Amplification-free RNA detection with CRISPR–Cas13

Shinoda, Hajime; Taguchi, Yuya; Nakagawa, Ryoya; Makino, Asami; Okazaki, Sae; Nakano, Masahiro; Muramoto, Yukiko; Takahashi, Chiharu; Takahashi, Ikuko; Ando, Jun; Noda, Takeshi; Nureki, Osamu; Nishimasu, Hiroshi; Watanabe, Rikiya

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.