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ABSTRACT CRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks and promote non-specific DNA cleavage after identifying specific DNA. To mitigate the off-target DNA cleavage of Cas12a, we previously developed aFrancisella novicidaCas12a variant (FnoCas12a^KD2P) by introducing double proline substitutions (K969P/D970P) in a conserved helix called the bridge helix (BH). In this work, we used cryogenic electron microscopy (cryoEM) to understand the molecular mechanisms of BH-mediated activation of Cas12a. We captured five structures of FnoCas12a^KD2Pat different states of conformational activation. Comparison with wild-type (FnoCas12a^WT) structures unravels a mechanism where BH acts as a trigger that allosterically activates REC lobe movements by tracking the number of base pairs in the growing RNA-DNA hybrid to undergo a loop-to-helical transition and bending to latch onto the hybrid. The transition of the BH is coupled to the previously reported loop-to-helix transition of the “lid”, essential for opening RuvC endonuclease, through direct interactions of residues of the BH and the lid. We also observe structural details of cooperativity of BH and “helix-1” of RuvC for activation, a previously proposed interaction. Overall, our study enables development of high-fidelity Cas12a and Cas9 variants by BH-modifications. 

Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine.