Engineering Guide RNAs for CRISPR-Based Biosensors

CRISPR-Cas systems, with their programmable nucleic acid-targeting capabilities, represent an ideal platform for constructing next-generation, highly sensitive biosensors. However, the clinical translation of these platforms is hindered by key limitations inherent to native single-guide RNAs (sgRNAs), including insufficient stability, potential immunogenicity, and off-target effects. To address these challenges, engineering sgRNAs has emerged as a central strategy to overcome such barriers and enhance overall biosensor performance. In this review, we provide a systematic overview of the field, beginning with the classification, molecular mechanisms, and structural features of representative CRISPR-Cas effector proteins to establish their foundational role as sensing elements. We then examine the specific limitations of native sgRNAs in biosensing applications. Building on this analysis, we highlight recent advances in sgRNA engineering strategies, which encompass three major approaches, including chemical modifications, structural remodeling, and modular functional integration. Furthermore, we review the integration of these engineered sgRNAs into advanced biosensor platforms, including microfluidic paper-based devices, centrifugal platforms, wearable patches, microneedles, and point-of-care testing (POCT) systems, and present a comparative table summarizing their performance in terms of detection signals, limits of detection, and other key metrics. Finally, we discuss persistent challenges such as the fine control of off-target effects, in vivo delivery bottlenecks, and system robustness in complex environments, and outline future directions toward amplification-free, multiplexed, and clinically translatable CRISPR-based biosensors. Overall, the engineering of sgRNAs offers a powerful means to systematically enhance the stability, specificity, and reliability of CRISPR-based biosensors, thereby accelerating their practical deployment in clinical diagnostics.