(365e) Preferential Trans-Cleaving Behavior of CRISPR-Cas12a Towards 3'-Overhang dsDNA Unlocks to Develop Ultrasensitive Hybrid DNA Reporter

The CRISPR-Cas12a system is generally considered to have no trans-cleavage activities towards blunt-end dsDNA substrates. The recent discovery of indiscriminate trans-cleavage of dsDNA substrates (e.g., λ DNA) promotes more studies on its actual mechanism. Here, we observed that a short toehold at the end of the blunt dsDNA could behave as a molecular switch, activating the substrate’s trans-cleaving activity. Additionally, we surprisingly discovered that the location of the overhang played a significant role: only a 3’-overhang could promote a complete trans-degradation of dsDNA substrate, while the 5’-overhang did not or only had a very mild effect. We hypothesized that the unique preferential trans-cleaving behavior of CRISPR-Cas12a towards 3’ overhang dsDNA might be governed by RuvC and Nuc domains. Utilizing this newly found behavior of Cas12a, we designed a hybrid DNA reporter, which sensitively detected ssDNA targets with a LOD of ~250 fM. This study not only casts a new insight into the mechanism understanding of Cas12a biology but also provided a new class of hybrid dsDNA reporters for more efficient CRISPR-Dx.