Development of a naked eye CRISPR-Cas12a and -Cas13a multiplex point-of-care detection of genetically modified swine

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dc.creatorWang, Y.
dc.creatorFu, L.
dc.creatorTao, D.
dc.creatorHan, X.
dc.creatorXu, B.
dc.creatorDeng, M.
dc.creatorLi, S.
dc.creatorZhao, C.
dc.creatorLi, X.
dc.creatorZhao, S.
dc.creatorGong, P.
dc.creatorYang, Y.
dc.creatorKhazalwa, Emmanuel M.
dc.creatorMa, Y.
dc.creatorRuan, J.
dc.creatorLi, C.
dc.creatorXie, S.
dc.date2023-07-21
dc.date2023-07-13T07:26:54Z
dc.date2023-07-13T07:26:54Z
dc.date.accessioned2026-06-27T16:32:35Z
dc.descriptionThe 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.
dc.identifierhttps://hdl.handle.net/10568/131143
dc.identifier.urihttp://hdl.handle.net/123456789/125961
dc.languageen
dc.publisherAmerican Chemical Society
dc.rightsLimited Access
dc.sourceWang, Y., Fu, L., Tao, D., Han, X., Xu, B., Deng, M., Li, S., Zhao, C., Li, X., Zhao, S., Gong, P., Yang, Y., Khazalwa, E.M., Ma, Y., Ruan, J., Li, C. and Xie, S. 2023. Development of a naked eye CRISPR-Cas12a and -Cas13a multiplex point-of-care detection of genetically modified swine. ACS Synthetic Biology 12(7): 2051–2060.
dc.subjectcrispr
dc.subjectdiagnosis
dc.subjectswine
dc.subjectgenetically modified organisms
dc.subjectbiomedical engineering
dc.titleDevelopment of a naked eye CRISPR-Cas12a and -Cas13a multiplex point-of-care detection of genetically modified swine
dc.typeJournal Article

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Instituto Internacional de Investigaciones Pecuarias (ILRI)