Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flex-ible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 50 pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 50 repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed sub-stantial flexibility for unbound apo-Cas12a. In contrast, crRNA 50 pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluo-rimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 50 pseudoknot. This RNP as-sembly mechanism may be rationalized by evolutionary pres-sure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.