Direct and noninvasive fluorescence analysis of an RNA-protein interaction based on a CRISPR/Cas12a-powered assay

Abstract

RNA-protein interactions (RPIs) play critical roles in gene transcription and protein expression, but current analytical methods for RPIs are mainly performed in an invasive manner, involving special RNA/protein labeling, hampering access to intact and precise information on RPIs. In this work, we present the first CRISPR/Cas12a-based fluorescence assay for the direct analysis of RPIs without RNA/protein labeling steps. Select vascular endothelial growth factor 165 (VEGF₁₆₅)/its RNA aptamer interaction as a model, the RNA sequence simultaneously serves as both the aptamer of VEGF₁₆₅ and crRNA of CRISPR/Cas12a system, and the presence of VEGF₁₆₅ facilitates VEGF₁₆₅/its RNA aptamer interaction, thus prohibiting the formation of Cas12a-crRNA-DNA ternary complex along with low fluorescence signal. The assay showed a detection limit of 0.23 pg mL^-1, and good performance in serum-spiked samples with an RSD of 0.4 %-13.1 %. This simple and selective strategy opens the door for establishing CRISPR/Cas-based biosensors for gaining intact information on RPIs, and shows widespread potential for other RPIs analysis.

Keywords: CRISPR/Cas12a; Fluorescence; RNA aptamer; RNA-protein interactions; VEGF(165).