Kinetic characterization of Cas9 enzymes

Bacterial adaptive immune systems employ clustered regularly interspaced short palindromic repeats (CRISPR) along with their CRISPR-associated genes (Cas) to form CRISPR RNA (crRNA)-guided surveillance complexes, which target foreign nucleic acids for destruction. Cas9 is unique in that it is composed of a single polypeptide that utilizes both a crRNA and a trans-activating crRNA (tracrRNA) or a single guide RNA to create double-stranded breaks in sequences complementary to the RNA via the HNH and RuvC nuclease domains. Cas9 has become a revolutionary tool for gene-editing applications. Here, we describe methods for studying the cleavage activities of Cas9. We describe protocols for rapid quench-flow and stopped-flow kinetics and interpretation of the results. The protocols detailed here will be paramount for understanding the mechanistic basis for specificity of this enzyme, especially in efforts to improve accuracy for clinical use.