trans Single‐Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction

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...

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Published in:Angewandte Chemie International Edition 2021-11, Vol.60 (45), p.24241-24247
Main Authors: Wei, Yangdao, Yang, Zhiqing, Zong, Chengli, Wang, Buhua, Ge, Xiaolin, Tan, Xiao, Liu, Xin, Tao, Zhenzhen, Wang, Peng, Ma, Chunxin, Wan, Yi, Li, Jinghong
Format: Article
Language:English
Subjects:
Cleavage
CRISPR
CRISPR-Associated Proteins - metabolism
CRISPR-Cas Systems
CRISPR-Cas14a1
Deoxyribonucleic acid
DNA
DNA Cleavage
DNA, Single-Stranded - chemistry
DNA, Single-Stranded - metabolism
Mutation
Nucleic acids
pathogenic bacteria
Point mutation
Ribonucleic acid
RNA
RNA - analysis
RNA - metabolism
RNA detection platform
Target detection
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Summary: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. A 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.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202110384