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. We recently published in CELL our studies where we combined cryoEM structures and single molecule FRET to provide a complete mechanistic understanding of endonuclease structural dynamics role in function and resetting. 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 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.

Conformational activation promotes CRISPR-Cas12a catalysis and resetting of the endonuclease activity

2019 Fall National Meeting

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