Integrating CRISPR/Cas Biosensors with Advanced Platforms: A Holistic Path Toward Preamplification-free, Multiplexed, and Continuous Molecular Monitoring.

The paradigm of molecular diagnostics has been transformed by the repurposing of CRISPR-Cas systems from being gene-editing tools to nucleic acid detection engines with remarkable specificity and programmability. Both the SHERLOCK and DETECTR platforms have shown high sensitivity and specificity; however, the requirement of a pre-amplification step to achieve clinically relevant detection limits adds another layer of complexity and cost and is also a potential source of contamination, precluding their use as true point-of-care (POC) tools. The next frontier for CRISPR diagnostics will be the design of biosensors that enable preamplification-free, multiplex, and continuous direct detection of targets. Achieving this goal will involve the very close integration of CRISPR biology with nano-biotechnology, microfluidics, orthogonal Cas enzyme systems, and artificial intelligence (AI). This review aims to provide a comprehensive overview of recent advancements and strategic thinking related to this integration. This review discusses how nanomaterials facilitate signal generation and transduction, how microfluidics automates, multiplexes, and miniaturizes "all-in-one" devices, and how orthogonal CRISPR systems can enable robust multiplexing. We will also probe into the emerging application of AI to accelerate guide RNA design and optimize the performance of CRISPR biosensors. Furthermore, the roles of orthogonality and nanomaterials in real-time, continuous molecular monitoring will be assessed. The review will finally discuss the transformative future applications of high-throughput biomarker discovery and theranostics potential through massively parallelized CRISPR sensing.