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bioRxiv · 10.1101/2024.01.04.574169

A split crRNA with CRISPR-Cas12a enables highly sensitive, selective, and multiplexed detection of RNA and DNA

Qiao, J.·Chen, Y.·Wang, X.·Yin, W.·He, B.·Liu, Y.
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Abstract

The complete 40-nt CRISPR RNA (crRNA) of Cas12a can be artificially divided into two parts, including the 20-nt scaffold RNA with fixed sequences and the 20-nt spacer RNA with variable sequences, respectively. Herein, we found that Cas12a protein, scaffold RNA, and spacer RNA can be reassembled into an active ribonucleoprotein (RNP) which has a trans-cleavage activity comparable to that of wild-type Cas12a RNP. By leveraging such split CRISPR-Cas12a system (SCas12a), we devised fast fluorescence and lateral flow assays for highly sensitive, selective, and multiplexed detection of miRNAs without the need for reverse transcription and pre-amplification, achieving a limit of detection (LoD) of 10 fM. Additionally, our SCas12a assay enables detecting long-stranded RNA without secondary structure, as well as distinguishing mature miRNA from its precursor (pre-miRNA) that comprises the same sequence of miRNA. Beyond RNA detection, SCas12a outperforms wild-type Cas12a in specificity towards DNA point mutations. In combination with recombinase polymerase amplification (RPA), we set up a one-pot assay to detect attomolar concentrations of human papillomavirus (HPV) in patient samples. In conclusion, this work provides a simple, cost-effective, yet powerful SCas12a-based rapid nucleic acid detection platform in various diagnostic settings. Significance StatementConventional Cas12a-based diagnostic methods cannot directly detect RNA targets. Here, we develop rapid fluorescence and lateral flow assays based on a split Cas12a system (called SCas12a), achieving amplification-free detection of RNA with high sensitivity and specificity. By supplying pooled activators, our method enables multiplexed detection of miRNA and DNA targets. In addition, SCas12a can discriminate mature miRNA from its pre-miRNA, which was achieved for the first time with CRISPR detection technology. Moreover, SCas12a outperforms wild-type Cas12a in specificity towards point mutation and can be combined with RPA to detect attomolar concentration of HPV in patient samples. Overall, this work offers a generic "split crRNA-activator" strategy for developing new CRISPR diagnostic tools.

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Qiao, J., Chen, Y., Wang, X., Yin, W., He, B., Liu, Y.. 2024-01-04. A split crRNA with CRISPR-Cas12a enables highly sensitive, selective, and multiplexed detection of RNA and DNA. https://doi.org/10.1101/2024.01.04.574169

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