文章基本信息

标题：Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2
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作者：Ahmed Mahas ; Tin Marsic ; Mauricio Lopez-Portillo Masson 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：28
DOI：10.1073/pnas.2118260119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance Thermostable Cas13 orthologues will expand and unlock the power of CRISPR systems for synthetic biology and biotechnology applications. We identified and characterized a thermostable Cas13 orthologue from Thermoclostridium caenicola (TccCas13a), which exhibits robust cis and trans catalytic activities over a broad range of temperatures from 37 to 70 °C. To demonstrate the utility of the thermostable Cas13 enzymes in enabling biotechnological applications, we coupled reverse transcription–loop-mediated isothermal amplification to TccCas13a to develop a one-pot assay for virus detection. The developed assay exhibits key point-of-care (POC) features, enabling its use for the detection of different pathogens. This platform was clinically validated and is suitable for multiplexed detection. TccCas13a will enable advancements in POC diagnostics, transcriptome engineering, editing, and targeted therapies. Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.
关键词：enCRISPR Cas13diagnosticsthermostable Cas13CRISPR diagnosticstranscriptome editing