CRISPR–Cas12a is a recent genome-editing system, which is promising for the diagnostics of viral infections, especially the novel SARS-COV-2 coronavirus that is rapidly spreading across multiple countries. Yet, the molecular basis of nucleic acid processing has remained elusive. Here, multi-microsecond molecular dynamics is used to characterize the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a.We show that DNA binding induces a “switch” in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable processing of the nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain – whose role is still controversial – could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prompt the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggest that REC2 could act as a “regulator” of the Nuc function, similar to what was observed previously for the HNH domain in the parent CRISPR-Cas9. Considering that the REC lobe is a key determinant in the system’s specificity (REF. XX), our findings provide a rational for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, these outcomes advance our mechanistic understanding of CRISPR-Cas12a and provides a ground for novel engineering efforts aimed at improved genome editing and viral detection.

DNA-induced dynamic switch triggers the activation of CRISPR-Cas12a

ACS Fall 2020 Virtual Meeting

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