The COVID-19 pandemic, caused by SARS-CoV-2, necessitates rapid and accurate diagnostic testing to control transmission. Current gold-standard quantitative real-time PCR (qRT-PCR) is slow and requires specialized equipment, limiting its accessibility. While rapid diagnostic tests (RDTs) like serological and antigen tests exist, they suffer from limitations in timing (antibodies appear late) and sensitivity (antigen tests), respectively. Isothermal amplification methods like RPA and LAMP offer speed but often produce false positives due to non-specific products. CRISPR-Cas systems offer a potential solution, but existing CRISPR-Dx for COVID-19 lack robustness to viral mutations and RNA editing, require long testing times (around 40 minutes), necessitate dual temperature protocols, typically only work with purified RNA, and often lack a built-in human internal control. This study aims to develop a CRISPR-based diagnostic assay addressing these limitations, incorporating features to improve robustness against viral mutations, reduce testing time, enable a single-temperature workflow, work directly with nasopharyngeal (NP) specimens, and incorporate a human internal control.

The literature extensively covers the challenges of rapid COVID-19 diagnostics. qRT-PCR, though accurate, is resource-intensive and time-consuming. Serological tests detect antibodies, but their appearance is delayed, limiting their utility for identifying infectious individuals. Antigen tests lack sensitivity. Isothermal amplification methods like LAMP and RPA provide faster results but struggle with non-specific amplification leading to false positives. CRISPR-Cas technologies have emerged as a promising alternative due to their potential for high sensitivity and specificity, but existing CRISPR-based COVID-19 tests have shortcomings in terms of robustness to mutations, speed, and ease of use. The authors cite numerous existing CRISPR-based COVID-19 diagnostic assays, highlighting their potential but also their individual limitations. A key concern is the lack of robustness to mutations in the SARS-CoV-2 genome and the effects of RNA editing by host enzymes, which can lead to false negative results. This literature review emphasizes the existing gaps and the need for an improved, more robust and rapid diagnostic test.

The study aimed to develop a robust and rapid CRISPR-based diagnostic assay for COVID-19. This involved several key methodological steps: 1. **Cas12a Enzyme and gRNA Selection:** Various Cas12a enzymes (LbCas12a, AsCas12a, and engineered variants enAsCas12a, enRR, enRVR) were evaluated in a fluorescence trans-cleavage assay using different guide RNAs (gRNAs) targeting various SARS-CoV-2 gene regions (N, O, and S genes). The assay involved comparing the collateral activity of the enzymes with perfectly matched and mismatched gRNAs at room temperature and 37°C to assess robustness against mutations. 2. **gRNA Optimization:** The study focused on enhancing the robustness of the assay against mutations. This involved testing different combinations of gRNAs with the Cas12a variants to identify the most effective combination for detecting SARS-CoV-2 regardless of single nucleotide variations (SNVs). 3. **Integration with RT-LAMP:** Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was integrated with the CRISPR-Cas12a system. The optimal RT-LAMP conditions, including primer concentrations, were determined to ensure high sensitivity and specificity. The effect of mismatches in LAMP primers on amplification efficiency was investigated and strategies to enhance robustness against mutations in the viral genome during amplification were explored (including the use of truncated primers and high-fidelity polymerase). 4. **Assay Optimization:** The study focused on optimizing the entire workflow, including the RT-LAMP and CRISPR-Cas12a reaction conditions. This involved investigating the impact of factors such as buffer conditions, enzyme concentrations, temperature, and chemical additives (glycine, taurine, DMSO) on the assay sensitivity and speed. Strategies such as the addition of swarm primers to optimize LAMP amplification were explored. 5. **Clinical Sample Testing:** The developed assay was evaluated using both purified RNA samples and unpurified nasopharyngeal (NP) swab specimens from patients with and without COVID-19 infection. The study included a comparison with qRT-PCR results to determine sensitivity and specificity. 6. **Internal Control Integration:** An internal control using LAMP primers targeting a human housekeeping gene (ACTB) was incorporated into the assay. Optimizations to ensure both the viral and human internal control worked successfully together were performed, including the addition of pyrophosphatase. 7. **Guide RNA Modifications:** The study explored modifications to improve the CRISPR detection module, testing various modified gRNAs such as those with 5' extensions and DNA-RNA hybrids. 8. **Quasi-one-pot Reaction:** The study explored a "quasi-one-pot" reaction, where the CRISPR reagents were added directly to the RT-LAMP reaction tube after amplification, eliminating the need for sample transfer and reducing the testing time. Guanidine was tested as a potential replacement for glycine to enhance LAMP kinetics. The methodology involved a combination of in vitro experiments using synthetic DNA and RNA templates, along with clinical evaluations using patient samples.

The key findings of the study are: 1. **Engineered AsCas12a (enAsCas12a) showed superior robustness to mutations:** Compared to other Cas12a variants, enAsCas12a demonstrated greater tolerance to single nucleotide mismatches in the gRNA target site, making it suitable for detecting SARS-CoV-2 variants. Using two gRNAs further improved the robustness. 2. **Hybrid DNA-RNA guides significantly improved reaction kinetics:** The use of hybrid DNA-RNA guides in the CRISPR detection module markedly improved the reaction rate, allowing detection in as little as 5 minutes at 60°C. 3. **enAsCas12a demonstrated wide operating temperature range:** The enzyme showed activity across a broad range of temperatures (37-65°C), enabling the entire RT-LAMP-CRISPR workflow to be performed in a single heat block at 60°C, further reducing testing time. 4. **Assay worked directly on unpurified NP specimens:** The assay successfully detected SARS-CoV-2 in unpurified nasopharyngeal specimens, eliminating the need for RNA purification and significantly shortening the testing time. 5. **Incorporation of a human internal control:** A human internal control (ACTB gene) was successfully integrated into the assay, ensuring that negative results were not due to insufficient sample input. This required the use of dual-color fluorescence detection. 6. **High sensitivity and specificity in clinical samples:** Using purified RNA samples, the assay demonstrated a limit of detection (LoD) of 50 copies/reaction (2 copies/µL), and 100% specificity and positive predictive value. With unpurified NP swabs, the LoD improved to 1000 copies/reaction (40 copies/µL), and specificity remained at 100%. The entire assay could be completed within 30 minutes, a substantial improvement over existing methods. 7. **Robustness to known mutations:** The assay successfully detected SARS-CoV-2 even in the presence of known mutations (S254F and N234N) in the viral genome, both in purified and unpurified samples.

This study successfully addresses critical limitations of existing CRISPR-based COVID-19 diagnostic tests. The use of enAsCas12a and dual gRNAs provides robust detection against viral mutations, while hybrid DNA-RNA guides significantly accelerate the reaction. The wide operating temperature range of enAsCas12a allows for a single-temperature workflow, and the ability to process unpurified NP samples increases the test's practicality. The incorporation of an internal control enhances reliability. The high sensitivity and specificity observed in both purified and unpurified clinical samples demonstrates the potential of this assay for widespread use. The findings highlight the importance of considering viral evolution and RNA editing in diagnostic test design and demonstrate that optimization strategies can significantly improve the speed, sensitivity, and robustness of CRISPR-based assays. The "quasi-one-pot" approach simplifies the workflow and minimizes potential errors. The study’s success in detecting low viral loads, even with known mutations, strengthens its potential for early disease detection and effective outbreak control.

This research presents a significantly improved CRISPR-based diagnostic assay for COVID-19, termed VaNGuard. The assay is robust against mutations, rapid (30-minute completion time), adaptable to unpurified clinical samples, and includes an internal control. The optimized methodology, utilizing engineered Cas12a, hybrid guides, and a streamlined workflow, offers a powerful tool for COVID-19 detection and has the potential to be adapted for future pandemic preparedness. Further research could explore the application of this technology to other viruses and the development of even more cost-effective and portable detection platforms.

While the study demonstrates high sensitivity and specificity, several limitations exist. The clinical evaluation with unpurified NP swabs showed some ambiguity in the Ct value boundary between positive and negative results, potentially due to the complex sample matrix and variability in viral RNA recovery during sample processing in the diagnostic laboratory. Additionally, the assay's performance with a broader range of mutations and RNA editing events needs further investigation. Although tested against other respiratory viruses, more extensive testing against various viral strains and other pathogens might be necessary for complete validation. Finally, while the cost is under $10 per test, large-scale manufacturing and distribution costs need further assessment.