Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Abstract

CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes 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 cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain 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 prime 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 suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system’s specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.

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

“…This indicates that the distance between WED and Nuc domain and the WED domain and REC lobe do not significantly change when Cas12a progresses through its activity cycle. This is in agreement with structural studies ( 2 , 21 ) and molecular dynamics simulations ( 50 ), in which only marginal changes in distances between these positions can be detected ( Supplementary Figure S7 ). Using the Cas12a Nuc-REC variant, a low FRET population with a FRET efficiency of 0.12 (± 0.04) and a high FRET population ( E = 0.97 ± 0.01) can be detected for the nucleic-acid free Cas12a.…”

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

“…This is in agreement with structural data for FnCas12a and AsCas12a (2,17,36–38, 20,21,30–35). Our finding that the ternary complex is more flexible than the binary complex agrees with recent molecular dynamics simulations that showed the increased flexibility of the REC and Nuc lobe in the ternary complex (43). We found that loading of the DNA target is more efficient when a ssDNA target is provided suggesting that the formation of a Cas12a-crRNA-dsDNA complex is energetically demanding.…”

“…This indicates that the distance between WED and Nuc domain and the WED domain and REC lobe do not significantly change when Cas12a progresses through its activity cycle. This is in agreement with structural studies (2, 21) and molecular dynamics simulations (43), in which only marginal changes in distances between these positions can be detected ( Figure S7). Using the Cas12a Nuc-REC variant, a low FRET population with a FRET efficiency of 0.12 (± 0.04) and a high FRET population (E = 0.97 ± 0.01) can be detected for the nucleicacid free Cas12a.…”

Establishing the allosteric mechanism in CRISPR‐Cas9

“…A similar allosteric control orchestrating interdependent domain dynamics was also identified in the activation of yet another CRISPR‐Cas system, viz. Cas12a, upon binding with the target dsDNA 65 . An allosteric regulation was also found to occur upon initial PAM recognition, governing the conformational mobility of the catalytic HNH domain 16,17 .…”

“…Cas12a, upon binding with the target dsDNA. 65 An allosteric regulation was also found to occur upon initial PAM recognition, governing the conformational mobility of the catalytic HNH domain. 16,17 Specifically, the recognition lobe was found to exert an allosteric control of the HNH activation through the REC2 and REC3 regions.…”