Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions

Abstract The widespread and versatile prokaryotic CRISPR–Cas systems (clustered regularly interspaced short palindromic repeats associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to type V system was added enzymes repertoire employed for gene editing purposes. is a bilobal enzyme composed of REC Nuc lobe connected by wedge, REC1 domain bridge helix (BH). We generated BH variants integrated biochemical single-molecule FRET (smFRET) studies elucidate role enzymatic activity conformational flexibility Francisella novicida Cas12a. demonstrate impacts trimming mismatch sensitivity resulting in with improved cleavage accuracy. smFRET measurements reveal hitherto unknown open closed state apo preferentially adopt state. Transition Cas12a-crRNA complex inefficient but semi-closed ternary can be adopted even if deleted its entirety. Taken together, these insights structural element influences catalytic transitions FnCas12a.