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

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.

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS‐CoV‐2 dual gene detection

“…However, the droplet microfluidic system is relatively complex throughout the process, which may limit its clinical applications. 20 22 In fact, this multi-diagnostic technology based on multiple enzymes has been used for various target detection in recent years, 23,24 particularly the platform established by Tian et al for the detection of SARS-CoV-2, 25 which is consistent with the present study in principle. However, as is well known, a convenient method of reading out the results is necessary for POCT, and test strips continue to play an important role in POCT detection.…”

Harnessing noncanonical trans-cleavage characteristics of Cas12 and Cas13a to enhance CRISPR-based diagnostics

“…A number of studies have sought to integrate various CRISPR-Cas systems, targeting multiplexed detection 22,23,24,25,26,27,28,29 or eliminating the need for pre-ampli cation prior to detection 38 . A fundamental condition for integrating CRISPR-Cas systems into a single detection assay is avoiding cross-activation of Cas enzymes by gRNA:DNA/RNA target duplexes from differing systems.…”

“…Overlooking these unique characteristics may compromise the e cacy of diagnostic systems. For example, leveraging Cas12a's canonical DNA-target activated trans-DNase activity along with Cas13a's RNA-target activated trans-RNase activity facilitated the development of a portable dual-gene detection platform that integrates both systems into a single assay 22,23,24,25,26,27,28,29 . However, Cas12a's atypical DNA target-activated trans-RNase activity may interfere with Cas13a's trans-RNase activity.…”