Structure-switchable branched inhibitors regulate the activity of CRISPR-Cas12a for nucleic acid diagnostics.

Background: In current years, the CRISPR (clustered regularly interspaced short palindromic repeats) based strategies have emerged as the most promising molecular tool in the field of gene editing, intracellular imaging, transcriptional regulation and biosensing. However, the recent CRISPR-based diagnostic technologies still require the incorporation of other amplification strategies (such as polymerase chain reaction) to improve the cis/trans cleavage activity of Cas12a, which complicates the detection workflow and lack of a uniform compatible system to respond to the target in one pot.

Results: To better fully-functioning CRISPR/Cas12a, we reported a novel technique for straightforward nucleic acid detection by incorporating enzyme-responsive steric hindrance-based branched inhibitors with CRISPR/AsCas12a methodology. The construction-transferable branched inhibitors coupled with a specific overhang flap induce spatial steric effects and result in the loss of the binding ability of Cas12a, which inhibits the activity of Cas12a. Target as the input signal would trigger the site-directed APE1 enzyme incision of the inhibitors, thus transforming the conformation of the inhibitors into split activators to illumine the CRISPR/AsCas12a catalyst system. At the same time, we found that APE1 could drive the enzymatic positive feedback circuit and exhibited considerably high amplification efficiency to enhance the detection ability of nucleic acids. Besides, our method provides universal platforms and can be realized in real-time and one-pot detection of HIV-1 DNA by replacing the inhibitors and crRNA with different target recognition sequences.

Significance And Novelty: Overall, due to the high programmability of the nucleic acid network, this work proposed a feasible way to use the steric hindrance-based inhibitors as a switchable element, decorating the CRISPR/Cas12a-based strategy equipment for molecular diagnostics. Besides, this strategy could offer a simple tool for detecting trace nucleic acid, which opens avenues for future clinical application.