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Summary: Bacterial adaptive immunity utilizes RNA-guided surveillance complexes comprising Cas proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single-guide RNA that base pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and the RuvC nuclease domains cleave opposing strands. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, and R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation. : Cas9 has revolutionized genome engineering applications. Gong et al. show that the rate-limiting step in Cas9 target cleavage is R-loop formation. R-loop formation is a two-step process, with RuvC adjustment occurring last. These results are consistent with an induced-fit mechanism for Cas9-mediated target cleavage. Keywords: Cas9, CRISPR, sgRNA, kinetics, R-loop, DNA cleavage