CRISPR-Chip shows potential for disease Dx, agricultural, research applications

CRISPR-based technologies have garnered significant attention as their potential for treating or curing various diseases has become more apparent. But the technology’s use as a research tool is also one of its most useful features — especially when it’s used to validate the efficiency and specificity of other CRISPR-based technologies.

Cardea Bio and Nanosens Innovations have taken the technology one step further. Earlier this year, the companies announced that they had teamed up to develop CRISPR-Chip — a CRISPR-Cas9-based biosensor diagnostic device using a graphene transistor. Now, they’ve demonstrated the chip’s utility in detecting Duchenne muscular dystrophy. And they’re also looking to develop additional disease diagnostics, agricultural tools, and research tools using the same basic idea.

Graphene is an allotrope of carbon. Its structure is a single layer of atoms in a 2D hexagonal lattice. In recent years, scientists have used graphene to develop semiconductors, conduct cancer research, and make lightweight fibers. The material is about 100 times stronger than the strongest steel, in proportion to its thickness. It conducts heat and electricity very efficiently and it’s nearly transparent. It’s also biocompatible.

People started theorizing about having a material that is in between a crystal and a metal, and is biocompatible. That means you can use it as a transistor like silicon, but it does not break down if you put [biological material] on it,” said Cardea CEO Michael Heltzen. “That means we can potentially have biology-based transistors — meaning a transistor [like the ones] in our computers and our cell phones, but instead of having an on and off gate that is electrical-based, we could have it based on whether the biology is there or if it’s not there.”

The advantage to this approach, Heltzen said, is that it allows a researcher to observe DNA in its natural state, without the need for amplification and optical instruments. Instead of breaking down the DNA, amplifying it, labeling it, and then shining an optical laser on it in order to read it (or as Heltzen puts it, “running it over with a bus three times“), the graphene-based chips are able to read the DNA in its native state and very quickly signal whether or not a specific mutation, protein, or other component is present.

“We have zoomed in optically on biology for so long that we couldn’t zoom in anymore, and what we started to do [with sequencing] was we started having biology bend or break so we could easily observe it,” he added. “You could tell a similar story about mass spec and a lot of other technologies that are optical. We make biology do certain things so we can easily observe it. That’s why you can’t do genomics and proteomics and transcriptomics on the same sample, because we do all of these things to it to observe one of the parameters.”

Cardea started when its Chief Technology Officer Brett Goldsmith, an expert on graphene, had the idea to combine graphene transistors with antigens in order to detect antibodies in biological samples. An antigen is bound to the surface of a graphene transistor, and when a biological sample is introduced on the chip, the binding interaction of the antibody and antigen cause a small charge that runs through the graphene, which then sends a positive signal to the readout on the chip.

Within five years, Heltzen said, the company’s R&D team had optimized the processes needed to mass-produce graphene-based antigen chips. They wanted to move on to nucleic acid detection as well, but realized that looking at DNA and RNA would be a bit harder to do than simple antibody detection.

Kiana Aran, an assistant professor and principal investigator at the Keck Graduate Institute of Applied Life Sciences, had a different problem. In 2011 or 2012, Aran started working with graphene to create biological transistors because she found the material was more sensitive than silicon.

I liked it because not only was it extremely more sensitive, but also you could functionalize it much easier than a silicon-based transistor, because you can modify the chemistry of the graphene surface and attach any type of biological molecules that you want. We could use that capacity to process biology at a very sensitive scale,” she said.

The idea of attaching a CRISPR-Cas complex to a graphene transistor seemed logical, she added, because the main function of CRISPR is to search for and find specific pieces of DNA. Aran’s chip uses deactivated Cas9 (dCas9) — the nuclease in this form is still able to search for and bind to specific stretches of DNA but is unable to cut. So, much like an antigen transistor, a CRISPR-based transistor searches a biological sample for a specific target using dCas9, and if the target is present, the charge that the biological interaction creates is sensed by the graphene, which then sends a signal to the readout on the chip. The genetic material is purified, but there’s no need for PCR amplification, Aran said.

The problem, however, was that she was spending an enormous amount of time fabricating graphene chips in order to test various CRISPR complexes and guide RNAs. A friend pointed her in the direction of Cardea, which had not only developed a mass production process for graphene chips by that point, but had also created a signal readout to attach to it. She and Heltzen cofounded Nanosens, and Cardea licensed its graphene transistors to the new company.

In a paper published in March in Nature Biomedical Engineering, Aran and her colleagues showed the possible utility of the CRISPR-Chip as a disease diagnostic. They demonstrated that they could rapidly and selectively detect a genomic DNA target sequence without the need for amplification.

They used CRISPR-Chip to analyze DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, as well as clinical DNA samples with two distinct mutations — deletions of exons 3 and 51 in the human dystrophin gene — which are commonly found in individuals with Duchenne muscular dystrophy. Within 15 minutes, the CRISPR-Chip signaled the presence of target genes at a concentration of 1.7 femtomolar and without the need for amplification. The chip’s limit of detection was lower than that of previously reported amplification-free technologies for the detection of target sequences, and its speed and simplicity showed it had the potential to be used as a point-of-care device in the future.


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