High-fidelity CRISPR/Cas13a trans cleavage-driven assembly of single quantum dot nanosensor for ultrasensitive detection of long noncoding RNAs in clinical breast tissues

Abstract

Long noncoding RNAs (lncRNAs) act as critical regulators in various cellular processes, and their dysfunction is implicated in carcinogenesis. Herein, we demonstrate high-fidelity CRISPR/Cas13a trans cleavage-driven assembly of single quantum dot (QD) nanosensor for ultrasensitive detection of long noncoding RNAs in clinical tissues. The presence of lncRNA can activate Cas13a/crRNA to collaterally cleave the substrate probes, producing a T7 promoter fragment that can initiate subsequent transcription amplification to generate efficient fluorescence resonance energy transfer (FRET). Taking advantage of excellent specificity of high-fidelity CRISPR/Cas13a system, high efficiency of transcription amplification, and near-zero background of single QD-based FRET, this nanosensor can achieve a detection limit of 1.65 aM, and it can differentiate target lncRNA from its mismatched members with single-base resolution. Moreover, it can measure lncRNA at the single-cell level, distinguish different subtypes of breast cancers, and assess the breast cancer progression. Notably, due to the programmability of crRNAs, this nanosensor can be extended to detect other nucleic acids (e.g., SARS-CoV-2 RNA, circRNA, miRNA, piRNA, and 16S rRNA) by simply altering the spacer region of crRNA, with great potential in lncRNAs-related molecular diagnostics.