Universal Amplification-Free RNA Detection by Integrating CRISPR-Cas10 with Aptameric Graphene Field-Effect Transistor

Abstract

Amplification-free, highly sensitive, and specific nucleic acid detection is crucial for health monitoring and diagnosis. The type III CRISPR-Cas10 system, which provides viral immunity through CRISPR-associated protein effectors, enables a new amplification-free nucleic acid diagnostic tool. In this study, we develop a CRISPR-graphene field-effect transistors (GFETs) biosensor by combining the type III CRISPR-Cas10 system with GFETs for direct nucleic acid detection. This biosensor exploits the target RNA-activated continuous ssDNA cleavage activity of the dCsm3 CRISPR-Cas10 effector and the high charge density of a hairpin DNA reporter on the GFET channel to achieve label-free, amplification-free, highly sensitive, and specific RNA detection. The CRISPR-GFET biosensor exhibits excellent performance in detecting medium-length RNAs and miRNAs, with detection limits at the aM level and a broad linear range of 10−15 to 10−11 M for RNAs and 10−15 to 10−9 M for miRNAs. It shows high sensitivity in throat swabs and serum samples, distinguishing between healthy individuals (N=5) and breast cancer patients (N=6) without the need for extraction, purification, or amplification. This platform mitigates risks associated with nucleic acid amplification and cross-contamination, making it a versatile and scalable diagnostic tool for molecular diagnostics in human health.

1. Introduction

Nucleic acid assays are essential for infectious disease diagnosis, genetic disease screening, and early cancer detection. Their sensitivity, specificity, and detection speed directly impact diagnostic accuracy, treatment efficacy, and public health security [1–4]. Rapid and precise nucleic acid detection is especially critical in contexts such as infectious disease outbreaks and personalized medicine. In recent years, recent progress in molecular biology and nanotechnology has accelerated the development of nucleic acid detection technologies, ranging from enzymatic amplification methods such as quantitative polymerase chain reaction (qPCR) and loop-mediated isothermal amplification (LAMP) [5–7] to enzyme-free amplification strategies like hybridization chain reaction and catalytic hairpin assembly [8–10], more recently, amplification-free direct detection methods [11–13]. Amplification-free strategies, though avoiding amplification-related contamination, are still challenging to detect ultra-low nucleic acid concentration [14, 15]. Thus, there is an urgent need for nucleic acid detection methods that integrate high sensitivity, efficiency, low cost, and operational simplicity to enhance their practical application in molecular diagnostics.

Recently, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have attracted significant academic and commercial interest for the development of CRISPR-based molecular diagnostics, owing to their exceptional nucleic acid-targeting capabilities [16–19]. In a typical type II CRISPR system, Cas9 proteins cleave double-stranded DNA (dsDNA) under the guidance of CRISPR RNA (crRNA). However, their strict dependence on protospacer adjacent motif recognition limits their applicability in nucleic acid detection [20, 21]. In contrast, type V (Cas12) and type VI (Cas13) CRISPR systems facilitate signal amplification through a unique trans-cleavage mechanism, wherein activation by the target DNA or RNA induces nonspecific cleavage of surrounding DNA or RNA. This process operates in a multiple flip-flop mode, enabling rapid sensor signal generation [22, 23]. However, it also leads to the degradation of the target DNA or RNA, causing ...