CRISPR/Cas13a-mediated interfacial cleaving of hairpin RNA reporter for PEAK1 nucleic acid sensing

Abstract

Dysregulation of PEAK1 (pseudopodium-enriched atypical kinase 1) plays a critical role in various cellular processes, including cell migration, proliferation and survival. Its aberrant expression or activity has been implicated in the pathogenesis of several diseases, particularly cancer. Early detection allows for the development and application of targeted therapies that can inhibit PEAK1 activity, potentially improving treatment outcomes. This study provides a new method for early diagnosis of PEAK1 by utilizing the specific RNA cleavage ability of Cas13a combined with electrochemical sensing technology. CRISPR/Cas13 has cis cleavage activity and can specifically recognize and cleave target RNA. Subsequently, its trans cleavage activity is activated to non-specifically cleave other single stranded RNAs, resulting in detectable signal changes. In the experiments conducted, high sensitivity for detecting PEAK1 mRNA was achieved by optimizing the interface cleavage of the hairpin reporter probe (ReRNA) molecule. The linear detection range is from 1 pg μL⁻¹ to 10 ng μL⁻¹, with a detection limit of 0.45 pg μL⁻¹. In addition, the results showed that the developed biosensor has good repeatability, reproducibility, and stability, which provides a novel method for the early screening of PEAK1.