Universal crRNA Acylation Strategy for Robust Photo‐Initiated One‐Pot CRISPR–Cas12a Nucleic Acid Diagnostics

Abstract Spatiotemporal regulation of clustered regularly interspaced short palindromic repeats (CRISPR) system is attractive for precise gene editing and accurate molecular diagnosis. Although many efforts have been made, versatile and efficient strategies to control CRISPR system are still desirable. Here, we proposed a universal and accessible acylation strategy to regulate the CRISPR–Cas12a system by efficient acylation of 2′‐hydroxyls (2′‐OH) on crRNA strand with photolabile agents (PLGs). The introduction of PLGs confers efficient suppression of crRNA function and rapid restoration of CRISPR–Cas12a reaction upon short light exposure regardless of crRNA sequences. Based on this strategy, we constructed a universal PhotO‐Initiated CRISPR–Cas12a system for Robust One‐pot Testing (POIROT) platform integrated with recombinase polymerase amplification (RPA), which showed two orders of magnitude more sensitive than the conventional one‐step assay and comparable to the two‐step assay. For clinical sample testing, POIROT achieved high‐efficiency detection performance comparable to the gold‐standard quantitative PCR (qPCR) in sensitivity and specificity, but faster than the qPCR method. Overall, we believe the proposed strategy will promote the development of many other universal photo‐controlled CRISPR technologies for one‐pot assay, and even expand applications in the fields of controllable CRISPR‐based genomic editing, disease therapy, and cell imaging.