A Secondary Checkpoint Refines CRISPR Precision

Using dynamic simulations, researchers have discovered that the presence of base mismatches in DNA triggers alterations in the HNH nuclease domain of Cas9. These modifications hinder its conformational activation, indicating a secondary checkpoint after DNA binding but before the cleavage event.

The mechanism appears to act as an additional fidelity check, ensuring only correct DNA sequences are cleaved. EvoCas9, a mutant variant of Cas9, shows even greater sensitivity to mismatches.

EvoCas9 deviates from the typical activation pathway, significantly shifting the HNH domain's positioning and disturbing the enzyme's allosteric communication. This response reinforces the secondary checkpoint's role in maintaining high gene-editing accuracy, suggesting that mutations in evoCas9 preserve and enhance this precision-enhancing mechanism.

The study not only provides a molecular basis for the high fidelity of CRISPR-Cas9 gene editing but also paves the way for the development of more accurate CRISPR technologies in the future.

Zhicheng Zuo led the study at Shanghai University of Engineering Science, and it was published in the Journal of Chemical Theory and Computation on Wednesday. You can read it here.