CRISPR-based molecular detection of SARS-CoV-2, its emerging variants, and diverse pathogens

Abstract

Pathogenic viruses such as SARS-CoV-2 (SCoV-2), continue to pose a significant threat to human civilization. The lessons learnt from SCoV-2 infections have highlighted the requirement for robust and readily available diagnostic tools in order to limit the virus transmission and prevent future pandemics such as COVID-19. RT-qPCR-based detection is routinely used for sensitive and accurate diagnosis, which requires a sophisticated instrument, laboratory setup, and technical expertise. Though RT-qPCR is highly reliable and considered the gold standard for pathogen detection, it is costly, time-consuming, and unaffordable for the masses. Therefore, other reliable methods for nucleic acid-based detection with sensitivity, specificity, and accuracy on-par with RT-qPCR are required. Recent advancement in CRISPR technology promises its development as a POC testing device, providing a high-end, instrument-free, portable, and cost-effective workflow. Further, COVID-19 pandemic has encouraged the development of next-generation CRISPR-based diagnostics with a provision for home-testing which has resulted in the development of portable and smart-phone integrated hand-held devices which can detect various pathogenic infections in a shorter time frame than RT-qPCR. For diagnosing the presence of SCoV-2, CRISPR-based diagnostics (SHERLOCK/DETECTR) are quicker (30–60 min), less expensive ($5–15/test), and portable than RT-qPCR (90–180 min; $10–50/test) demonstrating equivalent specificity (100%) and near-equivalent sensitivity (93-100% for CRISPR-based diagnostics vs 95-100% for RT-qPCR). For high-sensitivity centralized testing, RT-qPCR is still the gold standard, but CRISPR works well in point-of-care settings because it requires little equipment (like lateral flow strips or heating blocks) and allows multiplexing. CRISPR-based diagnostics breakthrough platform like CARMEN leverages microfluidic technology to test 5,000 plus samples in a single run, unlike RT-qPCR, which requires separate reactions for each target.In this review, the advancement in CRISPR technology such as SHERLOCK, DETECTR, and other Cas-9-based diagnostics are highlighted which exclusively focuses on the CRISPR-based diagnostics to detect SCoV-2 and its emerging VOCs, highlighting their advantages and limitations compared to the gold-standard RT-qPCR.