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Leveraging Synthetic Antibody–DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox
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    Leveraging Synthetic Antibody–DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox
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    • Elisa Paialunga
      Elisa Paialunga
      Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
    • Neda Bagheri
      Neda Bagheri
      Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
      More by Neda Bagheri
    • Marianna Rossetti
      Marianna Rossetti
      Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
    • Laura Fabiani
      Laura Fabiani
      Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
    • Laura Micheli
      Laura Micheli
      Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
    • Alejandro Chamorro-Garcia*
      Alejandro Chamorro-Garcia
      Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
      *Email: [email protected]
    • Alessandro Porchetta*
      Alessandro Porchetta
      Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
      *Email: [email protected]
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2025, 14, 1, 171–178
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    https://doi.org/10.1021/acssynbio.4c00541
    Published January 2, 2025
    Copyright © 2025 American Chemical Society

    Abstract

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    We report here the use of antibody–DNA conjugates (Ab–DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab–DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab–DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.

    Copyright © 2025 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00541.

    • Reagents and materials; additional experimental details and procedures; PAGE gels of the Ab–DNA, ELISA, CIA, and CIMA optimizations; standard commercial ELISA; and CIMA challenged in buffer (PDF)

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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2025, 14, 1, 171–178
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.4c00541
    Published January 2, 2025
    Copyright © 2025 American Chemical Society

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