RESUMO
Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.
Assuntos
Técnicas Biossensoriais/métodos , Proteína 9 Associada à CRISPR/metabolismo , DNA/genética , Polimorfismo de Nucleotídeo Único , Anemia Falciforme/genética , Anemia Falciforme/patologia , Técnicas Biossensoriais/instrumentação , Proteína 9 Associada à CRISPR/química , DNA/metabolismo , Genoma Humano , Grafite/química , Heterozigoto , Homozigoto , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Superóxido Dismutase-1/genética , Transistores EletrônicosRESUMO
Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening and disease modeling tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system (tumor-on-a-chip) that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions. Here we have validated this microphysiological system (MPS) platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment responses in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional monolayer culture and 3-dimensional spheroids.