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

The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12aandCas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPRis limited in single-tube multiplex detection applications due tothe lack of specific single-strand (ss) DNA-fluorescently quenched(ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavagemechanisms. We report the development of a sensitive and specificdual-gene Cas12a and Cas13a diagnostic system. To optimize the applicationfor field testing, we designed a portable multiplex fluorescence imagingassay that could distinguish test results with the naked eye. Herein,dual gene amplified products from multiplex recombinase polymeraseamplification (RPA) were simultaneously detected in a single tubeusing Cas12a and Cas13a enzymes. The resulting orthogonal DNA andRNA collateral cleavage specifically distinguishes individual andmixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow,fluorescent signal conversion reaction system, detectable with portableblue and ultraviolet (UV) light transilluminators. As a proof-of-concept,reliable multiplex RAVI-CRISPR detection of genome-edited pigs wasdemonstrated, exhibiting 100% sensitivity and specificity for theanalysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-typepig samples. This portable naked-eye multiplex RAVI-CRISPR detectionplatform can provide accurate point-of-care screening of geneticallymodified animals and infectious diseases in resource-limited settings.