Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Saturday Speaker Abstracts

DIRECT OBSERVATION OF CRISPR-CAS12 AS CONFORMATIONAL SAMPLING REVEALS HOW CONFORMATIONAL ACTIVATION PROMOTES CATALYSIS AND RESETTING OF THE ENDONUCLEASE ACTIVITY Nikos S Hatzakis 1,2 ; Stefano Stella 2 ; Pablo Mesa 2 ; Johannes Thomsen 1,2 ; Bijoya Paul 2 ; Pablo Alcon 2 ; Simon B Jensen 1,2 ; Bhargav Saligram 2 ; Matias E Moses 1,2 ; Guillermo Montoya 2 ; 1 University of Copenhagen , Chemistry & Nanoscience , Copenhagen , Denmark 2 University of Copenhagen , NovoNordisk center for Protein Research , Copenhagen , Denmark 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 combined cryoEM structures and single molecule FRET to provide a complete mechanistic understanding of endonuclease structural dynamics role in function and resetting (1). Cryo-EM readout provided the structures of intermediates of the cleavage reaction, and identified protein regions that sense the crRNA-DNA hybrid assembly triggering catalytic activation. Combined with single molecule readout of my group(2-4) and specifically FRET allowed us to directly observe the protein conformational dynamics along the entire reaction pathway. Parallel single molecule imaging provided the directionality of conformational transitions as well as the complete thermodynamic and kinetic characterisation of the conformational activation leading to function. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity degrading ssDNA molecules after activation and how other crRNAs displace the R-loop inside the protein after target DNA cleavage terminating indiscriminate ssDNA degradation. We proposed 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 recycle Cas12a specifically targeting new DNAs. 1. S. Stellaet al., Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity, Cell175, (2018) 1856.2. M. Liet al., Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase, J. Am. Chem. Soc.137, (2015) 16055.3. S. Veshaguriet al., Direct observation of proton pumping by a eukaryotic P-type ATPase, Science351, (2016) 1469.4. K. Bavishiet al., Direct observation of multiple conformational states in Cytochrome P450 oxidoreductase and their modulation by membrane environment and ionic strength, Sci Rep-Uk8, (2018).

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