Abstract
Among the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.
Highlights
Among the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers
Several outbreaks namely Spanish flu and Swine flu caused by H1N1 influenza virus[2,3,4], Acquired Immunodeficiency Syndrome (AIDS) caused by Human Immunodeficiency virus (HIV)[5], Zika disease caused by the Zika virus[6], Ebola Virus Disease (EVD) caused by the Ebola virus[7], Severe Acute Respiratory Syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS) caused by MERS coronavirus (MERS-CoV) have wreaked havoc on general population and had widespread economic implications in the world[8,9]
To harness the detectable viral RNA-stimulated DNase and RNase activities, we constructed two fluorescence reporters, an RNA oligo flanked by a fluorophore-quencher pair and a DNA oligo flanked by either the same or a different fluorophore-quencher pair (Fig. 1a)
Summary
Among the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. We describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. The traditional nucleic acid-based detection, based on Polymerase Chain Reaction (PCR) makes use of nucleic acid amplification methods that has many advantages over antigenbased and serological tests It is quantitative, highly sensitive and can be made available prior to the onset of a potential pandemic. We further showed the effectiveness of MORIARTY in rapid detection of SARS-CoV-2 virus with high sensitivity, thereby offering a Class I CRISPR-Cas based viral diagnostic
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
