P53-MDM2 interaction monitoring and inhibitors potency evaluation based on CRISPR-Cas12a sensing platform

Abstract

Protein-protein interactions (PPIs) monitoring is critical to reveal cellular fundamental mechanism and facilitate drugs discovery. Nevertheless, the facial and robust protein 53 (p53)-murine double minute2 (MDM2) interaction detection methods remain challenging. Herein, we developed a PPIs detection platform based on CRISPR-Cas12a sensing system for the first time for p53-MDM2 interaction monitoring and inhibitors potency evaluation, designated Cas-PPIor (Cas12a-mediated PPIs detector). Inspired by the conformational-dependent consensus DNA binding ability of p53, a double stranded DNA (dsDNA) probe was designed elaborately through incorporating Cas12a activation DNA sequence into the specific p53 DNA-binding sequence. Thus, the PPI event can then be transduced into the collateral cleavage activity of Cas12a. As both the Cas12a/CRISPR RNA (crRNA) complex and p53 can bind to the same dsDNA probe in a competitive manner, the activated Cas12a mediated fluorescent readout scaled negatively with the p53 binding. Accordingly, we monitored the p53-MDM2 interaction process as well as its inhibition by a small-molecule antagonist. Based on the sequence specific binding of wild p53, our designed Cas-PPIor achieved high accuracy for differentiation of wild-type p53 cells from p53 mutation or deletion cells. Through discriminating p53 levels from cellular matrix treated with enantiomers of Nutlin-3, the assay also demonstrated reliable capability to evaluate drugs antiproliferative potency. By integrating the DNA binding ability of proteins with intrinsic flexible programmability and outstanding sensitivity of Cas12a, the proposed Cas-PPIor methodology hold great potential to enable accurate and facial PPIs monitoring and inhibitors potency evaluations.