On March 25 2019,

Nature Biomedical Engineering published the Aran Lab’s research and development of a Graphene-enhanced Field Effect Biosensor that harnesses CRISPR’s genome-searching capability and graphene’s sensitivity for the detection of unamplified target genes.

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.

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.


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.

Watch a video about our system and read more about our biosensor in this article from Keck Graduate Institute.

Watch a video about our system and read more about our biosensor in this article from Keck Graduate Institute.