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 CRISPR 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, Ph.D., at the University of California, Berkeley. Following close behind, Sherlock Biosciences launched in March this year with an initial $35 million in funding and technology developed by Feng Zhang, Ph.D., 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 (SHERLOCK). Both CRISPR inventors are co-founders of the respective companies.
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—and that fight continues 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.
In 2015, Zhang and co-authors from the Broad, 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 June 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 RNA 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 told Clinical OMICs in an interview.
Once Cas13 recognizes and cuts its intended target, it releases a burst of energy and continues to cut other RNA 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 said. 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 gives off a color. “With CRISPR, you have this enzyme that’s basically spell-checking the base pairing,” said Trevor Martin, Ph.D., co-founder and CEO 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 the size of Cas9, making it the smallest CRISPR system found to date. In 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 of blood, spit, 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.
Martin says Mammoth plans to pair its diagnostic system, dubbed DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, with a smart phone application. He said a patient would be able to 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,” he said.
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 U.S., that means patients could get tested at their local pharmacies for things like flu or strep throat or even test themselves at home without going into a doctor’s office.
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,” said Rahul Dhanda, co-founder, president, and CEO of Sherlock Biosciences, which 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 (Finding Low Abundance Sequences by Hybridization), 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, Ph.D., 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 said, 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 said 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,” said Michael Heltzen, CEO of Cardea Bio, which has partnered with researchers at Berkeley and the Keck Graduate Institute to build a graphene-based CRISPR detector. “That’s essentially what whole-genome sequencing is.”
Meanwhile, Israeli biotech NovellusDx is developing a functional annotation for cancer treatment (FACT) assay that uses CRISPR technology licensed from the Christiana Care Health System in New Jersey. 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,” said Eric Kmiec, director of the Gene Editing Institute at Christiana Care. 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 said. “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.
“Our goal is to make sure this is used as broadly as possible in as many settings as possible,” Sherlock Biosciences’ Dhanda said.
NanoSens Innovations, based in San Diego, is another startup getting in the CRISPR diagnostics game. Co-founded by Kiana Aran, Ph.D., of the Keck Graduate Institute, the company has built a slightly more high-tech testing tool called CRISPR-Chip. The hand-held device combines thousands of Cas9 molecules with electronic transistors made of graphene manufactured by Cardea Bio, also headquartered in San Diego.
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 CRISPR-Chip. 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 recent paper in Nature Biomedical Engineering, Aran 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 (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 CRISPR-Chip without the need for specially trained lab technicians.
Aran said the CRISPR-Chip can also be programmed to look for a healthy gene or region of a gene. If the CRISPR-Chip doesn’t find its target, that would indicate a negative result, or a mutation in the gene.
In an interview, Aran said the CRISPR-Chip is “very close to a commercial system” but still needs to go through rigorous testing before it can be used as a health diagnostic. In the meantime, she thinks the tool will be useful for biotech and pharma companies pursuing CRISPR-based therapeutics. She said the CRISPR-Chip 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,” Aran said.