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CRISPR-Chip Shows Potential for Disease Dx, Agricultural, Research Applications

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NEW YORK – 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.

In a Nature Biomedical Engineering opinion column in June, researchers from the University of Freiburg and Imperial College London noted that the device should be amenable to the detection of multiple nucleic acids within a single sample by using complexes of dCas9 with different sgRNAs programmed to find specific target sequences. 

"Yet the implementation of an array of multiple [graphene-based field-effect transistor] gFETs on one single chip, which would be needed to spatially separate the different dCas9-sgRNA complexes, might increase the cost and complexity of the device," they wrote. 

But Heltzen and Aran said that they're already working on these issues, and trying to figure out the most elegant and efficient ways to build chips that could detect any required target — whether that involves multiplexing Cas enzymes together onto a single chip, multiplexing more than one gRNA into each CRISPR complex to look for more than one target at a time, or even linking a series of chips together to create a mini diagnostic machine of sorts.

"We're looking at other Cas enzymes. We're monitoring how they're working, how they're interacting [with the graphene]," Aran said. "We have so much capability that we have not exploited yet."

Heltzen also noted that although some diseases are marked by a mix of genetic mutations, it can take the presence of only one mutation to cause the disease. In such cases, it may not be necessary to develop a chip that can sense all the mutations for that given condition because signaling the presence of one would be enough to diagnose the disease. Further, some Cas enzymes act on RNA instead of DNA, which would be useful in certain applications. These nucleases are also being tested.

Additionally, the company is testing the chip's ability to detect a variety of diseases. Certain analytes are harder than others to detect without amplification, Heltzen said, so consequently, certain diseases may be harder to detect than others. But the company is working on possible ways to amplify DNA signals directly on the chip — previous studies have shown that Cas9 itself can be used to amplify target sequences, and that's also something Cardea is experimenting with, he added.

Eventually, the company would like to put a point-of-care disease diagnostic in doctors' offices, though that would also require approval from the US Food and Drug Administration.

Importantly, Heltzen emphasized, Cardea doesn't only want to make a product that it then sells. It also wants other organizations and companies to tell the company what they need and potentially partner with it. Cardea would then build specific CRISPR-graphene chips for those particular applications. The company has set up what it calls an innovation partnership program, under which it accepts suggestions from potential partners, and then sits down with other companies to systematically map out the needs of the customers. 

Heltzen compared his vision to what Intel has been able to do with computer chips. "What we set out to do is called 'Powered by Cardea.' It's inspired by Intel Inside," he said. "We would like to partner with a number of different companies and then have our little shiny sticker saying, 'Powered by Cardea,' the same way it says, 'Intel Inside' in a lot of laptops, and servers, and cell phones."

And the company has indeed started partnering with a number of different organizations that come to Cardea with information on what their own customers want and knowledge on the specific biological problems they're looking to solve. The value Cardea adds, Heltzen said, is the ability to create a detection technology that is almost instant and much simpler than existing optical technologies.

Aran and her team are also working on sample prep considerations for the chip. Generally, all that's needed is a purified biological sample, whether plasma from blood or something else. The company is working on developing a sample prep kit that could be sold with the chip for certain applications to help users purify samples as needed. 

Importantly, the CRISPR-Chip doesn't just have potential to diagnose diseases. It can also be used as a research tool, to test other CRISPR-based technologies — whether they're on-target or off-target — or to optimize the development of gRNAs and test binding strengths and fidelity. 

"We have innovation partnerships with therapeutics companies that are using [CRISPR-Chip technology] as a research and quality assurance tool for their work," Heltzen said.

They both emphasized the wide variety of possibilities that might be available in the future with the CRISPR-Chip, whether it's as a tool for assessing quality or even as a companion diagnostic for CRISPR-based therapies. 

"We're also looking at how each of these guide RNAs interact with the chromatic structure, because if you're doing editing, this is what the complex is interacting with," Aran said. "We're developing strategies to look into that and see if we can give some sort of an on- and off-targeting score for [various CRISPR] complexes. And of course, this would be a great companion diagnostic if you're doing some sort of editing and you want to know how much of your cells may have been edited in the process of the treatment. So, these are all possibilities that we will be developing with appropriate partners."

Even the agriculture industry could have its own CRISPR-Chip line. One of Cardea's innovation partners has asked the company to develop handheld transistors that farmers could use in their fields to diagnose plant diseases. This would obviate the need for mass use of pesticides, Heltzen said. But the project will take about five years of research to find the right biomarkers, create the right chips, and then develop a device that's durable enough to be used in the field.

Even the eventual disposal of the chips is something Cardea is looking at. In certain cases, as in the detection of certain diseases, the chips will be a one-time-use consumable. In other cases, there may be a way to wash the chip and use it again, Heltzen said. The company is even looking into a recycling program to reuse old chips, so that they don't end up creating a garbage problem.

Most important is that Cardea hasn't even started to show what it's capable of, Heltzen added, noting that eventually, the chip could be capable of dozens of applactions. "Anyone using any CRISPR system will have on- and off-target challenges until the guide RNA has been optimized. You can argue that [optimization] has to be done by someone else, but our perspective is that we can solve the problem and be a platform that is good at guide optimizing and even Cas optimizing, because we can see the differences," he added.

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