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Using CRISPR-Chip Technology, Cardea Bio Aims to Develop Lab-Free Multi-Omic Technology

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NEW YORK – When Cardea Bio launched the first product based on its CRISPR-Chip technology in September 2019, CEO Michael Heltzen said he envisioned a future in which users would be able to program the sensor with single-guide RNAs in order to search, edit, engineer, or curate genomes in a simple way, without the need for amplification or complex instruments.

The idea, Heltzen said at the time, was for a user to be able to "Google a genome," with all the speed and simplicity that the phrase implies. Cardea's newest projects seem to show that it may have found a way to make that happen.

The CRISPR-Chip is a CRISPR-Cas9-based biosensor diagnostic device that uses a graphene transistor to analyze DNA in its native state, without the need for amplification, labeling, or optical instruments. The chip is able to very quickly signal whether or not a specific mutation, protein, or other component is present.

The 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. The 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.

Cardea's plan has always been to become the Intel of the diagnostics world — the company has started partnering with a number of different organizations that come to it with information on what their own customers want and knowledge of the specific biological problems they're looking to solve, and it provides them with customized chips that are suited for that particular purpose. Much like Intel chips, which power many of the world's personal computers, Heltzen is aiming to show that his company's chips can fulfill a similar role for disease diagnostics, agricultural tools, and research tools.

Indeed, in a new study published on Monday in Nature Biomedical Engineering, Cardea Chief Scientific Officer Kiana Aran and colleagues from Keck Graduate Institute, Vilnius University, and elsewhere described their use of a CRISPR-Chip to distinguish single point mutations between wild-type and homozygous mutant alleles in sickle-cell disease and amyotrophic lateral sclerosis (ALS), in unamplified DNA samples in about one hour.

Aran and several colleagues had published a paper in Nature Biomedical Engineering in March 2019 showing that the CRISPR-Chip could detect Duchenne muscular dystrophy in DNA samples 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.

But this is the first time the chip has been able to detect single nucleotide polymorphisms, Heltzen said.

For their study, the researchers incorporated a novel Cas9 ortholog into the CRISPR-Chip alongside the dCas9, allowing them to distinguish between samples with different zygosity of a particular SNP. The Cas enzymes were used to detect SNPs in the disease model target genes. In the sickle cell model, they looked for the disease-associated point mutation in the HBB gene of patients with and without the disease. They also tested ALS as an additional disease model by searching for a SNP in the superoxide dismutase type 1 (SOD1) gene in genomic DNA extracted from human induced pluripotent stem cells from a healthy individual and an individual with familial ALS.

By using a guide RNA to target the healthy HBB allele, SNP-Chip CRISPR complexes containing both dCas9 and Cas9 were able to discriminate between genomic DNA samples from healthy patients and those with sickle cell disease, the researchers found. When they tested the technology in the presence of non-homogenous DNA samples containing different percentages of target versus non-target DNA, they were also able to demonstrate the quantitative nature of the technology.

Further, targeting a different mutation was as simple as designing a new gRNA, which they demonstrated by utilizing SNP-Chip to detect the single-nucleotide mutation implicated in ALS.

And while technologies have been developed for SNP genotyping in recent years that have removed the need for expensive and bulky optical equipment or have bypassed the need for amplification, the researchers noted, none have done both. The CRISPR-based SNP-Chip, however, managed to overcome both of these challenges, detecting the single-point mutations in these disease models without amplification and without optical equipment. The sample is introduced onto the chip, which is then placed into a reader. Once the reaction is finished, the reader can send the results to a user's laptop.

"The thing about the CRISPR-Chip universe is that because the sensor is so sensitive and because CRISPR is so powerful in searching, we can start doing these things without amplification," Heltzen explained. "In layman's language, that translates to without use of a laboratory — because the reason why we need to take genetics and genomics into a laboratory is we need to take the DNA fragments, rip it into small pieces, and then amplify it to get millions of the same base pairs. That's how PCR works. That's how next-generation sequencing works. Because this sensor is so sensitive and because CRISPR is so powerful in searching and combining those two, you don't need a lab anymore, and that is obviously something that will have big future implications."

What Heltzen is truly envisioning is bringing the power of CRISPR to the bedside within a generation, allowing doctors to see the state of their patients' health in near real-time, and being able to take samples, plug them into diagnostic machines powered by Cardea CRISPR-Chips, and get a result back as fast as the biological components take to react to each other.

"We believe that we can get to a state where the doctor can stand next to the patient and see what is going on in the patient right now," he said. "We would like to see that in life science and in the whole healthcare industry. And that is quite a paradigm shift we're talking about. Why should we not empower [doctors and patients] with all the different signals that the immune system is producing on a second-by-second basis, that the genome has available as a starting point, the proteomics signals that are going on in our body? Why should the doctor not have them instantly on the screen next to the patient? So, this is a new perspective, but we need to build multi-omics in an instant, real-time technology if we want to get there."

That's where the importance lies in stepping away from amplification and labeling, Heltzen further explained. Those techniques freeze the analyte in time, as opposed to providing the kind of real-time information that he believes is more valuable to a doctor looking for a complete picture of a patient's health.

"You can argue that CRISPR is the most advanced tool to be able to understand genetics and genomics — how they really work. It's not just about gene engineering. It's a new generation of insight tools," he said. "If you're blind and you don't have a data stream, then the power of being interactive goes away because you're left with this one-dimensional perspective of [the genome] sequence. And a genome, like any other molecular biology, is a 3D organism, and it's actually interactive, especially if you start engineering it."

As a real-time picture of human health is a key part of this vision of the future, it's perhaps not surprising that Cardea also looked to make an impact during the COVID-19 pandemic, willing to work with anyone who might have ideas about SARS-CoV-2's RNA, its proteins, its genes, the proteins of the immune system or patient DNA in correlation to certain outcomes, and to put its chips to work to measure these parameters as quickly and simply as possible, Heltzen said.

One project that is now coming to fruition has Cardea working as a subcontractor to a Defense Advanced Research Projects Agency (DARPA) grant awarded to the Georgia Tech Research Institute (GTRI), to develop a platform to detect airborne SARS-CoV-2 particles. Before the pandemic, GTRI had been developing a technology to take aerosols out of the air, by compressing air streams and basically squeezing the aerosols out in small drops, Heltzen said. The idea behind the SARS-CoV-2 biosensor platform that the partners are developing involves combining Cardea's chip technology with the GTRI aerosol platform, such that these small drops would be streamed onto a Cardea chip, which would test them for the virus in situ.

Such a system could be built into the air filtration system on airplanes, Heltzen said, or perhaps in schools or airports.

The company is also engaged in other COVID-related projects with other partners. Although Heltzen declined to provide specific details, he did say that the company has shown that it can use antibodies to the spike protein and antibodies to the N protein for detection, and different isotopes of the spike protein to detect different variants of SARS-CoV-2. He also said they're working on making a version of the CRISPR-Chip to specifically detect SARS-CoV-2 RNA.

"We can build a lot of different sensor technology for the pandemic," Heltzen said, adding that there is general interest in making sensors that can distinguish between COVID and the flu.

But these diagnostics and sensors — not to mention the real-time bedside healthcare monitoring system Heltzen has envisioned — are still to come. In the meantime, Cardea's first product to market is perhaps the purest definition of the Google-a-genome concept and could be one of the most useful to other CRISPR researchers.

In September, Cardea teamed up with CRISPR quality control testing firm CRISPR QC to launch CRISPR-BIND, a rapid and highly sensitive tool to characterize guide RNA and CRISPR-Cas interactions. Heltzen called it "the first layer of the onion" in terms of giving people who work with CRISPR the insight they need into what they're actually doing with the genomes they're engineering and making sure they're editing in the right places.

"It's easy to get started with CRISPR, but it's very difficult to master. So that's the canyon that we're trying to carry people over," he said.  

For example, Heltzen said, there are several bioinformatics programs available that will help a researcher construct and synthesize a guide RNA based on the specific sequence they'd like to edit. But if the guide doesn't work exactly as predicted, those bioinformatics programs don't tell the researcher why. As a result, the researcher will likely try one or two or five different versions of the guide RNA, and although one of them might work, it'll be hard to figure out why one version did and the others failed.

"And when it starts getting to a commercial scale, you can't just be using guesswork to try to power your way through this problem," Heltzen said. "It's fine for publication when you just need something to work. But if you, for example, are running a clinical trial in the future, it's not good enough. If you're standing in front of the FDA [you can't say], 'Well, we don't know why it works in some patients but not on others.'"

CRISPR-BIND answers those questions in real-time, he added. And not only does it work for all Cas enzymes, but Heltzen claims the next version of the technology will be able to compare the editing performance of different Cas enzymes or Cas orthologs to each other.

The company is also talking to different partners about building the system into various other CRISPR platforms — for example, into the incubators used to make cell lines — to serve as an instant quality control measure for genome engineering research and experimentation.

But Heltzen also believes the firm's technology could one day help usher in germline genome editing. In its September report on heritable human genome editing, the International Commission on the Clinical Use of Human Germline Genome Editing said the practice isn't yet safe or effective enough to be used in human embryos, citing in part the need for accurate and robust technology to assess both on- and off-target edits.

Right now, Heltzen said, the world — and the CRISPR community — is absolutely not ready for germline genome editing. If we are ever going to be ready for germline, he added, "there is a lot of quality control work in front of us, because we're getting ahead of our skis."

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