💡 Application Note: A critical analysis of reporter interactions in CRISPR-Cas12a LFA. The design strategies for eliminating false positives are directly applicable when using Due Bio’s optimized Lateral Flow Dipsticks.
Title: Advancing Lateral Flow Detection in CRISPR/Cas12a Systems Through Rational Understanding and Design Strategies of Reporter Interactions
Source: Biosensors (MDPI) (2025)
Abstract
CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a–LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions.