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

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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Fig. 1: CRISPR–Chip enables gene detection in less than 15 min.
Fig. 2: CRISPR–Chip is a liquid-gate field-effect transistor functionalized with CRISPR–dCas9.
Fig. 3: CRISPR–Chip selectively detects the gene target bfp.
Fig. 4: The gene-targeting dRNP unit effectively binds a selective gene locus in genomic DNA.
Fig. 5: CRISPR–Chip sensitivity and selectivity of the bfp target contained within whole genomic samples.
Fig. 6: CRISPR–Chip analysis of healthy and DMD clinical samples for DMD-associated dystrophin exon deletions.

Data availability

The authors declare that all data supporting the findings in this study are available within the paper and its Supplementary Information files.

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Acknowledgements

We acknowledge Cardea Bio for our use of their Agile R100 reader technology. We thank J. Corn (University of California, Berkeley) for providing us with the HEK-BFP cells. This work was primarily supported by Keck Start-up funding to the Aran Lab, by an Open Philanthropy Research Gift and by the Rogers Family Foundation to I.C.

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R.H. optimized the CRISPR–Chip design, performed the CRISPR–Chip DMD experiments, data collection and analysis, LOD optimization, HEK-BFP calibration methodologies in the presence and absence of contamination, and kinetic analysis, and prepared the manuscript. S.B. assisted in optimization of the CRISPR–Chip assay protocols, performed the MB-dRNP studies, DMD patient sample analysis, HEK-BFP PCR experiments and analysis, and prepared the manuscript. T.T. assisted with the initial CRISPR–Chip design, performed initial CRISPR–Chip protocols for HEK-BFP studies, and prepared the manuscript. T.d. performed the synthesis of sgRNA for the bfp and Scram studies, genomic purification and initial system design, and helped with manuscript preparation. J.E. contributed to the design of the DMD-based validation of CRISPR–Chip and provided the PCR and sequencing data for the DMD studies. M.S. contributed to the design of the DMD-based validation of CRISPR–Chip and assisted in manuscript preparation. N.A.W. and J.-Y.C. assisted T.D. with the synthesis of sgRNAs for bfp studies and assisted with sample preparation. J.N. and B.G. assisted with CRISPR–Chip data analysis and manuscript preparation. M.A. and J.P. assisted with manuscript preparation and data analysis. R.P. assisted with the design of threshold experiments, data analysis and CRISPR–Chip validation. N.M. supervised the synthesis of sgRNAs for the bfp and Scram studies. I.M.C. assisted with technology design, DMD validation and manuscript preparation. K.A. designed and developed the technology, planned and supervised the project, analysed, interpreted and integrated the data, and prepared the manuscript.

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Correspondence to Kiana Aran.

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K.A. is a co-founder of Nanosens Innovations, and R.P. is Vice President of Technology Development in the same company. The other authors declare no competing interests.

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Hajian, R., Balderston, S., Tran, T. et al. Detection of unamplified target genes via CRISPR–Cas9 immobilized on a graphene field-effect transistor. Nat Biomed Eng 3, 427–437 (2019). https://doi.org/10.1038/s41551-019-0371-x

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