CRISPR/Cas12a-Based APE1 Enzyme Cleavage Assay for Drug Resistance Analysis of Staphylococcus aureus-Related Pneumonia.

Abstract

Drug resistance analysis of Staphylococcus aureus is responsible for generating significant mortality and morbidity in numerous diseases. However, sensitive and accurate analysis of drug resistance of S. aureus remains a huge challenge. In this study, we present the development of a fluorescence biosensor based on the CRISPR/Cas12a system that enables label-free and ultrasensitive detection of the mecA gene in methicillin-resistant S. aureus (MRSA). The biosensor identified the mecA gene in MRSA using Cas12a/crRNA. This recognition triggered the trans-cleavage activity of Cas12a and the release of RNA1, which subsequently induced Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme-assisted target recycling and G-quadruplexes/Thioflavin T-based signal reaction. Based on this, the biosensor effectively detects the mecA gene with a low limit of detection of 212 aM and a high degree of selectivity, even toward single base mutations. Compared with the traditional CRISPR-Cas12a system-based methods, in which the signal amplification process is prone to generate nucleic acid sequence mismatch, which causes errors, the biosensor used APE1 to improve nucleic acid sequence recognition specificity to ensure that the RNA1 sequence released after Cas12a/crRNA cleavage can specifically guide the signal cycle. In addition to enhancing the CRISPR toolkit, the developed biosensor offers a novel method for the precise and sensitive identification of drug-resistant microbes that cause infections.