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HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation

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    HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation
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    • Linxian Li
      Linxian Li
      University of Chinese Academy of Sciences, Beijing, 100049, China
      Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China
      State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, Kaifeng, Henan 475004, China
      More by Linxian Li
    • Shiyuan Li
      Shiyuan Li
      Shanghai Tolo Biotechnology Company Limited, Shanghai, 200233, China
      More by Shiyuan Li
      Orcidhttp://orcid.org/0000-0002-6447-4745
    • Na Wu
      Na Wu
      University of Chinese Academy of Sciences, Beijing, 100049, China
      Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China
      School of Life Science and Technology, Shanghai Tech University, Shanghai, 201210, China
      More by Na Wu
    • Jiacheng Wu
      Jiacheng Wu
      School of Life Science and Technology, Shanghai Tech University, Shanghai, 201210, China
      More by Jiacheng Wu
    • Gang Wang
      Gang Wang
      State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, Kaifeng, Henan 475004, China
      More by Gang Wang
    • Guoping Zhao
      Guoping Zhao
      Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China
      Department of Microbiology and Li KaShing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China
      More by Guoping Zhao
    • Jin Wang*
      Jin Wang
      College of Life Sciences, Shanghai Normal University, Shanghai, 200234, China
      *E-mail: [email protected]
      More by Jin Wang
      Orcidhttp://orcid.org/0000-0003-4008-9284
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2019, 8, 10, 2228–2237
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    https://doi.org/10.1021/acssynbio.9b00209
    Published September 18, 2019
    Copyright © 2019 American Chemical Society
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    Abstract

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    The next-generation CRISPR-based molecular diagnostics has the merits of rapidness, accuracy, and portability. We discovered the Cas12a trans-cleavage activity against collateral single-stranded DNA (ssDNA) and employed the activity to develop a rapid nucleic acid detection system, namely HOLMES (one-hour low-cost multipurpose highly efficient system). Here, with the employment of thermophilic CRISPR-Cas12b, we create HOLMESv2 for four different applications: (1) specifically discriminating single nucleotide polymorphism (SNP); (2) simply detecting virus RNA, human cell mRNA and circular RNA; (3) conveniently quantitating target nucleic acids with a one-step system combined with LAMP amplification in a constant temperature, thus avoiding cross-contamination; (4) accurately quantitating target DNA methylation degree with the combination of Cas12b detection and bisulfite treatment. These results highlight the potential of HOLMESv2 as a promising platform for both molecular diagnostics and epigenetics applications.

    Copyright © 2019 American Chemical Society

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00209.

    • Oligonucliotides used in this study, comparison of HOLMESv2 and HOLMES; supplementary Figures S1–S10; Tables S1–S5 (PDF)

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    Cite this: ACS Synth. Biol. 2019, 8, 10, 2228–2237
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