期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 60, 期 45, 页码 24241-24247出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202110384
关键词
CRISPR-Cas14a1; DNA cleavage; pathogenic bacteria; RNA; RNA detection platform
资金
- National Key Research and Development Program of China [2018YFD0900704]
- National Natural Science Foundation of China [21621003, 41866002]
- Tsinghua University Initiative Scientific Research Program [2020Z99CFZ019]
- Tsinghua University Spring Breeze Fund [2020Z99CFZ019]
- Research Foundation of Hainan University [KYQD(ZR)1711, KYQD(ZR)1997]
- Open fund of Wenzhou Key Laboratory of Sanitary Microbiology [ZD202003KF04]
Cas14a1 exhibits a novel capacity where RNA can trigger its cleavage of DNA. The RNA-activated detection platform based on Cas14a1 shows high specificity for target RNAs with point mutations and can efficiently detect pathogenic nucleic acids, serving as a new tool for nucleic acid diagnostics.
As a CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here, we describe an unreported capacity of Cas14a1: RNA can trigger the trans ssDNA cleavage. This Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNA cleavage, ATCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNA-activation. Using ATCas-RNA via a fluorophore quencher-labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100 % accuracy with 25 milk samples. This platform can serve as a new tool for high-efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.
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