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.

Fig. 1 |. CRISPR–Chip enables gene detection in less than 15 min.

CRISPR–Chip exploits the gene-targeting capacity of CRISPR–Cas9 and the sensitivity of gFET to enable rapid detection of a gene target from the whole genomic sample without amplification. The dCas9 complexed with a target-specific sgRNA (referred to as dRNP) is immobilized on the surface of the graphene within the gFET construct. The immobilized dRNP scans the whole genomic DNA until it identifies its target sequence (complementary to the 5′ end of sgRNA), unzips the double helix and kinetically binds to the DNA target. The selective binding event of the target DNA to the dRNP complex modulates the electrical characteristics of the gFET and results in an electrical signal output within 15 min.

Fig. 2 |. CRISPR–Chip is a liquid-gate field-effect transistor functionalized with CRISPR-dCas9.

a, CRISPR–Chip is composed of a three-terminal gFET, which utilizes dRNP-functionalized graphene as a channel between the source and the drain electrodes, with a liquid gate that is in contact with the genomic sample. The binding of the dRNP to its target DNA results in modulation of graphene conductivity and Donnan potential, which results in a change in the electrical characteristics of the transistor. b, Schematics of CRISPR–Chip functionalization. The graphene surface of the gFET is first functionalized with a heterofunctional PBA linker comprised of a planar pyrene ring system that electrostatically interacts with the π-system of graphene. A carboxylate group (highlighted in light blue) at the terminal end of the hydrocarbon arm (highlighted in pink) of the PBA linker acts as the dCas9-tethering unit that covalently couples to dCas9, securing the nuclease to the surface of graphene. Any unfunctionalized PBA molecules are blocked with amino-polyethylene glycol 5-alcohol (PEG). Finally, sgRNA complementary to a gene of interest is introduced and complexes with dCas9 tethered to the graphene surface. Complexation of the sgRNA to dCas9 affords the functional gene-targeting dRNP unit and completes CRISPR–Chip functionalization.

Fig. 3 |. CRISPR–Chip selectively detects the gene target bfp.

To show selectivity, CRISPR–Chips were functionalized with dRNP-BFP (denoted with the red sgRNA) to target the bfp gene and dRNP-Scram (denoted with the grey sgRNA) as a negative control. a, bfp PCR products were analysed on both the bfp-targeting CRISPR–Chip (left) and the Scram-targeting CRISPR–Chip (right). b, The I response of the bfp-targeting CRISPR–Chip in the presence of the bfp PCR product was significantly higher (***P = 0.0002, two-tailed t-test, n = 3) than that generated by the Scram-targeting CRISPR–Chip. c, The dRNP-BFP-functionalized CRISPR–Chip representative real-time I response detected its target dsDNA in 2.5 min. Error bars represent s.d.

Fig. 4 |. The gene-targeting dRNP unit effectively binds a selective gene locus in genomic DNA.

a, A CRISPR-functionalized MB was synthesized to evaluate the binding capacity of the dRNP for its target gene contained within whole genomic samples. The CRISPR-functionalized MB was produced by first covalently attaching dCas9 to the MB surface, then incubating with sgRNA-BFP. Finally, genomic material was incubated with the functional CRISPR-beads for 30 min at 37 °C. b, The capture efficiency of the BFP-dRNP-functionalized CRISPR-beads was evaluated by gel electrophoresis to determine the amount of non-target HEK genomic material, or target HEK-BFP genomic material that was captured by the beads. The dRNP-BFP was significantly more able to bind and maintain its affinity to its target genomic material extract (HEK-BFP; ~54%) compared with non-target genomic material (**P = 0.002, two-tailed t-test, n = 3). Error bars represent s.d. See Supplementary Fig. 10 for full scans of the gels.

Fig. 5 |. CRISPR–Chip sensitivity and selectivity of the bfp target contained within whole genomic samples.

a, Solutions of HEK-BFP (left) and HEK DNA (right) were analysed with the dRNP-BFP-functionalized CRISPR–Chip. b, CRISPR–Chip demonstrated a significant change in signal output (***P = 0.0002, two-tailed t-test) when exposed to target HEK-BFP, which contained the bfp sequence, compared with the genomic sample lacking the bfp target. c, CRISPR–Chip sensitivity calibration curve in the presence of varied amounts of HEK-BFP (mean; n = 3). R² is the determination coefficient. d, Real-time CRISPR–Chip I response in the presence of varying concentrations of target HEK-BFP and the subsequent rinsing step (mean; n = 3). e, CRISPR–Chip selectivity for 900 ng HEK-BFP in the presence of varied concentrations of HEK DNA lacking the bfp gene target (mean; n = 3). f, Real-time CRISPR–Chip selectivity for 900 ng HEK-BFP in the presence of varied concentrations of HEK DNA lacking the bfp gene target (mean; n = 3). g, CRISPR–Chip selectivity test (NS, not significant (P > 0.05), two-tailed t-test) (mean; n = 3). h, Real-time CRISPR–Chip selectivity test (mean; n = 3). i, PCR selectivity normalized to the control (P > 0.05, two-tailed t-test). Error bars represent s.d.

Fig. 6 |. CRISPR–Chip analysis of healthy and DMD clinical samples for DMD-associated dystrophin exon deletions.

a, Schematic of the dystrophin gene with highlighted target exons. b, Top, I response obtained by CRISPR–Chip functionalized with dRNP-DMD3 in the presence of healthy and DMD clinical samples (*P = 0.017, one-tailed t-test). Bottom, schematic of sgRNA-DMD3, designed to target exon 3. x represents a false negative as confirmed by sequencing. c, Top, I response obtained by CRISPR–Chip functionalized with dRNP-DMD51 in the presence of healthy and DMD clinical samples (**P = 0.0014, one-tailed t-test). Bottom, schematic of sgRNA-DMD51, designed to target exon 51. d, CRISPR–Chip’s negative signal threshold was defined by testing CRISPR–Chips with samples lacking target exons at the highest concentrations of genomic sample obtainable from commercially available buccal swabbing methods to ensure that high sample concentrations would not lead to false positives. e, CRISPR–Chip results for the presence of targeted exons (+) within healthy and DMD clinical samples, as defined by a threshold of 1.73%. f, dRNP-DMD51-functionalized CRISPR–Chip’s I response in the presence of varied amounts of clinical sample A (mean; n = 3). g, Reproducibility of individual CRISPR–Chips functionalized with dRNP-DMD51 in the presence of clinical sample A. Error bars represent s.d.