CRISPR-Cas9 is best known for its powerful ability to make double-stranded breaks in DNA, allowing scientists to delete and edit genes with relative ease. But switch out Cas9 for another protein, and CRISPR becomes a programmable tool for detecting the presence of certain nucleic acid sequences.

This feature has startups eyeing the CRISPR toolbox for its use as a next-generation molecular diagnostic test, one that could be customized to virtually any disease, infection, or mutation and be administered at home, at the point of care or, in the event of a disease outbreak, to get a quick readout to allow faster response.

That’s the idea behind Mammoth Biosciences, which came out of stealth mode in April 2018 with intellectual property licensed from the lab of Jennifer Doudna, PhD, at the University of California, Berkeley. Following close behind, Sherlock Biosciences launched in March 2019 with an initial $35 million in funding and technology developed by Feng Zhang, PhD, and his colleagues at the Broad Institute of MIT and Harvard. Zhang’s company, based in Cambridge, MA, takes its name from its CRISPR platform, Specific High-sensitivity Enzymatic Reporter unLOCKing select SHERLOCK). Both CRISPR developers are co-founders of the respective companies.

co-founders of Mammoth Biosciences
The co-founders of Mammoth Biosciences include select from left to right) CDO Lucas Harrington, PhD; CEO Trevor Martin, PhD; CTO Ashley Tehranchi, PhD; scientific advisory board chair Jennifer Doudna, PhD; and CRO Janice Chen, PhD.

Since 2015, the two institutions—Berkeley and the Broad—have been involved in a bitter dispute over who made key discoveries that allowed CRISPR-Cas9 to be used in eukaryotic cells. That fight was recently rekindled and will likely continue in the courts. Leveraging CRISPR-Cas9, Doudna and Zhang also helped create companies in pursuit of CRISPR-based therapeutics. Now, the competition to commercialize CRISPR is heating up in the diagnostics space.

Similar to CRISPR gene editing, Mammoth and Sherlock’s CRISPR diagnostics platforms work by combining a guide RNA with a Cas enzyme. When CRISPR is used for cutting or editing, that enzyme is typically Cas9. Though Cas9 was the first protein to be optimized for CRISPR and became the most well known in the Cas family, Doudna and Zhang’s labs identified subsequent Cas enzymes in other bacterial species with slightly different properties—including Cas12a, Cas13, and Cas14.

Collateral activity

In 2015, Zhang and colleagues from the Broad Institute, the NIH, and Wageningen University in the Netherlands identified Cas12a as a new CRISPR system for recognizing and editing double-stranded DNA. Zhang and his colleagues then characterized Cas13 in 2016. A few months later, in the journal Nature, Doudna and her team described the use of Cas13 collateral cleavage activity for RNA detection.

“We realized that Cas13 has a somewhat strange property in that it cleaves RNAs that are recognized by the guide RNA, but it can also cleave other RNA molecules at the same time. That’s what we call collateral activity,” Zhang tells GEN in an interview.

Once Cas13 recognizes and cuts its intended target, it continues to cut other RNAs nearby. “Rather than recognizing one molecule and cleaving that molecule and stopping there, Cas13 recognizes one molecule and cleaves many, many more molecules. That cleavage is the amplification,” Zhang says. Cas12a also performs this collateral cleavage when it binds to a target, but it cuts DNA rather than RNA.

This CRISPR system is then attached to a reporter molecule. When Cas12a or Cas13 hits its target, the enzyme breaks apart the reporter molecule and releases a color. “With CRISPR, you have this enzyme that’s basically spell-checking the base pairing,” says Trevor Martin, PhD, co-founder and chief executive officer of Mammoth Biosciences. “You can get this really exquisite specificity and also high sensitivity from the collateral cleavage.”

More recently, Doudna’s lab identified another enzyme—Cas14—that can bind to single-stranded DNA. It’s just one-third of the size of Cas9, making it the smallest CRISPR system found to date. Last March, Mammoth Biosciences licensed the new tool from UC Berkeley.

Infectious disease detection

Unlike most molecular diagnostic tests, Mammoth and Sherlock are developing paper-based diagnostics without the need for PCR or next-generation sequencing. Instead, these CRISPR-based tests would be portable, as well as cheap to produce and buy. The paper strip is dipped into a patient sample—blood, saliva, or urine—and a line appears to indicate whether the target genetic sequence was detected or not. The tests could be performed in virtually any setting by anyone. For these reasons, both companies see huge potential to use these diagnostics for infectious disease testing.

CRISPR-based diagnostic application

CRISPR-based diagnostic applications are being developed that use paper strips or cards that provide visual cues, such as color changes, when pathogens or mutations are detected. [Mammoth Biosciences]

Martin says Mammoth plans to pair its diagnostic system, dubbed DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, with a smartphone application. A patient could take a test at home, upload a picture of the testing strip once it changes color, and receive a result from the app within 30 minutes. Ideally, the app would also be able to link a person to telemedicine services to schedule an appointment with a doctor or get a prescription once the app renders a result.

“One of the first promises of CRISPR diagnostics is allowing you to have a test that has molecular-style results in a rapid-style format,” Martin says.

Martin sees opportunity for CRISPR-based tests to improve accessibility to diagnostics and drive down testing costs by eliminating the need for centralized labs. In the United States, that means patients could get tested at their local pharmacies for diseases like flu or strep throat or even test themselves at home.

CRISPR diagnostics could also be programmed to detect pathogens like Ebola, Zika, or Escherichia coli in the field, or as a point-of-care diagnostic. Using these simple tests, scientists could monitor viral and bacterial disease outbreaks, as well as antibiotic resistance, in resource-poor areas. The quick turnaround time for results would be especially useful in an emergency during a disease outbreak when patients need to start receiving treatment immediately. Current molecular diagnostics and culture methods take hours or days to return results.

“The technology really is a platform. The exciting part for us is the broad applicability,” says Rahul Dhanda, co-founder, president, and chief executive officer of Sherlock Biosciences. The company is also developing a synthetic biology diagnostic platform, called Internal Splint-Pairing Expression Cassette Translation Reaction, or INSPECTR, that would be stable at room temperature.

At the Chan Zuckerberg BioHub—a collaborative effort by Berkeley, Stanford, and the University of California, San Francisco—researchers have created a CRISPR-based diagnostic tool that can rapidly identify common drug-resistant microbes. Called FLASH select Finding Low Abundance Sequences by Hybridization union the tool uses CRISPR-Cas9 enzyme to search through a patient’s metagenomic sample and cuts its target DNA on either side, separating the drug-resistant sequences from the rest of the microbial genome.

Emily Crawford, PhD, a scientist at the CZ Biohub Infectious Disease Initiative and adjunct assistant professor of microbiology and immunology at UCSF, said the test can be used to identify drug-resistant microbes in 24 hours. Standard culture-based methods take 48 to 72 hours or longer for slower-growing microbes. While metagenomic sequencing of microbial nucleic acid sequences is now being done in research settings, it’s not yet widely available.

The benefit of FLASH, Crawford says, is that it can be multiplexed to detect and reveal thousands of antimicrobial-resistant genes at once. In that sense, she said FLASH is highly complementary to the DETECTR and SHERLOCK system. Her group is currently collaborating with the Doudna lab to use FLASH and DETECTR in tandem for tuberculosis detection.

Cancer diagnostics and beyond

Both companies also want to use their platforms in oncology, though neither indicated what specific mutations they are targeting. The tests could potentially look for multiple mutations at once—and be faster and cheaper than tumor sequencing. Crawford says she also hopes FLASH will be used to find mutations in cancer.

“Imagine if you had to download all the information on the Internet and search it every time you wanted to find something online. That’s essentially what whole-genome sequencing is,” says Michael Heltzen, chief executive officer of Cardea Bio. In partnership with researchers at Berkeley and the Keck Graduate Institute, led by Kiana Aran, PhD, Cardea has built a graphene-based CRISPR detector, the CRISPR-Chip, the first transistor to search the genome for mutations select see Sidebar, “Genome Sensor Launched to read chapter 4.”). The results were published in March 2019 in Nature Biomedical Engineering.

Meanwhile, the Israeli biotech NovellusDx is developing a functional annotation for cancer treatment select FACT) assay that uses CRISPR technology licensed from the Christiana Care Health System in Delaware. The test is not meant to replace tumor sequencing, but rather supplement it. Using Cas12a, scientists were able to reproduce the genetic features of an individual patient’s tumor in a human DNA sample.

“What we developed was a way to take that DNA as if it were a blank canvas and recreate the mutagenic profile of the patient using CRISPR,” says Eric B. Kmiec, PhD, director of the Gene Editing Institute at Christiana Care in Delaware. That information is then put into computer algorithms that identify which signaling pathways are being activated or deactivated in a person’s tumor. The assay can also screen through cancer drugs and drug combinations to predict clinical results based on a patient’s results.

Kmiec thinks CRISPR diagnostics are more likely to come to market before CRISPR-based therapeutics. “I’m more optimistic right now about CRISPR influencing patient health through the diagnostic portal,” he says. “Of course, we and others want to develop gene editing as therapy. But the hurdles to get to patients are large and, in some cases, not known.”

Before that happens, companies will need to validate their CRISPR tests in randomized control trials against traditional diagnostics. Neither company provided details on when they plan to do that. Where CRISPR-based diagnostics could come to the market first is in a different industry altogether. Both companies plan to develop agriculture and manufacturing diagnostics, to test for contamination in food and water. The Zhang lab recently published an article in The CRISPR Journal that described the ability of SHERLOCK to detect plant genes.

Sherlock Biosciences’ Dhanda offers an apt summary: “Our goal is to make sure that this technology is used as broadly as possible in as many settings as possible.”

CRISPR Journal cover

Emily Mullin is a former freelance journalist and currently a staff writer with Medium.

This article was adapted from the July/August 2019 issue of Clinical OMICs.

Genome Sensor Launched

Cardea Bio, based in San Diego, is another startup gaining momentum in the CRISPR diagnostics game. The company, which just merged with Nanosens Innovations, has launched a high-tech testing tool called the Genome Sensor select formerly the CRISPR-Chip). This handheld device combines thousands of Cas9 molecules with electronic transistors made of graphene also manufactured by Cardea Bio.

Cardea's Genome Sensor
The alpha version of the Nanosens Genome Sensor, a handheld DNA search engine. [Cardea Bio]

The Cas9 proteins are deactivated so that they can bind to certain DNA sequences but not cut them. The binding creates an electrical charge on the surface of the graphene, which can be picked up by a digital biosensor in the device. The tool allows for detection of a specific genetic mutation from a patient’s DNA sample, without the need for amplification or sequencing, in about 15 minutes.

In a March 2019 paper in Nature Biomedical Engineering, Kiana Aran, PhD, of the Keck Graduate Institute, and her team tested the sensitivity of their CRISPR-Chip by using it to detect two common genetic mutations in blood samples from Duchenne muscular dystrophy select DMD) patients. The team is also testing it for sickle-cell disease, which is more difficult to detect, and hopes to increase the sensitivity so it can be used to identify infectious diseases as well.

Rapid genetic testing could allow doctors to start patients on treatment sooner than they can currently. It could also quickly identify genetic variations that make some people unresponsive to certain drugs—like the blood thinner Plavix—to help doctors personalize treatment plans. A nurse or physician can take a blood sample and process it with the Genome Sensor without the need for specially trained lab technicians.

Aran said the Genome Sensor can also be programmed to look for a healthy gene or region of a gene. If the Genome Sensor doesn’t find its target, that would indicate a negative result, or a mutation in the gene.

Launched under the Nanonsens brand, the Genome Sensor still needs to prove itself as a diagnostic. In the meantime, Aran thinks the device will be useful for biotech and pharma companies pursuing CRISPR-based therapeutics. The Genome Sensor can be used to test and monitor gene editing efficiency and help speed CRISPR therapies to the clinic. “We’re trying to make a quality control tool to help companies design better CRISPR complexes and make sure they actually do what they’re supposed to do,” she said.