On-site SNP discrimination of Monkeypox viral DNA at room temperature using dCas9-enhanced extended-gate field-effect transistor

Abstract

During the COVID-19 pandemic, there was heightened concern about the sudden spread of viral diseases. As the threat of coronavirus transmission began to diminish, a subsequent outbreak caused by monkeypox virus (MPXV) emerged in Europe and North America. Notably, MPXV infection tends to be transmitted through sexual contact, increasing the difficulty of tracing the infection route through passive testing. As a result, there is a pressing need for cost-effective, precise, and rapid on-site screening techniques to detect MPXV. Recently, the CRISPR system, known as a genome editing tool, has been used as a rapid on-site diagnostic tool for detecting nucleic acids mostly through the use of Cas12a and Cas13a. Intriguingly, deficient Cas9 (dCas9) can recognize specific doublestranded DNA (dsDNA) sequences and bind to complementary DNA sequences. Here, we devised an innovative digital field-diagnosis platform that can be readily employed in clinical settings. This platform combines an ultrasensitive field-effect transistor (FET) with the dCas9 system, enabling quantitative analysis of biomarkers. Our dCas9-EG-FET can directly detect viral dsDNA without any amplification or pretreatment. When the dCas9 of our dCas9-EG-FET system recognizes the specific target viral DNA, the EG-FET system provides voltage differences by using FET features that measure the ion concentration on the surface of the FET sensor. Furthermore, it takes only 20 min to detect MPXV DNA using our dCas9-EG-FET system at room temperature (RT), and this system can be used to effectively distinguish single-point mutations with high sensitivity. Our findings suggest that the use of a CRISPR-Cas-based biosensor could lead to an effective strategy for screening for infectious diseases.