Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR-based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification-free nucleic acid detection via harnessing the trans-cleavage mechanism of Cas13a and ultrasensitive graphene field-effect transistors (gFETs). CRISPR Cas13a-gFET achieves the detection of SARS-CoV-2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS-CoV-2 samples, including those with low viral loads (Ct value > 30). Overall, these findings establish our CRISPR Cas13a-gFET among the most sensitive amplification-free nucleic acid diagnostic platforms to date.

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

“…For example, upon binding to ssDNA or ssRNA, the trans -cleavage mechanism can release a fluorescence reporter from the quenched fluorescence probe [ 9 , 12 , 20 , 70 , 71 , 72 , 73 , 74 , 75 , 76 ]. Different CRISPR-Cas systems have been utilized for multiplex SARS-CoV-2 detection, including the Cas9 system (recognizes dsDNA) [ 77 , 78 ], Cas12 (recognizes both ssDNA and dsDNA) [ 9 , 71 , 72 , 75 , 76 , 79 ], and Cas13 (recognizes ssRNA) [ 12 , 20 , 71 , 74 , 80 , 81 ]. Additionally, amplification techniques, such as RT-PCR [ 9 , 71 , 72 , 76 ], RT-LAMP [ 73 , 77 , 79 ], and RT-RPA [ 9 , 20 , 75 , 78 ], are often combined with CRISPR-based detection to enhance the limit of detection (LOD) in viral detection assays.…”

Nano for CRISPR

“…Nanotechnology offers advantages to CRISPR-based nucleic acid detection assays. For example, graphene and gold nanoparticles have been used to sense signal changes resulting from target DNA cleavage on field-effect transistors; , reporter oligonucleotides labeled with gold nanoparticles can be readily detected by the eye in laminar flow assays, and DNA hydrogels labeled with fluorophores can be employed as assay substrates . Liposomal nanoparticles that employ isothermal amplification reagents can also be used to detect pathogen RNA in extracellular vesicles secreted by diseased cells, which can be directly captured from serum or plasma for point-of-care applications …”

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

“…In 2022, a dual enzyme amplification scheme that combines target-induced Cas activation with a following release of the enzymatic reporter—horseradish peroxidase (HRP)—into solution, has achieved quick, convenient (25 °C) and sensitive (~10 fM) detection of nucleic acids, without the need for PCR [ 74 ]. CRISPR/Cas13a-graphene field-effect transistors (gFETs) method, may be one of the most sensitive amplification-free diagnostic systems so far and has been validated for the detecting of SARS-CoV-2 and respiratory syncytial virus as low as 1 aM [ 73 ].…”