Search

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. • PPIs detection platform based on CRISPR-Cas12a was developed for the first time. • The protein binding event can be detected with cleavage activity of Cas12a. • The p53-MDM2 interaction and its inhibition by antagonist was monitored. • The designed Cas-PPIor achieved high accuracy for cells in different p53 states. [ABSTRACT FROM AUTHOR]

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans -cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 μL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility. • Orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay was proposed. • Lateral flow strips with two target lines were designed for SARS-CoV-2 detection. • The detection sensitivity reached 10 copies per test. • Nasopharyngeal swab clinical samples were tested with high accuracy. • The whole dual genes detection procedure can be finished within 1 h. [ABSTRACT FROM AUTHOR]