Smart crRNA engineering in CRISPR/Cas12a biosensing: From performance enhancement to application expansion

Abstract

The CRISPR/Cas12a system has emerged as a core tool in the fields of gene-editing and molecular diagnostics due to its high specificity, flexible programmability, and unique trans-cleavage activity. In this system, the crRNA serves as a crucial guiding component, with concise structure and remarkable plasticity, making it an ideal regulatory element to precisely control Cas12a activity and optimize system performance. However, the conventional CRISPR/Cas12a system still has limitations in sensitivity, controllability, and specificity that constrain its application potential in complex biological environments. To overcome these limitations, various engineered crRNA strategies have been developed to construct “smart guide RNA” responsive to specific endogenous or exogenous signals, thereby transforming the system into a dynamically regulatable multifunctional molecular platform. This review provides a systematic overview of recent advances in crRNA engineering for the CRISPR/Cas12a system, focusing on key strategies including photocontrolled, chemically modified, conformational-gated, length-regulated, and split approaches. Furthermore, their design principles, mechanisms of action, and applications in molecular diagnostics, gene regulation, and biosensing are discussed. These strategies achieve precise control of Cas12a activity across temporal, spatial, and logical dimensions by introducing external signal-responsive modules, regulating molecular conformation and physicochemical properties, optimizing structural parameters, and reconstructing functional domains. Finally, the review further summarizes the major challenges faced by crRNA engineering in practical applications and discusses its future perspectives. This provides theoretical references and research approaches for the design and application of next-generation high-performance, intelligent CRISPR tools.