A Sensitive Technique Unravels the Kinetics of Activation and Trans‐Cleavage of CRISPR‐Cas Systems

Abstract Activation of the CRISPR‐Cas13a system requires the formation of a crRNA‐Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR‐Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real‐time measurements of the dynamically formed active CRISPR‐Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants ( k on and k off ) and dissociation constant ( K d ) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real‐time trans ‐cleavage rates of the CRISPR‐Cas system and obtaining the real‐time concentrations of the active CRISPR‐Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7–37 °C) with fast trans ‐cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).