Conformational regulation of CRISPR-associated nucleases

•Cas nucleases are orthosterically and allosterically regulated.•DNase activation involves several molecular checkpoints.•Cas nucleases rely on conformational control to activate DNase.

Adaptive immune systems in bacteria and archaea rely on small CRISPR-derived RNAs (crRNAs) to guide specialized nucleases to foreign nucleic acids. The activation of these nucleases is controlled by a series of molecular checkpoints that ensure precise cleavage of nucleic acid targets, while minimizing toxic off-target cleavage events. In this review, we highlight recent advances in understanding regulatory mechanisms responsible for controlling the activation of these nucleases and identify emerging regulatory themes conserved across diverse CRISPR systems.