In recent years， Cas9 has revolutionized the genome-editing field and enabled a broad range of applications from basic biology to biotechnology and medicine. Cas9 specificity is dictated by base pairing of the guide RNA to the complementary DNA strand， however to initiate hybridization， a short protospacer adjacent motif (PAM) sequence is required in the vicinity of the target sequence. The PAM is recognized by the Cas9 protein and varies between Cas9s. There are thousands of type II CRISPR-Cas9 sequences available in sequence databases. To characterize the PAM recognition diversity provided by Cas9 orthologs， we developed a phylogeny-guided bioinformatics approach and streamlined our experimental procedures for Cas9 expression and RNP complex assembly using cell lysates and in vitro translation mixtures. This approach could be easily adapted for the characterization of other CRISPR-Cas nucleases that require PAM sequences and generate double-strand breaks following target recognition.