Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview

•PAM sequences are uniquely associated with each Cas9 protein.•PAM is required to initiate base-pairing between guide RNA and DNA target.•High-throughput screening approaches allow PAM identification.•Cas9 with novel PAM requirements may expand toolbox for genome editing. Recently the Cas9, an RNA guided DNA endonuclease, emerged as a powerful tool for targeted genome manipulations. Cas9 protein can be reprogrammed to cleave, bind or nick any DNA target by simply changing crRNA sequence, however a short nucleotide sequence, termed PAM, is required to initiate crRNA hybridization to the DNA target. PAM sequence is recognized by Cas9 protein and must be determined experimentally for each Cas9 variant. Exploration of Cas9 orthologs could offer a diversity of PAM sequences and novel biochemical properties that may be beneficial for genome editing applications. Here we briefly review and compare Cas9 PAM identification assays that can be adopted for other PAM-dependent CRISPR-Cas systems.