CRISPR/Cas13a Trans-Cleavage-Triggered Catalytic Hairpin Assembly Assay for Specific and Ultrasensitive SARS-CoV-2 RNA Detection

New coronavirus (SARS-CoV-2), which has caused the coronavirus disease 2019 (COVID-19) pandemic, has brought about a huge burden on global healthcare systems. Rapid and early detection is important to prevent the spread of the pandemic. Here, an assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA), termed as Cas-CHA, was developed for ultrasensitive and specific detection of SARS-CoV-2 RNA. Upon specific recognition of the target, the CRISPR/Cas13a collaterally cleaved a well-designed hairpin reporter and triggered the CHA reaction. Under optimized conditions, the assay detected the SARSCoV-2 RNA with a wide range of 100 aM to 100 nM and realized a low detection limit of 84 aM. At the same time, the whole detecting process could be completed within 35 min. More importantly, the assay was able to distinguish SARS-CoV-2 RNA from common human coronaviruses and analyze in saliva samples. By the flexible design of crRNA, the assay was expanded to detect other viruses. The clinical sample analysis verified that the proposed assay held a great potential for practical testing.

Automated summary

The development of a Cas-CHA assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA) enables ultrasensitive and specific detection of SARS-CoV-2 RNA. The assay can detect a wide range of concentrations and has a low detection limit. It can distinguish SARS-CoV-2 RNA from other human coronaviruses and analyze samples from saliva. The proposed assay holds great potential for practical testing.