STRUCTURE AND FUNCTION OF Lb2CAS12A AND ITS RNA-DNA COMPLEXS UNCOVER THE CRRNA CAPTURING AND DNA CLEAVAGE MECHANISMS AND ITS APPLICATIONS

Abstract

The CRISPR-Cas protein, Cas12a, processes precursor crispr (cr) RNA and cleaves invading genetic material. We report the crystal structures of apo Lachnospiraceae bacterium MA2020 (Lb2)Cas12a and the Lb2Cas12a-crRNA complex, and cryo-EM structures of Lb2Cas12a-crRNA-DNA heteroduplex in different conformations along with functional studies. Apo Lb2Cas12a is in the unique elongated open conformation and its single-stranded (ss) DNA cleavage activity is triggered by Mn2+ accompanied by a “Lid” movement, while the crRNA, crRNA-DNA duplex bound forms are in the closed/intermediate conformations. The Lb2Cas12a recognition lobe displays the unique mobility of 102 Å movement and 103 rotation aiding to position the RNA-DNA duplex near the catalytic site. Further, we present an in vitro single crRNA array strategy to detect multiple-genes, reducing the probability of detection escape, and demonstrated with SARS-CoV-2, MERS, and DENV. Our findings provide insight for crRNA capture, crRNA-independent ssDNA cleavage, open/close conformation of Cas12a for activity and a clinically relevant method for nucleic acid detection.