Bead-based approaches for increased sensitivity and multiplexing of CRISPR diagnostics

Abstract CRISPR-based diagnostics have emerged as a promising tool for fast, accurate and portable pathogen detection. There has been rapid progress in pre-amplification processes and CRISPR-related enzymes used in these approaches, but the development of reporter systems and reaction platforms has lagged behind. In this paper, we develop bead-based techniques to address these gaps. First, we develop a novel bead-based split-luciferase reporter system with up to 20× sensitivity compared with standard fluorescence-based reporter design in CRISPR diagnostics. Second, we develop a highly deployable, bead-based platform capable of detecting nine distinct viral targets in parallelized, droplet-based reactions, with sensitivity reaching as low as 2.5 copies per µl of input RNA. We demonstrate the enhanced performance of both approaches on synthetic and clinical sample sensitivity, speed, multiplexing and deployability.