CRISPR-Cas Systems: A Promising Tool for Viral Disease Detection and Therapy

The review addresses the urgent need for rapid, accessible, and scalable diagnostics and antivirals in response to emerging and re-emerging viral outbreaks (e.g., SARS-CoV, MERS-CoV, SARS-CoV-2). Traditional PCR-based diagnostics, while sensitive, are limited by cost, infrastructure, turnaround time, and personnel requirements. High viral diversity, rapid mutation rates, and limited numbers of approved antivirals and vaccines underscore the necessity for programmable, sequence-informed platforms that can be quickly adapted to new pathogens and variants. CRISPR-Cas systems, particularly class 2 effectors Cas12 and Cas13 with programmable guide RNAs and characteristic collateral cleavage activities, offer a promising foundation for rapid diagnostics and potential antiviral strategies that require only sequence information and minimal specialized equipment.

The paper synthesizes advances in CRISPR-Cas diagnostics and antivirals. It outlines: 1) Background on gold-standard PCR and isothermal alternatives (LAMP, RPA, NASBA) to simplify amplification and reduce equipment needs. 2) Molecular biology of CRISPR-Cas effectors used in diagnostics: Cas9 (genome editing), Cas12 (dsDNA recognition with trans-ssDNA collateral activity), and Cas13 (ssRNA recognition with trans-ssRNA collateral activity and intrinsic crRNA processing). 3) Collateral cleavage discovery and exploitation: Cas12 and Cas13 trans-cleavage on nonspecific ssDNA/ssRNA reporters enables fluorescence or lateral-flow readouts. 4) Diagnostic platforms leveraging collateral activity: SHERLOCK (Cas13 + RPA/T7), DETECTR (Cas12 + RPA/LAMP), HOLMES (Cas12 + PCR), STOPCovid (Cas12b + RT-LAMP), SHINE (Cas13 one-pot), DISCoVER (Cas13 + LAMP microfluidics), and FIND-IT (Cas13 + Csm6 tandem with microfluidics). 5) Multiplexing via orthogonal Cas enzymes (LwaCas13a, LbuCas13a, PsmCas13b, CcaCas13b, AsCas12a) with distinct reporter motif preferences; tandem amplification of signals using Csm6; high-throughput microfluidics (CARMEN) for thousands of reactions. 6) Applications to diverse viruses: ZIKV, DENV, influenza, Ebola, Lassa, HPV16/18, and SARS-CoV-2, including variant genotyping. 7) CRISPR beyond diagnostics: Cas13-based antiviral targeting of viral RNAs (HIV-1, DENV, LCMV, influenza, SARS-CoV-2), design considerations for crRNAs, delivery strategies (AAV, mRNA, polymers), and current efficacy/safety observations. 8) Future directions: discovery of smaller effectors (Cas14/CasX), thermostable orthologs, phage-derived systems, and integration into point-of-care devices with simplified sample processing.

This is a narrative review. The authors collate and synthesize biochemical characterizations of Cas12/Cas13 orthologs, engineering of CRISPR-based detection platforms, and clinical/analytical validation studies reported across the literature. They summarize assay chemistries (amplification strategies such as RPA, LAMP, PCR; transcription steps; reporter designs), readouts (fluorescence, lateral flow, colorimetric, microfluidic), performance metrics (limits of detection, sensitivity, specificity, turnaround times), and deployment considerations (lyophilization, ambient operation, sample inactivation/lysis, single-tube workflows). No new experiments or systematic search protocol are described; rather, the review integrates key milestones and comparative features to map the field’s development and practical applications.

- Collateral activity underpins CRISPR diagnostics: Cas13 trans-ssRNA and Cas12 trans-ssDNA cleavage of labeled reporters enable sensitive detection in fluorescence or lateral-flow formats. LbuCas13a shows robust trans-RNase activity with ~10^4 turnovers per target; ssRNA cleavage ~80× faster than crRNA processing. - SHERLOCK v1 (Cas13 + RT-RPA + T7) achieves attomolar sensitivity comparable to RT-qPCR/droplet digital PCR; detected ZIKV/DENV at ~2 aM; lyophilized reagents detected unamplified RNA at 20 fM and ~3 aM with pre-amplification (slightly reduced to ~20 aM after paper spotting/lyophilization). - SHERLOCK v2 couples HUDSON for nuclease inactivation and direct use of saliva/urine; detects single copy/µl of ZIKV/DENV with 100% specificity and sensitivity versus RT-qPCR; supports serotyping and pan-flavivirus panels; multiplexing improved via orthogonal Cas enzymes and Csm6 boosting (~3.5× sensitivity gain). - Cas12a DETECTR: LbCas12a detects HPV16/18 DNA at pM levels; with RPA pre-amplification, achieves aM sensitivity; performance comparable to PCR on clinical anal swabs. - Multiplexing: Orthogonal motif preferences (e.g., LwaCas13a-U, PsmCas13b-A, CcaCas13b-GA, AsCas12a-ssDNA) enable triplex/quadruplex detection in single reactions; AsCas12a required pre-amplification due to weak collateral activity (>10 nM input otherwise). Detection limits as low as ~200 zM reported using optimized RPA and Cas13 variants. - Tandem nuclease amplification: Cas13 collateral products (2′,3′-cyclic phosphate oligos) activate Csm6, enabling stronger, faster signals (FIND-IT platform detected SARS-CoV-2 at ~30 copies/µl in ~20 min, Ct ~33). - High-throughput CRISPR: CARMEN microfluidics pairs color-coded droplets to screen up to ~5,000 reactions; supports pan-viral detection and influenza A HA/NA subtyping. - SARS-CoV-2 diagnostics: • SHERLOCK clinical validation: 154 NP/throat swabs—100% specificity/sensitivity (fluorescence); 100% specificity/97% sensitivity (lateral flow); 380 negative pre-op samples—100% concordance with RT-qPCR. • STOPCovid v1/v2 (Cas12b + RT-LAMP): v1 sensitivity 93.1%, specificity 98.5% on 202 positive/200 negative NP swabs; v2 detected ~1/30 of CDC RT-qPCR detection threshold. • DETECTR (LbCas12a + RT-LAMP): ~95% agreement with RT-qPCR; ~45 min turnaround; amenable to microfluidic cartridges and freeze-dried reagents. • DISCoVER (LbuCas13a + LAMP microfluidic saliva test): ~95% sensitivity, 100% specificity; ~40 copies/µl detection; 100% PPV, ~93% NPV. • SHINE v1/v2: one-pot Cas13 RT-RPA; v1 on 60 NP samples—90% sensitivity, 100% specificity in ~50 min; v2 runs at 37°C with lyophilized reagents, ~90% sensitivity and 100% specificity within 90 min; variant-of-concern genotyping by Cas12a panel (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, D614G) showing 100% agreement with sequencing on 32 positives. - Single-nucleotide resolution: Strategic guide mismatches enable discrimination of SNPs/serotypes (ZIKV isolates, DENV serotypes, HPV16/18 SNPs) and HIV drug-resistance mutations (27 validated). - Sample processing for POC: Modified HUDSON, proteinase K + heat, room-temperature lysis, magnetic bead RNA extraction; single-tube workflows via physical separation or thermophilic Cas12b integration (HOLMESv2, STOPCovid) reduce contamination risk. - Antiviral applications: Cas13 (a/b/d) reduces viral RNA/replication in cell and animal models (HIV-1, DENV, LCMV, influenza, SARS-CoV-2); PAC-MAN (Cas13d) targeted conserved coronavirus regions; mRNA-encoded Cas13a reduced viral loads in rodents; collateral off-target effects in typical cellular settings appear minimal, though collateral in glioma overexpression context observed. - Future effectors: Cas14a (CasX) are smaller (~400–700 aa), PAM-independent, ssDNA-targeting with collateral activity, facilitating SNP detection and potential AAV packaging; phage-encoded CRISPR systems and hyperactive LbuCas13a expand diagnostic/therapeutic scope.

The review demonstrates that CRISPR-Cas12/Cas13 diagnostic platforms directly address limitations of traditional RT-qPCR by enabling rapid, low-cost, and portable detection with minimal instrumentation, while maintaining high sensitivity/specificity. The inherent programmability allows rapid retargeting to new pathogens and variants, and orthogonal/tandem nuclease strategies enable multiplexing and enhanced sensitivity suitable for outbreak surveillance and point-of-care use. Integration with isothermal amplification, simplified sample processing, lyophilized reagents, and microfluidics reduces turnaround times (often ≤60 minutes) and operational complexity, making mass screening feasible. Additionally, Cas13 antiviral strategies illustrate the therapeutic potential of RNA-targeting CRISPR to degrade viral genomes or transcripts, with early evidence of efficacy and limited off-target effects in many contexts. Together, these findings support CRISPR’s role as a versatile platform for both detection and intervention across diverse viral threats.

CRISPR-Cas12 and Cas13 orthologs, through their collateral cleavage properties and programmability, have catalyzed the development of rapid, sensitive, and adaptable diagnostic platforms (e.g., SHERLOCK, DETECTR, STOPCovid, SHINE, DISCoVER, FIND-IT), many validated clinically for SARS-CoV-2 and other pathogens. Advances in orthogonal enzyme selection, tandem nuclease amplification (Csm6), microfluidics (CARMEN), and single-tube workflows support multiplexing, high throughput, and deployment beyond centralized labs. On the therapeutic front, Cas13-based antivirals show promise for targeting RNA viruses, though delivery, potency, and safety require further optimization. Future research should prioritize: discovery of smaller, thermostable effectors (e.g., Cas14/CasX) for AAV delivery and POC integration; improved reagent stability and ambient operation; standardized single-tube, amplification-free assays; robust variant genotyping panels; optimization of guide design to minimize resistance; deeper characterization of collateral effects in vivo; and scalable, user-friendly devices for decentralized testing and surveillance.

- As a narrative review, no systematic search strategy or quantitative meta-analysis is described; selection bias is possible. - Many reported performance metrics derive from heterogeneous studies with varying sample types, processing, and endpoints, limiting direct comparability. - Several CRISPR diagnostics still rely on pre-amplification (RPA/LAMP), which can introduce contamination risk and complicate true single-pot workflows. - Lateral flow formats may have slightly reduced sensitivity relative to fluorescence readouts. - Field deployability depends on reagent lyophilization stability, ambient operation, simple lysis, and ruggedized devices—areas still under active development. - For therapeutics, key challenges remain in delivery (e.g., AAV/mRNA/polymer systems), sustained expression, minimizing off-target/collateral effects, potency across infection stages, and resistance management.