CRISPR-Cas biosensors have emerged as a transformative technology for nucleic acid detection, offering high specificity, sensitivity, and rapid results. These systems leverage the programmable nature of CRISPR-associated (Cas) proteins to identify target sequences, making them ideal for diagnosing infectious diseases such as SARS-CoV-2. A key feature enabling their utility is collateral cleavage activity, where upon target recognition, the Cas enzyme indiscriminately degrades nearby single-stranded nucleic acids. This property amplifies detection signals and facilitates integration with various readout mechanisms, including electrochemical and optical systems.

Collateral cleavage is central to CRISPR-based diagnostics. For example, Cas12 and Cas13 proteins exhibit trans-cleavage behavior, where activation by a target RNA or DNA triggers non-specific cutting of reporter molecules. These reporters are often fluorescent or chemically labeled oligonucleotides, producing measurable signals upon cleavage. In fluorescent readouts, collateral activity releases quenched fluorophores, generating a quantifiable light signal. Electrochemical approaches leverage cleavage-induced changes in conductivity or redox reactions, enabling portable and low-cost detection. The combination of CRISPR specificity with signal amplification through collateral cleavage allows detection limits as low as attomolar concentrations, rivaling traditional PCR in sensitivity while eliminating the need for thermocycling.

Optical and electrochemical readouts are the most widely adopted for CRISPR diagnostics. Optical systems typically rely on fluorescence or colorimetric changes. For instance, lateral flow assays incorporate gold nanoparticles or colored dyes that become visible when reporters are cleaved, providing a simple yes/no result. Fluorescence-based platforms offer higher sensitivity, often quantified using handheld fluorimeters. Electrochemical biosensors translate collateral activity into electrical signals via electrodes functionalized with nucleic acid reporters. Cleavage alters the electrode surface, modulating current or impedance. These systems are advantageous for point-of-care applications due to their miniaturization potential and compatibility with smartphone-based analysis.

Point-of-care applications of CRISPR biosensors have gained prominence during the COVID-19 pandemic. Portable devices such as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) demonstrate the feasibility of rapid, equipment-free testing. Recent advancements include handheld CRISPR diagnostic devices integrating microfluidics and smartphone detection. For example, some systems use disposable cartridges for sample preparation, CRISPR reaction, and optical readout, delivering results within 30-60 minutes. These innovations address critical needs in resource-limited settings, where access to centralized laboratories is constrained.

Despite their promise, CRISPR biosensors face challenges, particularly off-target effects and limited multiplexing capability. Off-target activity occurs when Cas proteins cleave sequences resembling the target, leading to false positives. Strategies to mitigate this include improved guide RNA design, engineered high-fidelity Cas variants, and stringent reaction conditions. Multiplexing, or simultaneous detection of multiple targets, remains technically challenging due to signal crosstalk and the need for orthogonal CRISPR systems. Recent progress involves using distinct Cas proteins or combining CRISPR with other amplification methods to enable multi-pathogen detection.

Recent handheld CRISPR devices highlight the technology’s potential for decentralized diagnostics. Examples include battery-operated systems with integrated heating modules for isothermal amplification and CRISPR detection, enabling field deployment. Some devices employ paper-based fluidics to simplify sample handling, while others leverage smartphone cameras for signal quantification. These developments underscore a shift toward user-friendly, scalable diagnostic tools that could revolutionize infectious disease surveillance.

In summary, CRISPR-Cas biosensors represent a paradigm shift in nucleic acid detection, combining precision with adaptability for point-of-care use. Collateral cleavage activity enables sensitive and versatile readouts, while ongoing advancements in device miniaturization address practical challenges. However, overcoming off-target effects and achieving robust multiplexing are critical for broader adoption. As handheld CRISPR diagnostics evolve, their impact on global health, particularly in pandemic response and low-resource settings, is poised to grow significantly.