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.