Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA

Yichuan Chen, Xinping Wang, Junqi Zhang, Qingyuan Jiang, Bin Qiao, Baoxia He, Wenhao Yin, Jie Qiao () and Yi Liu ()
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Yichuan Chen: Wuhan Polytechnic University
Xinping Wang: Hubei University
Junqi Zhang: Hubei University
Qingyuan Jiang: Hubei University
Bin Qiao: Zhengzhou University
Baoxia He: Affiliated Cancer Hospital of Zhengzhou University and Henan cancer hospital
Wenhao Yin: BravoVax Co. Ltd.
Jie Qiao: Wuhan Polytechnic University
Yi Liu: Hubei University

Nature Communications, 2024, vol. 15, issue 1, 1-12

Abstract: Abstract The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system’s specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings.

Date: 2024
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DOI: 10.1038/s41467-024-52691-x

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