The specificity of the Cas9 nuclease is determined by the 20-nt guide sequence within the sgRNA. For the S. pyogenes system, the target sequence (e.g., 5′-GTCACCTCCAATGACTAGGG-3′) must immediately precede (i.e., be 5′ to) a 5′-NGG PAM, and the 20-nt guide sequence base pairs with the opposite strand to mediate Cas9 cleavage at ~3 bp upstream of the PAM (Figs. 1 and 4a top strand example). Note that the PAM sequence is required to immediately follow the target DNA locus, but that it is not a part of the 20-nt guide sequence within the sgRNA.
