Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a

CRISPR/Cas12a has been believed to be powerful in moleculardetectionand diagnostics due to its amplified trans-cleavage feature. However,the activating specificity and multiple activation mechanisms of theCas12a system are yet to be elucidated fully. Herein, a synergisticactivator effect is discovered, which supports an activationmechanism that a synergistic incorporation of two short ssDNA activatorscan promote the trans-cleavage of CRISPR/Cas12a, while either of themis too short to work independently. As a proof-of-concept example,the synergistic activator-triggered CRISPR/Cas12a system has beensuccessfully harnessed in the AND logic operation and the discriminationof single-nucleotide variants, requiring no signal conversion elementsor other amplified enzymes. Moreover, a single-nucleotide specificityhas been achieved for the detection of single-nucleotide variantsby pre-introducing a synthetic mismatch between crRNA and the helperactivator. The finding of synergistic activator effectnot only provides deeper insight into CRISPR/Cas12a but also may facilitateits expanded application and power the exploration of the undiscoveredproperties of other CRISPR/Cas systems.

Automated summary

A synergistic activator effect is discovered, where the incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a. This finding provides deeper insight into CRISPR/Cas12a and may facilitate its expanded application and exploration of other CRISPR/Cas systems.