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 Reprints and permissions information is available at www.nature.com/reprints. * kiana_aran@kgi.edu. Correspondence and requests for materials should be addressed to K.A. Author contributions 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.

CRISPR/Cas13a‐Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification‐Free miRNA Diagnostics

“…In the recent past, studies, employing CRISPR‐associated methods for the detection of nucleic acids, have been utilized using different CRISPR‐associated (Cas) effectors . Varying Cas effectors were used for targeting different nucleic acids, like the Cas9 effector for the detection of double‐stranded DNA (dsDNA), along with the Cas12 for the detection of single‐stranded DNA (ssDNA) . The microbial CRISPR effector Cas13a (previously named C2c2), displays, in contrast to the other Cas effectors, a triggered cleavage capability of nontarget single‐stranded RNAs (ssRNAs) in the surrounding .…”

“…Moreover, until now amplification‐free CRISPR‐powered biosensing methods are rare and only one approach, employing Cas9, exists for an amplification‐free detection of dsDNA, based on a graphene field‐effect transistor (gFET) . For the detection of dsDNAs, several incubation steps lead to the final functionalized CRISPR‐Chip, having a readout time of down to 15 min, compared to our stop‐flow amplified readout process of roughly 9 min (Figure S17, Supporting Information).…”

Biochemically diverse CRISPR-Cas9 orthologs

“…In addition to guide RNA (gRNA) recognition, a sequence motif, termed the PAM (protospacer adjacent motif), is required for the initiation of Cas9-guide RNA target binding and cleavage 6,7 . Easily reprogrammed to recognize new DNA sequences, it has been widely adopted for use in a multitude of applications to edit genomic DNA, modulate gene expression, visualize genetic loci, or detect targets in vitro [8][9][10][11][12][13][14][15] . To date, just a handful of variants are used for these applications [16][17][18] with the Streptococcus pyogenes (Spy) Cas9 being used most widely 2 .…”

Highly specific enrichment of rare nucleic acids usingThermus thermophilusArgonaute

“…In recent years, researchers have identified various enzymes from archaea and bacteria that can be programmed with nucleic acids to cleave complementary strands, among which CRISPR-Cas have attracted considerable attention as genome-editing tools [1][2][3] . In medical diagnostics, CRISPR-associated nucleases have been used to (1) amplify reporter signals during nucleic acid detection [4][5][6][7][8] , (2) detect unamplified targets immobilized on graphene field-effect transistors 9 , and (3) enrich rare alleles to enhance detection limits of oncogenic sequences [10][11][12] . Despite remarkable progress, the applications of CRISPR-Cas are restricted due, among other things, to their reliance on the protospacer-adjacent motif (PAM), which is absent in many sequences of interest [10][11][12] .…”