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

Journal Article
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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.

Citation

Wang, 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.

Authors

  • Wang, Y.
  • Fu, L.
  • Tao, D.
  • Han, X.
  • Xu, B.
  • Deng, M.
  • Li, S.
  • Zhao, C.
  • Li, X.
  • Zhao, S.
  • Gong, P.
  • Yang, Y.
  • Khazalwa, Emmanuel M.
  • Ma, Y.
  • Ruan, J.
  • Li, C.
  • Xie, S.

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