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


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.


  1. 1.

    Yuen, R. K. C. et al. Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder. Nat. Neurosci. 20, 602–611 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Iyer, G. et al. Genome sequencing identifies a basis for everolimus sensitivity. Science 338, 221 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    Waddell, N. et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 518, 495–501 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Wu, D. et al. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon. Anal. Chim. Acta 957, 55–62 (2017).

    CAS  Article  Google Scholar 

  5. 5.

    Ermini, M. L., Mariani, S., Scarano, S. & Minunni, M. Direct detection of genomic DNA by surface plasmon resonance imaging: an optimized approach. Biosens. Bioelectron. 40, 193–199 (2013).

    CAS  Article  Google Scholar 

  6. 6.

    Bao, Y. P. et al. SNP identification in unamplified human genomic DNA with gold nanoparticle probes. Nucleic Acids Res. 33, e15–e15 (2005).

    Article  Google Scholar 

  7. 7.

    Bartlett, J. M. S. & Stirling, D. in PCR Protocols (eds Bartlett, J. M. S. & Stirling, D.) 3–6 (Humana Press, 2003).

  8. 8.

    Cao, L. et al. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications. Biosens. Bioelectron. 90, 459–474 (2017).

    CAS  Article  Google Scholar 

  9. 9.

    Furlan, I., Domljanovic, I., Uhd, J. & Astakhova, K. Improving design of synthetic oligonucleotide probes by fluorescence melting assay. ChemBioChem 20, 587–594 (2019).

    CAS  Article  Google Scholar 

  10. 10.

    Busse, N. et al. Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes. Oncotarget 8, 12620–12636 (2017).

    Article  Google Scholar 

  11. 11.

    Gootenberg, J. S. et al. Nucleic acid detection with CRISPR–Cas13a/C2c2. Science 365, 438–442 (2017).

    Article  Google Scholar 

  12. 12.

    Li, S.-Y. et al. CRISPR–Cas12a-assisted nucleic acid detection. Cell Discov. 4, 20 (2018).

    Article  Google Scholar 

  13. 13.

    Pardee, K. et al. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 165, 1255–1266 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    Chen, J. S. et al. CRISPR–Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018).

    CAS  Article  Google Scholar 

  15. 15.

    Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  16. 16.

    Zheng, C. et al. Fabrication of ultrasensitive field-effect transistor DNA biosensors by a directional transfer technique based on CVD-grown graphene. ACS Appl. Mater. Interfaces. 7, 16953–16959 (2015).

    CAS  Article  Google Scholar 

  17. 17.

    Reddy, D., Register, L. F., Carpenter, G. D. & Banerjee, S. K. Graphene field-effect transistors. J. Phys. Appl. Phys. 44, 313001 (2011).

    Article  Google Scholar 

  18. 18.

    Mekler, V., Minakhin, L. & Severinov, K. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation. Proc. Natl Acad. Sci. USA 114, 5443–5448 (2017).

    CAS  Article  Google Scholar 

  19. 19.

    Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).

    CAS  Article  Google Scholar 

  20. 20.

    Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    Article  Google Scholar 

  21. 21.

    Georgakilas, V. et al. Noncovalent functionalization of graphene and graphene oxide for energy materials, biosensing, catalytic, and biomedical applications. Chem. Rev. 116, 5464–5519 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Ohshima, H. & Ohki, S. Donnan potential and surface potential of a charged membrane. Biophys. J. 47, 673–678 (1985).

    CAS  Article  Google Scholar 

  23. 23.

    Bergveld, P. A critical evaluation of direct electrical protein detection methods. Biosens. Bioelectron. 6, 55–72 (1991).

    CAS  Article  Google Scholar 

  24. 24.

    Schasfoort, R. B. M., Bergveld, P., Kooyman, R. P. H. & Greve, J. Possibilities and limitations of direct detection of protein charges by means of an immunological field-effect transistor. Anal. Chim. Acta 238, 323–329 (1990).

    CAS  Article  Google Scholar 

  25. 25.

    Kaisti, M. Detection principles of biological and chemical FET sensors. Biosens. Bioelectron. 98, 437–448 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Palazzo, G. et al. Detection beyond Debye’s length with an electrolyte-gated organic field-effect transistor. Adv. Mater. 27, 911–916 (2015).

    CAS  Article  Google Scholar 

  27. 27.

    Lerner, M. B. et al. Large scale commercial fabrication of high quality graphene-based assays for biomolecule detection. Sens. Actuators B Chem. 239, 1261–1267 (2017).

    CAS  Article  Google Scholar 

  28. 28.

    Lepvrier, E., Doigneaux, C., Moullintraffort, L., Nazabal, A. & Garnier, C. Optimized protocol for protein macrocomplexes stabilization using the EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, zero-length cross-linker. Anal. Chem. 86, 10524–10530 (2014).

    CAS  Article  Google Scholar 

  29. 29.

    Johnsson, B., Löfås, S. & Lindquist, G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal. Biochem. 198, 268–277 (1991).

    CAS  Article  Google Scholar 

  30. 30.

    Gao, N. et al. Specific detection of biomolecules in physiological solutions using graphene transistor biosensors. Proc. Natl Acad. Sci. USA 113, 14633–14638 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Afsahi, S. et al. Novel graphene-based biosensor for early detection of Zika virus infection. Biosens. Bioelectron. 100, 85–88 (2018).

    CAS  Article  Google Scholar 

  32. 32.

    Liu, B. et al. Parts-per-million of polyethylene glycol as a non-interfering blocking agent for homogeneous biosensor development. Anal. Chem. 85, 10045–10050 (2013).

    CAS  Article  Google Scholar 

  33. 33.

    Glaser, A., McColl, B. & Vadolas, J. GFP to BFP conversion: a versatile assay for the quantification of CRISPR/Cas9-mediated genome editing. Mol. Ther. Nucleic Acids 5, e334 (2016).

    CAS  Article  Google Scholar 

  34. 34.

    Richardson, C. D. et al. CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat. Genet. 50, 1132–1139 (2018).

    CAS  Article  Google Scholar 

  35. 35.

    Deboer, T. R., Wauford, N., Chung, J.-Y., Torres Perez, M. S. & Murthy, N. A cleavage-responsive stem-loop hairpin for assaying guide RNA activity. ACS Chem Biol. 13, 461–466 (2018).

    CAS  Article  Google Scholar 

  36. 36.

    Lee, K. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889–901 (2017).

    Article  Google Scholar 

  37. 37.

    Amoasii, L. et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 362, 86–91 (2018).

    CAS  Article  Google Scholar 

  38. 38.

    Den Dunnen, J. T. et al. Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet. 45, 835–847 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Aartsma-Rus, A., Ginjaar, I. B. & Bushby, K. The importance of genetic diagnosis for Duchenne muscular dystrophy. J. Med. Genet. 53, 145–151 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Dumont, N. A. et al. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nat. Med. 21, 1455–1463 (2015).

    CAS  Article  Google Scholar 

  41. 41.

    Allen, D. G., Whitehead, N. P. & Froehner, S. C. Absence of dystrophin disrupts skeletal muscle signaling: roles of Ca2+, reactive oxygen species, and nitric oxide in the development of muscular dystrophy. Physiol. Rev. 96, 253–305 (2016).

    CAS  Article  Google Scholar 

  42. 42.

    Mendell, J. R. et al. Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann. Neurol. 71, 304–313 (2012).

    CAS  Article  Google Scholar 

  43. 43.

    Chamberlain, J. S., Gibbs, R. A., Rainer, J. E., Nguyen, P. N. & Thomas, C. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16, 11141–11156 (1988).

    CAS  Article  Google Scholar 

  44. 44.

    Tang, Z. et al. A dynamic database of microarray-characterized cell lines with various cytogenetic and genomic backgrounds. G3 (Bethesda) 3, 1143–1149 (2013).

    Article  Google Scholar 

  45. 45.

    Myers, R. H. Huntington’s disease genetics. NeuroRx 1, 255–262 (2004).

    Article  Google Scholar 

  46. 46.

    Koeberl, D. D. et al. Mutations causing hemophilia B: direct estimate of the underlying rates of spontaneous germ-line transitions, transversions, and deletions in a human gene. Am. J. Hum. Genet. 47, 202–217 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Giannelli, F. et al. Gene deletions in patients with haemophilia B and anti-factor IX antibodies. Nature 303, 181–182 (1983).

    CAS  Article  Google Scholar 

  48. 48.

    D’Agata, R. et al. Direct detection of point mutations in nonamplified human genomic DNA. Anal. Chem. 83, 8711–8717 (2011).

    Article  Google Scholar 

  49. 49.

    International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature 431, 931–945 (2004).

  50. 50.

    Long, G. L. & Winefordner, J. D. Limit of detection. A closer look at the IUPAC definition. Anal. Chem. 55, 712A–724A (1983).

    CAS  Article  Google Scholar 

  51. 51.

    Muhammad, A. et al. A screen printed carbon electrode modified with carbon nanotubes and gold nanoparticles as a sensitive electrochemical sensor for determination of thiamphenicol residue in milk. RSC Adv. 8, 2714–2722 (2018).

    CAS  Article  Google Scholar 

  52. 52.

    Wu, W. et al. Low-cost, disposable, flexible and highly reproducible screen printed SERS substrates for the detection of various chemicals. Sci. Rep. 5, 10208 (2015).

    CAS  Article  Google Scholar 

  53. 53.

    Shams, N. et al. A promising electrochemical sensor based on Au nanoparticles decorated reduced graphene oxide for selective detection of herbicide diuron in natural waters. J. Appl. Electrochem. 46, 655–666 (2016).

    CAS  Article  Google Scholar 

  54. 54.

    Shams, N. et al. Electrochemical sensor based on gold nanoparticles/ethylenediamine-reduced graphene oxide for trace determination of fenitrothion in water. RSC Adv. 6, 89430–89439 (2016).

    CAS  Article  Google Scholar 

  55. 55.

    Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  Article  Google Scholar 

  56. 56.

    Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).

    Article  Google Scholar 

  57. 57.

    Huang, Y. et al. Nanoelectronic biosensors based on CVD grown graphene. Nanoscale 2, 1485–1488 (2010).

    CAS  Article  Google Scholar 

  58. 58.

    Boughanem, H. & Macias-Gonzalez, M. High-Throughput Isolation of Genomic DNA From Buccal Swab on the Eppendorf epMotion® 5075 VAC (Eppendorf, 2016).

  59. 59.

    Storhoff, J. J. et al. Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system. Biosens. Bioelectron. 19, 875–883 (2004).

    CAS  Article  Google Scholar 

  60. 60.

    Jung, Y. L., Jung, C., Park, J. H., Kim, M. I. & Park, H. G. Direct detection of unamplified genomic DNA based on photo-induced silver ion reduction by DNA molecules. Chem. Commun. 49, 2350–2352 (2013).

    CAS  Article  Google Scholar 

  61. 61.

    Storhoff, J. J., Lucas, A. D., Garimella, V., Bao, Y. P. & Müller, U. R. Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nat. Biotechnol. 22, 883–887 (2004).

    CAS  Article  Google Scholar 

  62. 62.

    Kalyanasundaram, D. et al. Rapid extraction and preservation of genomic DNA from human samples. Anal. Bioanal. Chem. 405, 1977–1983 (2013).

    CAS  Article  Google Scholar 

  63. 63.

    Rodriguez, N. M., Wong, W. S., Liu, L., Dewar, R. & Klapperich, C. M. A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples. Lab Chip 16, 753–763 (2016).

    CAS  Article  Google Scholar 

  64. 64.

    Stine, R., Mulvaney, S. P., Robinson, J. T., Tamanaha, C. R. & Sheehan, P. E. Fabrication, optimization, and use of graphene field effect sensors. Anal. Chem. 85, 509–521 (2013).

    CAS  Article  Google Scholar 

  65. 65.

    Tuite, E. & Norden, B. Sequence-specific interactions of methylene blue with polynucleotides and DNA: a spectroscopic study. J. Am. Chem. Soc. 116, 7548–7556 (1994).

    CAS  Article  Google Scholar 

  66. 66.

    Lau, H. Y. et al. Specific and sensitive isothermal electrochemical biosensor for plant pathogen DNA detection with colloidal gold nanoparticles as probes. Sci. Rep. 7, 38896 (2017).

    CAS  Article  Google Scholar 

  67. 67.

    Jiang, F. & Doudna, J. A. CRISPR–Cas9 structures and mechanisms. Annu. Rev. Biophys. 46, 505–529 (2017).

    CAS  Article  Google Scholar 

  68. 68.

    Gill, R. T., Garst, A. & Lipscomb, T. E. W. Nucleic acid-guided nucleases. US patent 10,011,849 (2018).

  69. 69.

    Gill, R. T., Garst, A. & Lipscomb, T. E. W. Nucleic acid-guided nucleases. US patent 9,982,279 (2018).

  70. 70.

    Goldsmith, B. R. et al. Digital biosensing by foundry-fabricated graphene sensors. Sci. Rep. 9, 434 (2019).

    Article  Google Scholar 

  71. 71.

    Gao, L. et al. Repeated growth and bubbling transfer of graphene with millimetre-size single-crystal grains using platinum. Nat. Commun. 3, 697–699 (2012).

    Article  Google Scholar 

  72. 72.

    Kulkarni, G. S. & Zhong, Z. Detection beyond the Debye screening length in a high-frequency nanoelectronic biosensor. Nano Lett. 12, 719–723 (2012).

    CAS  Article  Google Scholar 

  73. 73.

    Munje, R. D., Muthukumar, S., Selvam, A. P. & Prasad, S. Flexible nanoporous tunable electrical double layer biosensors for sweat diagnostics. Sci. Rep. 5, 14586 (2015).

    CAS  Article  Google Scholar 

  74. 74.

    Wang, C., Yan, Q., Liu, H. B., Zhou, X. H. & Xiao, S. J. Different EDC/NHS activation mechanisms between PAA and PMAA brushes and the following amidation reactions. Langmuir 27, 12058–12068 (2011).

    CAS  Article  Google Scholar 

  75. 75.

    Everaerts, F., Torrianni, M., Hendriks, M. & Feijen, J. Biomechanical properties of carbodiimide crosslinked collagen: influence of the formation of ester crosslinks. J. Biomed. Mater. Res. A 85, 547–555 (2008).

    Article  Google Scholar 

  76. 76.

    Dang, Y. et al. Optimizing sgRNA structure to improve CRISPR–Cas9 knockout efficiency. Genome Biol. 16, 280 (2015).

    Article  Google Scholar 

  77. 77.

    BcMag Carboxy-Terminated Magnetic Beads (Bioclone, 2004).

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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).

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