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

Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

“…Here we perform single-molecule fluorescence assay to monitor conformational dynamics of the Cas12a ternary complex during TS cleavage following the initial cleavage of NTS of DNA. Recently, several groups have utilized similar methodological approaches to monitor the molecular interaction between Cas12a RNP and target DNA by using labeled target DNA and crRNA ( 35 – 37 ) and the interdomain dynamics of Cas12a protein by using labeled Cas12a ( 31 , 41 ). Using this assay, here we identified the features of intermediates that form during conformational rearrangements in the TS cleavage reaction to complete dsDNA cleavage and revealed its underlying mechanism based on a kinetic analysis of the conformational dynamics.…”

Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

“…Wildtype FnCas12a and FnCas12a variants were expressed and purified as previously described (24). In short, the Cas12a gene from Francisella novicida (Addgene #69975) was cloned into a pGEX-2TK expression vector.…”

“…Electrophoretic mobility shift assays of ternary complexes (Cas12a-crRNA-target DNA) were conducted as previously described (24). The pre-formed binary complex (200 nM) was added in 7.5-fold (75 nM) excess to 10 nM of fluorescently labeled target DNA (10 nM).…”

“…The crRNA furthermore interacts with the RuvC and REC domains (11, 23, 33, 34). Upon loading of target DNA to the Cas12a-crRNA complex, the Nuc and REC lobes transition from a closed to an open conformation (11, 15, 24).…”

“…Previous studies showed that mutations in the BH have drastic effects on the cleavage activity and accuracy of Cas12a (24, 35). However, FnCas12a variants with full deletion of the BH are still able to bind crRNA and target DNA and to undergo the conformational transition upon crRNA and DNA binding (24). According to crystal structures of the binary Cas12a-crRNA complex and the ternary Cas12a-crRNA-target DNA complex (11, 15, 18, 23, 33, 34, 36), the BH is connected to and acts in concert with the adjacent helix of the RuvC domain (termed helix 1).…”