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

Abstract

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 SARS-CoV-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.

Application of CRISPR/Cas13a-based biosensors in serum marker detection

“…In contrast to the isothermal amplification of RPA or RT-RPA, which requires enzymes to perform the reaction, catalytic hairpin assembly (CHA) is an isothermal amplification that does not require enzymes. Yang et al 31 designed a special hairpin report structure, that is, five uracil ribonucleotides (rU) easily cleaved by Cas13a were introduced into the hairpin structure. After the hairpin structure is cut by Cas13a, it will trigger the catalytic hairpin assembly (CHA) reaction, thus generating a large number of fluorescence signals.…”

miRNA Detection for Prostate Cancer Diagnosis by miRoll-Cas: miRNA Rolling Circle Transcription for CRISPR-Cas Assay

“…CRISPR-based diagnostic tests are able to detect specific genetic mutations that are associated with certain diseases, such as cancer or genetic disorders. , Cas13a has been used for nucleic acid target recognition, signal generation, and amplification. After being activated by the nucleic acid of interest ssRNA, Cas13 cleaves short single-stranded reporter oligonucleotides by trans -cleavage, , separating the fluorophore from the quencher normally labeled at the opposite end of the reporter molecule and producing a measurable fluorescence signal . CRISPR-Cas13a-based methods are relatively simple, efficient, and cost-effective compared to the traditional diagnostic methods.…”

“…42 CRISPR-based diagnostic tests are able to detect specific genetic mutations that are associated with certain diseases, such as cancer or genetic disorders. 43,44 trans-cleavage, 45,46 separating the fluorophore from the quencher normally labeled at the opposite end of the reporter molecule and producing a measurable fluorescence signal. 47 CRISPR-Cas13a-based methods are relatively simple, efficient, and cost-effective compared to the traditional diagnostic methods.…”