Cardea combined with CRISPR enables amplification-free digital genomics


Cardea Bio Inc. and Nanosens Innovations Inc., Keck Graduate Institute and UC Berkeley detect two deleted DMD exons in 15 minutes down to 1.7 fM sensitivity

Image of graphene biosensor, electrodes and circuitry from Nature Biomedical Engineering https://www.nature.com/articles/s41551-019-0371-x
Image of graphene biosensor, electrodes and circuitry from Nature Biomedical Engineering March 2019

We are currently living in an exciting time in molecular diagnostics, as the fields of chemical fluorescence, optics, electronics, microfluidics, biochemistry, genomics and nanofabrication intersect and produce surprising synergies. For example, at the recent Advances in Genome Biology and Technology conference in Marco Island, FL a new sponsor called Codexis (Redwood City, CA) has an offering for the life sciences vendor: a directed evolution platform they call CodeEvolver for custom enzyme development. Say you are using a proprietary enzyme that is very expensive or perhaps restricted for your use due to patent restrictions: they will start with a native enzyme, and the biochemical end-point parameters you require for the specific application, and they will evolve that native enzyme into an optimized, custom one.

Okay this process takes time and money, and this service has been offered to other vertical industries (think biopharmaceutical processing or the food and beverage industry) and now Codexis is helping life science tools and diagnostic companies. Here is a combination of molecular biology, biochemistry, and industrial chemical engineering (to produce these enzymes at scale) with unique value.

Some background on Cardea Bio

For those of you unfamiliar with Cardea, I wrote them up with this post in the context of Personalized Medicine last October. In late January of 2019 they published this paper in Nature Scientific Reports titled “Digital Biosensing by Foundry-Fabricated Graphene Sensors”, with a particular emphasis upon industrial manufacturing. Imagine a set of single-atom-thick graphene sheets being applied to a disposable microelectronic chip in the manufacturing context, something that has not been done before, in spite of hundreds of millions (and even billions) of dollars being spent in the academic world on nanotechnology and engineered materials (the EU’s Graphene Flagship is one of several examples).

One of Cardea’s early access academic partners Keck Graduate Institute already published on using the chip and electronic reader to study heterochronic parabiosis, where two animals of different ages are joined surgically and experiments are performed to determine which system-wide proteins have age-specific effects on the older animal. This experiment has demonstrated superior sensitivity and minimal sample input requirements compared to other label-free protein analysis technologies (surface plasmon resonance, biolayer interferometry, the list goes on).

The latest news from Cardea is their partnership with Nanosens and the product CRISPR-Chip, allowing for an electronic readout to for people working with CRISPR gene-editing, and it can furthermore be used to exploit potential genetic diagnostics applications.

The race for CRISPR diagnostics

When I attended the AMP Europe 2018 conference last spring (April 2018) in Rotterdam, an interesting presentation was given by a Broad Institute researcher named Omar Abudayyeh. In true conference fashion, their group published this CRISPR diagnostics paper in Science only the day before using a new class of Cas enzymes Cas13, Cas12a and Csm6. They improved the original concept through multiplexing (to four-fold), a sensitivity increase to 2 attomolar, increasing the signal 3.5-fold, and figuring out how to translate the assay to a lateral-flow assay (LFA) readout platform, a mature technology. (The Supplemental Information helpfully points out the costs of their reagents is “<$0.60”). The Broad Institute published a handy explainer of their technology here, and only last week (March 21 2019) the diagnostic company Sherlock Biosciences was announced with a $35M initial financing round.

Not to be outdone, in the same issue of Science the Berkeley group of Jennifer Doudna published this paper describing the functions of CRISPR-Cas-12a, and a company called Mammoth Biosciences raised $23M last summer.

The use of CRISPR for sensitive and inexpensive genetic diagnostics is enormously appealing. Instead of trying to deploy a real-time thermal cycler in the field, or trying to figure out the best way to deploy a USB-powered single-molecule sequencer, a dip-and-read lateral flow device is an attractive alternative.

A few applications for PONT

The acronym ‘POCT’ stands for Point-Of-Care-Test, often in the context of tests that do not have the same level of complexity nor need for as extensive training. Point-of-Care represents a setting where a given patient may be waiting for a healthcare provider to return the results of a rapid strep test – tests are typically done where the patient is receiving care.

The Point-Of-Need (PON) is in a general environment, wherever the need may be. It could be in a lake or stream where suspected coliform contamination occurs, or on a farm where livestock are raised and salmonella needs to be quickly tested. It could be at-home, to test for a strep infection.

There are multi-billion-dollar worldwide markets for manufacturers of microbiology testing equipment and reagents in a central laboratory context, and that central laboratory business model will continue; however, the PONT for genetic tests in the field or at-home represents an enormous opportunity.

Doing away with amplification

Through use of an enzymatically inactivated CRISPR-Cas complex tethered to the Cardea detector, it detects the 20 base-pair recognition sequence as determined by the guide RNA resulting in changes to the electrical properties of the biosensor.

Screencapture from video showing the graphene chip being loaded
Screencapture from video showing the graphene chip being loaded

The aforementioned CRISPR methods still use RPA (recombinase polymerase amplification), which while feasible as an isothermal amplification method, requires a fixed temperature incubation adding complexity and cost. Purified nucleic acid, however, is still required for the Cardea-Nanosens method.

Direct detection of target DNA

The paper, “Detection of unamplified target genes via CRISPR/Cas9 immobilized on a graphene field-effect transistor”, was published in Nature Biomedical Engineering and showed data from the device they call CRISPR-Chip to detect within 15 minutes a DMD deletion from unamplified genomic DNA down to 1.7 femtomolar sensitivity, or 3.3 ng/uL of genomic DNA. The paper states room for improving sensitivity by optimizing the disabled Cas9 density, the geometry of the graphene channel, and the field-effect transistor parameters, and also the potential use of planar additives such as methylene blue.

The use of cell-line DNA is curious to me, although upon further reflection given the limited sensitivity it makes sense they would choose to tackle a target for CRISPR therapeutics.

Cardea Bio and Nanosens Inc. put together the following video with a nice presentation of the technology and animations of how the technology works.

Description of the Nanosens CRISPR-Chip courtesy of Cardea Bio and Nanosens

What is next for the CRISPR-Chip

One attractive application the senior author Dr. Kiana Aran indicates for future application is to use this platform for detecting off-target CRISPR edits that currently uses whole-genome sequencing, which has limited sensitivity, helping to make CRISPR based therapeutics safer and more effective in the future. This CRISPR QC application represents a brand new digital biology method of looking at how we understand and interact with genomes in a much faster and user-friendly manner.

Nanosens Innovation is currently in early access, and you can contact them here for further information. They have put together a CRISPR-Chip QC Brochure here (PDF) around the off-target QC application.

References

  1. Haijan R and Aran K et al. Nature Biomed Engr 2019. Detection of unamplified target genes via CRISPR/Cas9 immobilized on a graphene field-effect transistor.
  2. Cardea Bio website.
  3. Nanosens Innovation website.
  4. Keck Graduate Institute website of Dr. Kiana Aran.

About Dale Yuzuki

A sales and marketing professional in the life sciences research-tools area, Dale currently is employed by Sysmex-Inostics USA as the Director of Marketing. He will help Sysmex-Inostics build out their liquid biopsy franchise (OncoBEAM and Plasma SafeSEQ) with market planning, positioning and branding as well as thought leadership, opinion-leader management, and sensing of market trends. He also represents Sysmex at tradeshows and other events. For additional biographical information, please see my LinkedIn profile here: http://www.linkedin.com/in/daleyuzuki and also find me on Twitter @DaleYuzuki.

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