The Evolution of CRISPR Technology from Editing to Diagnostics

This year marks the 10-year anniversary of when CRISPR-Cas9 gene editing was first revealed to the world. Much has happened since then, including wide rollout in many sectors, development of CRISPR-based therapeutics and the discovery that the technology can also be used to develop highly accurate and affordable diagnostics.

The discovery that using CRISPR plus a guide enzyme known as Cas9 could allow targeted gene editing was published in a paper in Science in June 2012 authored by University of California, Berkeley, biochemist Jennifer Doudna and microbiologist Emmanuelle Charpentier, then based at Umeå University, Sweden, along with colleagues.

At a similar time, a molecular biologist at the Broad Institute, Feng Zhang, and his team were also working on developing CRISPR-Cas9 gene editing technology. While the Berkeley team filed for a patent for the technology a few months before Zhang and colleagues, a long-running argument and patent dispute continues between the two groups about who invented the technology first.

Clustered regularly-interspaced short palindromic repeats (CRISPR) are a family of DNA sequences found in bacteria and archaea. Derived from fragments of bacteria-eating viruses called phages, the previously infected microbes use these sequences to detect and destroy DNA from new phage virus attacks. With the help of suitable guide enzymes, such as Cas9, this bacterial ability can be harnessed to carry out precise genetic edits in many different genomes.

Since the initial discovery, use of CRISPR gene editing technology has exploded around the world for many different uses both in and out of the clinic, in academia, and in industry. Doudna and Charpentier, as well as Lithuanian CRISPR pioneer Virginijus Siksnys, were recognized for their discovery and awarded the Kavli Prize in Nanoscience in 2018, followed by the Nobel Prize in Chemistry in 2020  for Doudna and Charpentier.

As well as ever expanding uses, including genome editing, gene therapy, epigenetic modulation, and many others, the technology itself is also evolving. No longer limited to just the Cas9 enzyme as a guide, many others have been developed and are now being used for different purposes. Discovered in 2015 and 2016, respectively, Cas12 binds strongly to a DNA target, remains bound and then non-discriminately cleaves single stranded DNA in the vicinity, whereas Cas13 binds an RNA target before similarly cleaving nearby single stranded RNA.

“When some of these enzymes were first discovered, this collateral cleavage activity was a bit of a concern. But it turns out, you can harness that for diagnostic purposes,” Princeton University associate professor and group leader Cameron Myhrvold, who has experience working in this area, told Inside Precision Medicine.

The discovery of these new Cas enzymes really catalyzed researchers to investigate how CRISPR technology could be used in diagnostics and over the last few years several biotech companies have been set up to explore this potential. COVID-19 has further spurred on the use of CRISPR in diagnostics. However, it remains to be seen whether and in what form the current consumer, investor and industry interest will continue once the pandemic is officially over.

Exploiting the Power of New Cas Enzymes

One of the first companies to explore the potential of CRISPR technology for diagnostic applications was Mammoth Biosciences. Founded in 2017, by Doudna and two of her graduating PhD students Janice Chen and Lucas Harrington, as well as Trevor Martin from Stanford University, the company was set up, at least initially, to explore how Cas12 and Cas13 could be used in diagnostics. “A lot of the work that was foundational to starting Mammoth was done in the lab of my professor, Jennifer Doudna, at UC Berkeley, together with one of my co-founders, Lucas Harrington, and our colleagues there,” explained, current CTO, Chen in an interview. “One of the really unexpected findings was that you could use CRISPR, not just for gene editing, but also for gene detection. That was a new opportunity in the field and opened up this opportunity for molecular detection using CRISPR.”

The company has developed a platform called DNA endonuclease-targeted CRISPR trans reporter (DETECTR) using Cas12 (previously known as Cpf1). The CRISPR nucleases are programmed to find a specific sequence in the DNA, using a guide RNA. When the sequence is found, Cas12 also cuts a single stranded fluorescent reporter sequence in the reaction, which signals that the pathogen of interest has been found. The system was tested on two similar types of human papilloma virus (HPV), HPV16 and HPV18, and was able to distinguish between them within an hour. While the company has diversified since 2017 and is now also developing therapeutics, use of CRISPR for diagnostic purposes is still one of its key areas.

“What we know is that we can do much higher multiplexing than what we see in currently available commercial products,” Mammoth VP of Product Management in Diagnostics Adriana Dantas Lemberg told Inside Precision Medicine.

“CRISPR is cleaner, more elegant and less prone to interference than the sample preparation that is required on any PCR reaction. So, I think that’s the greatest advantage that we’re going to see.”

The second big player in the field of CRISPR diagnostics is Sherlock Biosciences. The company was set up in 2019 to commercialize earlier findings from Feng Zhang and colleagues at the Broad Institute, showing the potential of CRISPR-Cas13 (previously known as C2c2) for detection of RNA sequences. Jim Collins is a professor at MIT and a core founding faculty member of the Wyss Institute at Harvard University. He is also a co-founder of Sherlock, along with Zhang, David Walt, another Harvard professor, and a number of other researchers.

“I think it goes back to the spring of 2016. My team had developed a paper-based diagnostic for Zika… we showed you could use CRISPR-Cas9 to differentiate between different strains,” explained Collins. “When that paper came out, Feng Zhang, my colleague, here at MIT and the Broad, reached out and shared that his team was working on a different CRISPR enzyme, Cas13, and asked if I would be interested in collaborating with his group on potentially using this enzyme to create a new diagnostic platform.”

This marked the start of Sherlock, named after its platform – Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK). Similar to DETECTR, the platform works by targeting RNA sequences of interest and cleaving and activating a fluorescent reporter sequence to indicate a positive result.

Unlike Mammoth the company has remained focused on diagnostics, but is now trying to make its products even easier and more cost effective to use by increasing its focus on point of care, as opposed to laboratory-based testing, and has a new fast test in development. “It works isothermally at ambient temperatures and therefore requires no heat, no electricity whatsoever. That technology can detect from sample to answer in just under an hour,” explained new CEO, Bryan Dechairo, who moved to the company from his position as Executive Vice President of Clinical Development at Myriad Genetics last year.

Sherlock had started work on diagnostics for sexually transmitted infections before the pandemic started, specifically in the areas of chlamydia and gonorrhea. “We have now continued those programs and are moving them forward, especially now that our chemistries really meet the market demand for speed, accuracy and costs,” says Dechairo.

Combining Transistors With CRISPR

Making use of new Cas enzymes is not the only way to create a CRISPR-based diagnostic tool. Kiana Aran, an associate professor and group leader at the Keck Graduate Institute in California, is also CSO at Cardea Bio. Previously named Nanomedical Diagnostics, the company was set up in 2013 to develop electrical biosensors using graphene.

After joining the Keck Institute in 2017, Aran also began work with what is now Cardea Bio to produce the company’s Biosignal Processing Unit (BPU). This is a next generation electrical transistor that uses graphene instead of silicon and can pick up a variety of biological signals. “Graphene is much more biology friendly,” explains Aran, “biology doesn’t impact it and its super sensitive.”

The BPU can be adapted to host a number of different detector molecules including CRISPR. As described in a 2019 Nature Biomedical Engineering publication, Aran and colleagues added immobilized and catalytically deactivated CRISPR-Cas9 on the chip allowing targeting of specific genetic sequences.

The first proof of concept for the technology involved testing DNA samples with two distinct deletion mutations from individuals with Duchenne muscular dystrophy. Within 15 minutes the chip identified samples with the correct target sequence, without the need for a DNA amplification step.

However, the Cas9 enzyme they used initially was not able to pick up smaller mutations. “In 2021, we worked with Dr Virginijus Siksnys who was one of the pioneers in CRISPR… He’s a close collaborator of mine,” said Aran. Siksnys introduced Aran to a different Cas9 variant that was more sensitive to single point mutations and was able to pick up those causative for sickle cell disease and amyotrophic lateral sclerosis (ALS).

“They have a library of different Cas enzymes, and each of them have a different functionality. Some can operate at higher temperature, some are more sensitive to single point mutations, and a whole wide range of other characteristics. He helped us select an enzyme that was best for our application.”

These are just some possible uses for the technology, explains Aran, who adds that she and her colleagues hope it will be useful in fields such as cancer. “For liquid biopsy, if you’re detecting free circulating DNA or reciprocating RNA, you’re not just interested in detecting ‘yes’ or ‘no’, you’re interested in testing how much of it you have so you can see if therapy has been effective.”

The Cardea technology also has other uses linked with CRISPR, as it can test how effective different parts of the reaction are with different complexes and enzyme combinations. Just last month, Cardea launched a spin off company called CRISPR-QC, which plans to focus on helping researchers and companies using CRISPR technology, for example, those developing medical gene editing therapies, to improve and monitor the quality of their experiments.

The COVID Effect

Since 2020 the world has changed. We have all gone through a global pandemic and the importance of vaccines, therapeutics and effective diagnostics has never been clearer. Diagnostics historically has been a difficult business to master. “We know that diagnostics drive 70% of the decision making in clinical settings. But only 2% of the total budget and healthcare systems are spent in diagnostics,” explained Dantas Lemberg.

However, since the beginning of the pandemic, different types of diagnostic tests such as PCR, RT-LAMP (loop-mediated isothermal amplification) and antigen tests have become a common discussion topic over the dinner table. Like many companies in the life science space, companies with a focus on diagnostics or related areas were quick to begin developing tests for SARS-CoV-2. Sherlock achieved the first ever FDA approval, albeit an Emergency Use Authorization (EUA) rather than a standard approval, for CRISPR in a medical setting with its rapid CRISPR SARS-CoV-2 test kit in May 2020. Earlier this year, Mammoth also achieved an EUA for its high-throughput SARS-CoV-2 test that combines CRISPR technology with laboratory automation for diagnostic testing.

Many new startups also decided to try their luck at developing COVID vaccines, drugs and diagnostics. One such company is Proof Diagnostics. In early 2020, Feng Zhang, Jonathan Gootenberg and Omar Abudayyeh, all of whom are also Sherlock founders, decided to launch a new company to commercialize and build a new type of point of care, easy to use CRISPR diagnostic based on their SHERLOCK testing in one pot (STOP) assay.

“What’s been a silver lining in this really terrible two and a half years is you’ve seen a lot more investment from the public sector and from the private sector go into the space,” says Proof CEO, Sid Shenai. “Five years ago, not a whole lot of investors were very focused on diagnostics.”

Cameron Myhrvold has also been applying his expertise to developing CRISPR-based COVID tests over the last few years, using the knowledge he gained as a PhD student in the lab of virologist and Broad Institute professor Pardis Sabeti (also a Sherlock co-founder). Myhrvold has worked with Cas13 since becoming a postdoc and now has his own lab at Princeton with a focus on using CRISPR to study viral and host RNA. Before starting at Princeton, Myhrvold worked with Sabeti and team to help develop a technique to test for multiple pathogens at the same time called Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN). By combining CARMEN with Cas13, Myhrvold and colleagues were able to test more than 4500 targets on a single array.

During the pandemic, Myhrvold and his team adapted the earlier CARMEN work to help test for different strains of SARS-CoV-2. Microfluidic (m)CARMEN, combines CRISPR-based diagnostics and microfluidics and can test for up to 21 viruses including variants of SARS-CoV-2. “The work has been translated a lot faster than I could ever have imagined,” says Myhrvold.

“Omicron has a unique combination of mutations that differentiated it from Delta and from all of the previous variants. When the Omicron variant started spreading across Massachusetts, our assay, even though we didn’t design it for Omicron, was ready to go. We were able to use it, both in some colleges in the Boston area, as well as in partnership with the Department of Public Health in Massachusetts, in order to track the spread over time.”

Predicting the Future

Molecular diagnostics was and still is dominated by old and well-validated techniques such as PCR (polymerase chain reaction), or reverse transcriptase (RT)-PCR. However, CRISPR technology does have advantages over older techniques. For example, it is highly specific and can be carried out without the time-consuming thermal amplification steps and expensive machinery PCR requires.

Due to the pandemic a lot of time and investment has been poured into developing new diagnostic technology, including tests powered by CRISPR. However, whether the COVID ‘silver lining’ will last remains to be seen.

“In a diagnostics company, you need to sell product and your value is a direct multiple of the revenue that you generate, whereas in a therapeutics company, you can sell hope for decades,” says Collins. “Without getting to a product, you can’t do that in a diagnostic space.”

While the increased accuracy offered by CRISPR diagnostics is, at least on paper, very attractive, the widespread use of lateralflow antigen tests during the pandemic has taught us that convenience and minimal cost can win over increased accuracy. Even competing in the more accurate ‘gold standard’ testing space occupied by PCR and RT-PCR tests could prove difficult, as these tests are so well established.

Eric Rhodes is the CEO of ERS Genomics, a company formed by Charpentier to license patents owned by the University of California, University of Vienna, and Emmanuelle Charpentier (CVC). “I think there will be applications where you want to use something like CRISPR, that it might have an advantage over the amplification required for PCR technology. But I don’t see it supplanting PCR technology and sequencing technologies, for
a variety of reasons,” says Rhodes.

Myhrvold is hopeful for the progress of CRISPR diagnostics, but acknowledges that a number of challenges need to be overcome before more widespread rollout can become a reality. “One of those is sample processing… it is challenging to make sure that you have something of good quality going into the test itself. A lot can go wrong at the very beginning, which then makes it impossible to get a good result no matter how good your technology is,” he explains.

“The other big one, I think, is manufacturing, which is a challenge for a lot of these new technologies. You need that mass production to drive down the cost. You can use something at a smaller scale, but it’s not really going to matter if the cost is now prohibitively expensive.”

It’s important to remember that it is early stages for CRISPR-based diagnostics, which have only been around for 5-6 years and are still in the early stages of development. “There is first generation CRISPR diagnostics, there’s been a second generation… and there’s this next generation of gene editing diagnostics that we’re trying to be a leader in,” says Shenai.

Dantas Lemburg is also hopeful for the future. “I really think it’s time for a change. I think the world today is more much more prone to accepting new technology. And I think CRISPR has demonstrated its capabilities, what it can do. I really believe we’re creating a next generation of molecular testing.”