A team of academic and industry researchers in California has developed a new technique that further demonstrates the utility of CRISPR-Cas technology in the diagnostic space. The researchers have developed a novel graphene-based biosensor, named CRISPR-Chip, that enables digital detection of a specific genetic mutation from a patient’s DNA sample, without the need for amplification or sequencing.

The hand-held device utilizes the sequence-specific gene-targeting properties of deactivated Cas9 complexed with a specific single guide RNA and immobilized on a graphene-based field effect transistor.

“We have, together with our partners, developed the first transistor that uses CRISPR to search your genome for potential mutations,” says senior author Kiana Aran, PhD, assistant professor in the department of bioengineering at University of California (UC), Berkeley, and the Keck Graduate Institute (KGI). “You just put your purified DNA sample on the chip, allow CRISPR to do the search, and the graphene transistor from Cardea reports the result of this search in 15 minutes.”

The CRISPR-Chip is reported in a March 25th paper entitled, “Detection of unamplified target genes via CRISPR/Cas9 immobilized on a graphene field-effect transistor” in Nature Biomedical Engineering. The CRISPR-Chip, following last week’s launch of the (unrelated) Sherlock Biosciences, focused on CRISPR diagnostics, is solidifying the hard tack CRISPR-Cas is taking into this arena.

Senior author Kiana Aran, PhD, of the University of California, Berkeley

The CRISPR-Chip collaboration includes researchers from UC Berkeley, KGI, Scripps College, and Claremont Mckenna College, as well as two young bioscience companies—Cardea Biosciences and Nanosens Innovations—both in San Diego.

Described in an accompanying video released by Nanosens, the CRISPR-Chip prototype overlays thousands of CRISPR-Cas molecules on a graphene transistor, which is sensitive to electrically charged materials such as DNA. If the CRISPR-Cas molecules fail to detect a target gene in the sample, the DNA will be released without binding. However, when a perfect sequence match is found, the capture of the sample sequence binding will create an additional charge on the graphene surface, which is sensed by CRISPR-Chip and read out as an electrical signal.

In the new report, Aran’s team used CRISPR-Chip to analyze DNA samples collected from HEK293T cell lines expressing the bfp gene, which encodes for blue fluorescent protein, a gene commonly used for validation of CRISPR-Cas gene editing. They also utilized clinical samples from patients with Duchenne muscular dystrophy (DMD) which is caused by mutations in the dystrophin gene. Although mutations can occur across all 79 exons in the gene, the most frequent deletions occur at exons 2–10 and exons 45–55. The team used CRISPR-Chip to detect mutations at two of these commonly deleted exons (exon 3 or exon 51.) To do this, two CRISPR-Chip constructs were incubated with tissue derived genomic material from male DMD patients with identified, known DMD mutations. CRISPR-Chip was able to specifically detect the deletions without any pre-amplification.

“We need about 1/10 of the amount of DNA from a cheek swab,” notes Aran. She says the technology is “pretty exciting, because we’re skipping most of the lab processing that’s usually performed on those DNA samples.” The hardware required is about the size of a large cell phone, and can be run from a laptop, and in the future as an app on a mobile device.

“The sensitivity in technical terms is 3.3 nanograms per microliter,” Aran tells GEN. “Of course, sensitivity and specificity for an individual diagnostic test needs to be measured in a clinical trial, which we have not done yet.”

Clever CRISPR

The platform is “very clever,” comments Stuart Lindsay, director of the Center for Single Molecule Biophysics in the Biodesign Institute at Arizona State University, who was not involved with the study. “The novelty here lies in using CRISPR to do the genomic searching. It would be very easy and pretty conventional to detect a target by hybridization on the surface of any type of chemfet [chemical-sensing field effect transistor] and here it happens to be a graphene-based chemfet. But the search function of the CRISPR allows it to spool through the genome looking for the target, which is why unamplified DNA can be used.”

Despite its promise, one DNA molecular electronics expert (who requested anonymity) told GEN that the article has a relatively thin amount of experimental data that raises doubts that this is working reproducibly. The expert would like to see evidence that the system can detect conformational changes in a single molecule, noting that it performs an ensemble binding measurement and that the signal the chip detects is from the contribution of maybe millions of large DNA molecules interacting with the surface.

But, Brett Goldsmith, PhD, CTO of Cardea and co-senior author on the study asserts that  the “data [are] among the best and most reproducible I’ve seen from a nanoelectronic sensor.”

Regarding detection, Goldsmith notes that “with [this] system, you could absolutely PCR amplify DNA to the point that you could measure from any starting pre-amplified concentration. You would also have the same background problems any DNA amplification process brings. This is exactly what everyone else does, but very few people talk about what can be done to detect DNA without amplification (because most techniques can’t do it).” He calls into question the assumption that it is always a good idea to amplify DNA and notes that they think that DNA amplification like PCR should only be used when it’s absolutely necessary, because there are a lot of practical problems to using it outside of a research lab.

The lab tool will be ready this year, according to Aran, who says her team is already producing devices with Cardea and using them together with KGI. They are talking to several industry partners about potential partnerships. Aran says they have seen a lot of interest in the lab version of the CRISPR-Chip but that moving into the clinical space is more complicated. “A clinical tool requires clinical trials, a mature regulatory strategy, and an understanding of the billing practices in diagnostics,” Aran said.

“It is very important that we are smart and careful about how we use the opportunities, that we, the world, have been given via having access to gene-editing,” Aran notes. She adds that they are very focused on the on-target vs. off-target problem that “a lot of people seem to be ignoring or at least not addressing directly.”

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