Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay |
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| Authors: | Yongya Li Hayam Mansour Colton J F Watson Yanan Tang Adam J MacNeil Feng Li |
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| Institution: | Key Laboratory of Green Chemistry & Technology of Ministry of Education, College of Chemistry, Analytical & Testing Center, Sichuan University, 29 Wangjing Road, Chengdu Sichuan 610064 China.; Department of Chemistry, Centre for Biotechnology, Brock University, St. Catharines Ontario L2S 3A1 Canada.; Department of Cell Biology, National Research Center, 12622 Egypt ; Department of Health Sciences, Brock University, St. Catharines Ontario L2S 3A1 Canada |
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| Abstract: | Herein, we describe an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced indiscrimitive single-stranded DNase activity of Cas12a for the quantitative profiling of gene expression at the mRNA level and detection of proteins with high sensitivity and specificity. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows for flexible assay design. A binding-induced primer extension reaction is used to generate a predesigned CRISPR-targetable sequence as a barcode for further signal amplification. Through this dual amplification protocol, we were able to detect as low as 1 fM target nucleic acid and 100 fM target protein isothermally. The practical applicability of this assay was successfully demonstrated for the temporal profiling of interleukin-6 gene expression during allergen-mediated mast cell activation.Herein, we develop an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced collateral cleavage activity of Cas12a for the quantitative profiling of gene expression and detection of proteins with high sensitivity and specificity. |
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