Bridging the sensitivity gap in amplification-free CRISPR diagnostics: An engineering-driven redesign

The CRISPR/Cas system, with its programmable nucleic acid recognition and cleavage capabilities, has emerged as a powerful platform for next-generation molecular diagnostics. However, the intrinsic trans-cleavage activity natural Cas effector proteins remain limited, which constrains the direct detection of low-abundance targets in amplification-free diagnostic formats. This review focuses on the sensitivity bottleneck in amplification-free CRISPR diagnostics and provides a structured overview of recent engineering strategies aimed at enhancing CRISPR performance. These strategies are organized around five key components, including Cas protein, crRNA, DNA activator, reporter probe, and reaction buffer systems. By engineering these modules, recent studies have improved reaction efficiency, signal output, and assay robustness, thereby reducing reliance on traditional nucleic acid pre-amplification and lowering contamination risk. We further discuss how the interplay among these components influences overall system performance and highlight remaining challenges in improving sensitivity in complex biological matrices. Finally, we outline future directions, including improved system integration, enhanced compatibility with real-world samples, and more effective translation toward clinical applications for infectious disease diagnosis and trace biomarker detection.