CRISPR-based applications in next-generation diagnostic and pathogenesis in enteroviruses

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) is a versatile gene-editing technology that is widely used in life science research to improve human health. The advancement of CRISPR technologies provided comprehensive information on the pathogenesis of emerging infectious pathogens and their potential applications in rapid detection. In this study, two CRISPR-Cas technologies were being employed in an attempt to improve the diagnosis and elucidate the pathogenesis in enteroviruses. Enteroviruses are a group of highly contagious emerging viruses which are associated with a wide spectrum of human diseases. Although the Enterovirus B (EV-B) species consists of the largest number of serotypes among the Enterovirus genus, the existing virus entry receptors have not fully recapitulated the pathogenesis and virus tropism of EV-B. In the first part of my study, CRISPR-Cas9-based genome-wide functional screenings were conducted to discover potential host factors for the pathogenesis of EV-B. Beta-2-microglobulin (B2M) was identified as an important factor for echovirus 9 (E9) replication. The knockout of B2M conferred stronger protection against E9 infection in rhabdomyosarcoma cells and a delayed viral replication was observed in Calu-3 cells. B2M was also involved in facilitating viral replication for certain EV-B serotypes, including echoviruses and coxsackievirus A9 (CV-A9), but not CV-B. However, B2M knockout failed to confer protection against Enterovirus A, C, and D, suggesting that it is a specific host factor for effective viral replication for some EV-B serotypes. EV-D68 is the predominant non-polio enterovirus serotype circulating in Hong Kong and is occasionally associated with severe and fatal complications. In the second part of my study, integration of multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR-Cas12a was introduced for the development of a rapid diagnostic tool for EV-D68 infection. The multiplex RT-LAMP assay demonstrated excellent sensitivity and specificity for EV-A to EV-D. Using the EV-D68-specific crRNAs, the CRISPR-Cas12a successfully distinguishes EV-D68 from the isothermal amplified enterovirus-positive specimens. This established CRISPR-based detection method could serve as the promising candidate for the next-generation diagnostic platform in the setting of point-of-care testing. In conclusion, the CRISPR-Cas9 approach was a feasible method for genome-wide functional screening for critical host factors of enteroviruses, with the successful identification of B2M as an essential host factor for effective viral replication in some EV-B serotypes. As for diagnostics, integration of isothermal amplification and CRISPR-Cas12a technology for rapid EV-D68 diagnosis could be a promising new trend. These CRISPR technologies have extended current knowledge on host-enterovirus interaction and the development of rapid enterovirus detection.