Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2023-07-11 DOI:10.1021/acssynbio.3c00089

Yuan Wang, Lanting Fu, Dagang Tao, Xiaosong Han, Bingrong Xu, Manfei Deng, Sheng Li, Changzhi Zhao, Xinyun Li, Shuhong Zhao, Ping Gong, Yu Yang, Emmanuel Mulaya Khazalwa, Yunlong Ma, Jinxue Ruan*, Changchun Li* and Shengsong Xie*,

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引用次数: 1

Abstract

The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green–red–yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.

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转基因猪CRISPR-Cas12a和-Cas13a多重检测方法的建立

快速视觉CRISPR (RAVI-CRISPR)检测使用Cas12a和Cas13a酶在样品中进行精确的基因检测。然而，由于缺乏特异性的单链dna荧光猝灭(ssDNA-FQ)和rna荧光猝灭(ssRNA-FQ)报告基因切割机制，RAVI-CRISPR在单管多重检测中的应用受到限制。我们报告了一种敏感和特异性双基因Cas12a和Cas13a诊断系统的发展。为了优化现场测试的应用，我们设计了一种便携式多重荧光成像检测方法，可以用肉眼区分测试结果。本研究使用Cas12a和Cas13a酶在单管中同时检测多重重组酶聚合酶扩增(multiple recombinase polymerase amplification, RPA)的双基因扩增产物。由此产生的正交DNA和RNA侧枝切割，使用绿-红-黄荧光信号转换反应系统，用便携式蓝光和紫外线(UV)光透照器检测，特异性地区分单个和混合的ssDNA-FQ和ssRNA-FQ报告子。作为概念验证，证明了基因组编辑猪的可靠多重RAVI-CRISPR检测，对CD163敲除、乳铁蛋白(LF)敲入和野生型猪样本的分析具有100%的灵敏度和特异性。这种便携式裸眼多重RAVI-CRISPR检测平台可以在资源有限的环境中提供准确的转基因动物和传染病的即时筛查。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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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.