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

Woerle, Elisabeth and Jakob, Leonhard and Schmidbauer, Andreas and Zinner, Gabriel and Grohmann, Dina (2021) Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions. NUCLEIC ACIDS RESEARCH, 49 (9). pp. 5278-5293. ISSN 0305-1048, 1362-4962

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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.

Item Type: Article
Uncontrolled Keywords: RNA-GUIDED ENDONUCLEASE; R-LOOP COMPLEX; CRYSTAL-STRUCTURE; STRUCTURAL BASIS; SINGLE-MOLECULE; PAM RECOGNITION; CRISPR; CPF1; CLEAVAGE; CAS9;
Subjects: 500 Science > 570 Life sciences
600 Technology > 615 Pharmacy
Divisions: Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Mikrobiologie (Archaeenzentrum) > Prof. Dr. Dina Grohmann
Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann, formerly Prof. Seifert)
Depositing User: Dr. Gernot Deinzer
Date Deposited: 05 Jul 2022 06:32
Last Modified: 05 Jul 2022 06:32
URI: https://pred.uni-regensburg.de/id/eprint/45586

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