Abstract
There are many current major limitations of the point-of-care (POC) diagnostics for infectious diseases such as low sensitivity and specificity, and the need for complex infrastructure that makes it difficult to deploy in resource-poor environments. The CRISPR-Cas system is then a programmable, sequence-specific detector with great potential for nucleic acid detection, and in particular Cas12, Cas13 and Cas14 have emerged as highly promising. In this review, the major principles and applications of major CRISPR-based diagnostics platforms, along with the unique collateral cleavage activity of CRISPR, are highlighted, and how this activity makes pathogen identification rapid and accurate. The following new developments are explored, including integration with isothermal amplification methods (RPA and LAMP) that improve the sensitivity of the assays and lateral flow readouts and smartphone integration that make these assays truly field-deployable. The review also highlights practical application of these technologies towards translation into POC application such as in the detection of antimicrobial resistance genes, viral and bacterial as well as parasitic infections. Despite these great advances, some challenges exist and are discussed critically: achieving high-order multiplexing in a single reaction, the absence of true quantification as compared to digital PCR and the regulatory approval issues to date, which have prevented many clinical applications from becoming broadly adopted. Going forward, the review presents a vision for the future where POC diagnostics based on CRISPR has a pivotal role in pandemic readiness and decentralized diagnostics. These platforms offer the promise of rapid, accurate, lab light testing at the bedside, or in a community setting, thereby potentially revolutionizing the care of infectious diseases, decreasing reliance on centralized laboratories, and increasing global health equity, especially in low and middle income countries where traditional diagnostics are not readily available