Leveraging Synthetic Antibody-DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2025-01-02 DOI:10.1021/acssynbio.4c00541

Elisa Paialunga, Neda Bagheri, Marianna Rossetti, Laura Fabiani, Laura Micheli, Alejandro Chamorro-Garcia, Alessandro Porchetta

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Abstract

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.

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我们在此报告利用抗体-DNA 结合物（Ab-DNA）激活 CRISPR-Cas12a 酶的旁路裂解活性。我们的研究结果表明，抗体-DNA共轭物能有效触发CRISPR-Cas12a的附带切割活性，使抗体介导的识别事件转化为荧光输出。我们利用抗体 DNA 作为 Cas12a 的激活剂开发了两种不同的免疫测定方法：基于 CRISPR 的免疫传感测定（CIA）和基于 CRISPR 的免疫磁测定（CIMA），前者用于检测 SARS-CoV-2 穗状 S 蛋白，其灵敏度优于传统的酶联免疫吸附测定（ELISA）。值得注意的是，CIMA 成功检测了未稀释唾液中的 SARS-CoV-2 spike S 蛋白，在 2 小时的检测中，检测限 (LOD) 为 890 pM。我们的结果凸显了将基于 Cas12a 的信号放大与抗体检测方法相结合的好处。Ab-DNA共轭物的潜力与CRISPR技术相结合，为免疫测定中使用的传统酶提供了一种有前途的替代方法，并能促进用于检测非核酸目标的多功能CRISPR分析平台的发展。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.