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Amplification-free nucleic-acid testing

Detection of unamplified target genes via CRISPR–Cas9 immobilized on a graphene field-effect transistor

This issue highlights a nanopatterned microfluidic chip for the detection of circulating exosomes in patient samples, a microfluidic assay for the quantification of the metastatic propensity of breast cancer cells, an amplification-free electrical biosensor for nucleic acids, virtual staining of unlabelled tissue sections via deep learning, and a quantitative microimmunohistochemistry assay for the grading of immunostains on tumour tissues.

The cover illustrates an electrical biosensor for nucleic acids that relies on the binding of target sequences to Cas9 immobilized on a graphene field-effect transistor.

See Hajian, et al. Nature Biomedical Engineering 3, 427–437 (2019)

Abstract:

Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR–Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR–Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR–Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR–Chip expands the applications of CRISPR–Cas9 technology to the on-chip electrical detection of nucleic acids.

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