The 21st century has witnessed a surge in sudden viral outbreaks, such as SARS-CoV, MERS-CoV, and SARS-CoV-2, largely attributed to increased human encroachment into wildlife habitats. This heightened risk of zoonotic transmission necessitates advanced diagnostic and antiviral strategies that are rapid, affordable, and easily deployable. Current gold-standard molecular diagnostics, primarily PCR-based, are time-consuming, requiring specialized equipment and personnel, hindering widespread surveillance. The limited number of approved vaccines and antivirals further accentuates the urgency for innovative approaches. The high sequence diversity among viruses, coupled with their rapid mutation rates, poses a significant challenge in developing universally effective diagnostics and therapeutics. Existing methods such as PCR-based assays, while sensitive and specific, suffer from limitations in cost, processing time, and the requirement for sophisticated equipment, restricting their widespread use. Antigen-based tests offer an alternative but fall short in sensitivity and specificity. The development of effective antiviral drugs is also a lengthy process. This review explores CRISPR-Cas systems, a bacterial adaptive immune system component, as a potential game-changer in addressing these limitations, offering opportunities for rapid development of virus diagnostics and potential antiviral therapies.

The literature extensively documents the challenges in existing viral diagnostics and antiviral development. PCR-based methods, though the gold standard, are limited by cost, time constraints, and infrastructure needs. Isothermal amplification techniques, such as LAMP, RPA, NASBA, and nicking enzyme amplification, offer alternatives to PCR, but improvements in sensitivity and ease-of-use are still needed. Existing antiviral drug development is time-consuming and relies heavily on understanding viral and host target proteins. While many small-molecule inhibitors exist, the identification and repurposing process is lengthy. Nucleic acid-based antivirals such as siRNAs show promise in vitro and in animal models but lack clinical approval. This review highlights the advantages of CRISPR-Cas systems in overcoming these limitations.

This review examines the biochemical properties of Cas12 and Cas13 orthologs, focusing on their use in viral diagnostics and antiviral therapies. It explores the mechanisms of target recognition and cleavage by these enzymes, emphasizing their collateral activity – the nonspecific cleavage of single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) upon target recognition. This collateral activity forms the foundation of many CRISPR-based diagnostic assays. The review delves into various diagnostic technologies that exploit this collateral activity, including SHERLOCK (Specific High-sensitivity Enzymatic Reporter Unlocking) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter), comparing their sensitivity, specificity, multiplexing capabilities, and ease of use. The application of these technologies to SARS-CoV-2 detection is highlighted, detailing the development of various platforms and their performance characteristics, including detection limits, turnaround times, and agreement with gold-standard PCR methods. The review also covers different sample processing methods, including HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases) which simplifies sample preparation for direct detection. Methods for multiplexed high-throughput virus detection using CRISPR-Cas, such as CARMEN (Combinatorial Array Reactions for the Multiplex Evaluation of Nucleic Acids), are also discussed. The potential of Cas13 as an antiviral agent is explored, reviewing studies demonstrating its ability to target and degrade viral mRNA, reducing viral replication and infectivity. This includes discussions on various delivery methods and strategies for minimizing off-target effects. Finally, the review explores future perspectives in the field, including the potential use of smaller Cas enzymes and advancements in multiplexing capabilities.

This review presents several key findings: 1. CRISPR-Cas systems, particularly Cas12 and Cas13, offer significant advantages over traditional PCR-based methods in viral diagnostics due to their speed, affordability, and ease of use. 2. The collateral activity of Cas12 and Cas13, which involves nonspecific cleavage of ssDNA or ssRNA upon target recognition, is exploited in numerous diagnostic platforms. 3. SHERLOCK and DETECTR, among other CRISPR-based diagnostic platforms, show high sensitivity and specificity in detecting various viruses, including SARS-CoV-2, with detection limits reaching the attomolar range. 4. Multiplexing capabilities, allowing for simultaneous detection of multiple viral targets or variants, have been significantly advanced using orthogonal Cas enzymes and microfluidic technologies. 5. The ability to perform CRISPR-based detection directly on crude samples, using techniques such as HUDSON, reduces processing time and complexity, facilitating point-of-care applications. 6. Cas13, an RNA-targeting enzyme, exhibits potential as a direct antiviral agent by specifically degrading viral mRNA, although further research is needed on its in vivo efficacy and delivery mechanisms. 7. Advancements in Cas enzyme characterization, including the identification of smaller enzymes such as Cas14, and optimization of CRISPR RNA (crRNA) design offer pathways to improve sensitivity, specificity, and multiplexing capabilities. 8. The combination of isothermal amplification techniques such as LAMP and RPA with CRISPR-Cas detection significantly enhances sensitivity, enabling detection at very low viral loads. 9. Visual detection methods, including lateral flow assays, expand the accessibility of CRISPR diagnostics beyond well-equipped laboratories.

This review highlights the transformative potential of CRISPR-Cas systems in viral diagnostics and antiviral therapies. The superior speed, affordability, and simplicity of CRISPR-based assays compared to traditional PCR-based methods address many of the limitations of current technologies. The ability to perform multiplexed assays, detect low viral loads, and use simpler sample processing techniques, all while maintaining high sensitivity and specificity, greatly enhances the capacity for rapid outbreak response and widespread surveillance. The emerging potential of Cas13 as a direct antiviral agent adds another layer to the impact of CRISPR technology. However, further research is needed to fully realize the clinical potential of Cas13, particularly regarding its in vivo efficacy, delivery mechanisms, and mitigation of off-target effects. The continuous discovery and characterization of new Cas orthologs, coupled with advancements in microfluidic technologies and crRNA design, will drive further innovation and improved performance in both diagnostics and therapeutics.

CRISPR-Cas systems represent a significant advancement in viral disease detection and therapy. Their programmability, ease of use, and high sensitivity and specificity make them ideal for rapid diagnostic testing, particularly in point-of-care settings. The potential of Cas13 as a direct antiviral agent further expands the therapeutic applications of CRISPR technology. Future research focusing on optimizing delivery methods, minimizing off-target effects, and exploring the potential of newly discovered Cas enzymes will continue to enhance the capabilities of CRISPR-Cas systems in combating viral diseases.

While CRISPR-Cas technology offers immense promise, certain limitations remain. The in vivo efficacy of Cas13-based antiviral therapies needs further investigation to confirm its clinical potential and overcome challenges associated with delivery and potential off-target effects. Further optimization of crRNA design is crucial to enhance specificity and minimize the risk of viral escape mutations. The widespread adoption of CRISPR-based diagnostics depends on the development of robust, user-friendly, and affordable point-of-care devices.