Designing a dry-lab pipeline for multiplex CRISPR μPAD assays against dengue and chikungunya

Abstract

This study demonstrate the design of a dry-lab multiplex CRISPR diagnostics pipeline for Dengue (DENV) and Chikungunya (CHIKV) in this manuscript. The pipeline integrates sequence input with CRISPR assay design through multiple critical steps: conserved-window discovery, primer design for RT-RPA, Cas12a guide discovery, and ?PAD timing estimation. To provide an in silico platform for an easily re-producible pipeline to bridge the gap between genetic sequences and readily implementable multiplex diagnostics designed for ?PADs. Here the propose method bypass the wet-lab testing in the early stages and generates the design assets such as ranked primers, guide sequences and ?PAD timing for prototyping the device. This pipeline can facilitate rapid point-of-care diagnostics development for resource limited settings while bridging the translation gap between molecular diagnostics and practical device constraints. This method is very tunable, intuitive and delivers results ready for downstream experimental work directly.