To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleav-age. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (al-beit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in ‘seed ’ sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (posi-tions 13–18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit mod-est) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA ex-pression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9– gRNA interaction and cleavage beyond the general paradigm of site determination based on the ‘seed’ sequence and PAM
