Cooperative activation of CRISPR-Cas12a based on optimization of nucleic acid hybridization kinetics and thermodynamics: More specific, flexible and controllable

Abstract

The high efficiency of CRISPR-Cas12a is highly beneficial for rapid and sensitive detection of low-concentration markers. However, its sloppy identification and uncontrollable high activity lead to a significant loss in specificity, flexibility, and expandability. In this paper, we present a cooperative activation strategy that is both simple and powerful, based on the kinetic and thermodynamic mechanisms of nucleic acid hybridization reactions. Using the EGFR T790M and L858R mutant as models, we study base by base in detail and demonstrate that the cooperative activation strategy has strong general properties that can be applied to various sequence systems. Utilizing the sub-stable state of hybridization to effectively improve the specificity of single-base mismatches of activators, and also dramatically improves the design flexibility and utilization efficiency of crRNAs by cooperating with different sequences. In addition, this strategy also creatively uses the strand displacement reaction to terminate the trans-activity at any time, and more importantly, such inhibition is reversible, which undoubtedly greatly improves the controllability of the CRISPR-Cas12a system, and enables it to have a more and more abundant functional expansion space. Overall, our strategy has obvious advantages, which will unleash the potential for expanding and deepening the application of CRISPR technology in molecular biology.