Abstract
CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows “CRISPR-based amplification-free digital RNA detection (SATORI)”, by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.
Highlights
CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics
As a proof-of-concept experiment, we sought to detect a target single-stranded RNA (ssRNA) in our microchamber device, using Leptotrichia wadei Cas13a (LwaCas13a) and the CRISPR RNA, which were used in SHERLOCK9 (Fig. 1a)
The fluorescence intensity significantly increased throughout the array (Fig. 1b, c), indicating that the LwaCas13a–crRNA1 complexes recognized the tgRNA1 and cleaved the fluorophore quencher (FQ) reporters in trans in the microchambers
Summary
CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. While reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used as a “gold standard” method, CRISPR-based nucleic-acid detection, such as SHERLOCK and DETECTR, have recently been attracting keen attention as rapid and sensitive methods[4,5,6,7,8,9,10] These CRISPRbased methods comprise a pre-amplification process of target nucleic acids and a subsequent detection mediated by CRISPR–Cas enzymes, such as Cas12a or Cas13a, via fluorescent or colorimetric readout. The pre-amplification process increases the time to detection (by at least several tens of minutes), and could cause false-negative or -positive results due to amplification errors[12,13] To overcome these challenges, we combined the CRISPR– Cas13-based nucleic-acid detection system[9] and our microchamber technology[14,15], to develop a platform that enables accurate and rapid detection of ssRNA at a single-molecule level, termed SATORI (CRISPR-based amplification-free digital RNA detection). SATORI enabled rapid and sensitive detection of the N-gene RNA and whole genomic RNA from SARS-CoV-2, thereby highlighting the potential of SATORI as a powerful new class of rapid and robust viral diagnostics
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