Programmable switching of CRISPR/Cas12a activity by adjusting guide RNA conformation for non-nucleic acid marker analysis and logic gate applications

Abstract

During the course of disease progression, changes in nucleic acid and non-nucleic acid marker contents can serve as indicators for disease diagnosis. The clustered regularly spaced short palindromic repeat (CRISPR)/Cas12a system, as a natural amplifier, has been widely exploited to construct amplified detection platforms for the detection of various analytes owing to its advantages of simplicity, high activity, and strong specificity. Nowadays, most of the focus is on the use of nucleic acid chains as response elements to activate Cas12a. Consequently, the development of switchable crRNA-based detection methods is urgently needed to expand such a switch's application in the quantification of non-nucleic acid analytes. A powerful and universal fluorescent sensing platform for the detection of non-nucleic acid analytes based on programmable switching of CRISPR/Cas12 activity via modulating crRNA conformation is proposed. In this system, two states, one in which the crRNA sequence is partially caged with a specially designed blocker DNA (“locked state”) and the other where native crRNA is regained after the blocker DNA gets lost (“unlocked state”), are modulated through target recognition. When a target is present, the function of the CRISPR/Cas12a system can be rapidly reinstated by switching locking to unlocking to cleave fluorescent reporters for signal output. As a proof-of-concept example, it was demonstrated that this platform could detect three analytes, namely adenosine triphosphate (ATP), glutathione (GSH), and formamidopyrimidine-DNA glycosylase (Fpg), with high specificity and good sensitivity (LOD_ATP = 0.046 μM, LOD_GSH = 0.1 μM, and LOD_Fpg = 0.2 U mL⁻¹). Moreover, by importing a programmable module, a set of CRISPR/Cas12 system-based logic gates was successfully constructed. Our constructed switchable crRNA-based sensing platform is universal, in which the target binding event is converted into the tunable trans-cleavage of ssDNA reporters via Cas12a, and thus, this platform could be expanded for detecting a wide spectrum of analytes by redesigning the corresponding sequence for target recognition. With its properties of universality, robustness, convenience, and programmability, the present platform indeed provides a useful and practical tool for molecular diagnosis and biomedicine research.