 2015; Zetsche et al. 2015) and recently, protein engineering has been used to alter the PAM sequences of several Cas9 nucleases (Kleinstiver et al. 2015a, b) (as discussed elsewhere in this issue).
