Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity

• Published in: Cell 2018-12, Vol.175 (7), p.1856-1871.e21
• Main Authors: Stella, Stefano, Mesa, Pablo, Thomsen, Johannes, Paul, Bijoya, Alcón, Pablo, Jensen, Simon B., Saligram, Bhargav, Moses, Matias E., Hatzakis, Nikos S., Montoya, Guillermo
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Catalysis; CRISPR-Cas; CRISPR-Cas Systems; cryo-EM; DNA Cleavage; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - genetics; Francisella - chemistry; Francisella - genetics; Gene Editing; genome editing; protein-RNA-DNA complex; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - genetics; single-molecule FRET
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2018.10.045

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs. [Display omitted] •Structural landscape of Cas12a in the intermediate state by cryo-EM•smFRET shows the thermodynamic and kinetic characterization of Cas12a•The crRNA-DNA hybrid formation triggers the opening of the catalytic cleft•The displacement of the R-loop after target DNA cleavage shuts down the nuclease The cryo-EM structures of Cas12a and smFRET reveal the activation mechanism for target DNA cleavage and indiscriminate ssDNA degradation. The displacement of the R-loop by crRNA suggests a mechanism to stop unspecific ssDNA degradation, resetting the endonuclease to target a new DNA sequence.