The Type II CRISPR system is one of the best characterized26–28,37,38, consisting of the nuclease Cas9, the crRNA array that encodes the guide RNAs and a required auxiliary trans-activating crRNA (tracrRNA) that facilitates the processing of the crRNA array into discrete units26,28. Each crRNA unit then contains a 20-nt guide sequence and a partial direct repeat, where the former directs Cas9 to a 20-bp DNA target via Watson-Crick base pairing (Fig. 1). In the CRISPR-Cas system derived from Streptococcus pyogenes (which is the system used in this protocol), the target DNA must immediately precede a 5′-NGG PAM27, whereas other Cas9 orthologs may have different PAM requirements, such as those of S. thermophilus (5′-NNAGAA22,26 for CRISPR1 and 5′-NGGNG28,37 for CRISPR3) and Neisseria meningiditis (5′-NNNNGATT)39.
