A Guiding Light for CRISPR

CRISPR has captured imaginations and investor interest, with a growing number of companies now developing therapies based on genome editing. We’ve also just seen the world’s first approvals for a CRISPR/Cas9 edited medicine (Casgevy; approved by the UK’s MHRA in November and by the US FDA in December 2023). Caribou Biosciences has been working in the area for over a decade and has attracted considerable attention because of its CRISPR chRDNA technology, which can improve the precision of genome edits and reduce off-target events. The company was founded by Rachel Haurwitz (CEO of Caribou) and Jennifer Doudna (joint winner of the 2020 Nobel Prize in Chemistry for her work in gene editing with Emmanuelle Charpentier).

Here, one of Caribou’s earliest hires, Paul Donohoue (now Associate Director of Platform Discovery), gives us insight into the early days of the company and how the technology was developed. 

How did your experience at the University of California Davis influence the early part of your career?
 

I grew up in Davis. When I was in high school, one of my science teachers had contacts with a lot of labs at the university and had convinced them to take on undergrad interns. Through this program, I ended up at UC Davis in the lab of Dave Wilson, who was a structural biologist. Dave paired me up with a postdoc student, Eric di Luccio. Eric taught me the fundamentals of science, from how to pipette to molecular biology, cloning, protein expression, protein purification, and some early nuggets around X-ray crystallography and structural biology. 

It was really challenging but I was really into the work. It was satisfying to work on super hard problems, such as trying to get E. coli to express a human protein and then purify it to the level that it could be used in solving the protein structure. From these challenges, I learned to appreciate simple things, such as seeing a single clean protein band on an SDS-PAGE gel at the end of a purification process.

I applied to attend college at Davis, and Dave also offered me part-time paid work in the lab. It was basic stuff, such as washing dishes, preparing media, and buffers, but I was also able to continue with protein research with Dave. I ended up working in the lab for the next two years of my undergraduate degree, learning more about protein biochemistry and structural biology. As that wrapped up, another principal investigator, Irwin Segel, who had heard about me from Dave, offered me work in his lab.

Irwin was another formative mentor for me. He had been involved in science for decades and had written one of the earliest books describing enzyme kinetics back when it was a nascent discipline. He was not one to suffer people who weren’t driven or scientifically curious. He really imparted a lot of those values on to me, and he also imparted to me an understanding and appreciation of enzyme kinetics, which complemented the structural biology insight and protein chemistry I had learned from Dave’s lab.

I was very fortunate to have these opportunities and valuable mentors who were invested in me so early on. 

Did you join the pharma industry straight out of university?
 

No – I went into the wine industry! I was really interested in the applied side of science – and at the age of 21 I was developing a burgeoning interest in wine. I ended up in a science job for the Kendall-Jackson Winery. It was a really informative experience, but it wasn’t the type of scientific environment that inspired me. Having that exposure redirected me back to an early research focus and I then went into biofuels, which was fascinating – until the company shut down.

While job hunting, a recruiter reached out about a biotech position researching CRISPR in relation to an opening at Caribou Biosciences. I started reading CRISPR papers from Caribou’s CEO, Rachel Haurwitz, who had worked in the lab of Jennifer Doudna – Nobel Prize winner in Chemistry in 2020 for her work on CRISPR. Many of Rachel’s papers were structural biology based; she was solving the protein structures of Cas proteins, and then coupling this knowledge with fundamental enzyme kinetic characterization of the Cas protein. It reminded me of my work with Dave and Irwin, and I decided this was the environment I wanted to be in. 

At the time, Caribou was in an incubator space with just three employees; Rachel, Andy May as the Chief Scientific Officer, and an undergrad intern, who was a computational biologist. Andy was also a structural biologist and, during the job interview, we spent a lot of time geeking out about x-ray crystallography and protein chemistry, and how CRISPR systems worked and could theoretically be applied. There was a lot of energy and excitement – and I was hired for the role. This was back in 2013 and I’m still with the company today.

What is the story behind chRDNA?
 

In the early days of Caribou, we focused on understanding the basic functional properties of CRISPR-Cas systems and how we could better control their gene editing function. We were particularly interested in understanding the interaction between the Cas9 protein and its guide RNA. Guide RNAs are really interesting molecules because they have lots of secondary structures. We wanted to figure out what elements of the secondary structure were important for driving Cas9- targeting of DNA. 

We performed a lot of structural mutations in the guide RNA, including truncating the secondary structures, making them bigger, making sequence changes, and even outright deleting some of the secondary structural elements to see how it impacted the Cas9’s ability to target DNA. In time, we understood what parts of the guide were the most important in allowing the Cas9-guide RNA complex to carry out its function.

Something that was interesting to me as I looked at the way the Cas9 protein interacted with the guide RNA was that there wasn’t much direct readout of the 2' hydroxyl group on the sugar backbone of the guide RNA backbone by the Cas9 protein side chains. So I started to wonder whether the guide RNA had to be all RNA? Could we go in and replace some of these RNA bases with DNA? 

Andy and I decided to try it. We put together some initial designs of hybrid guides that had DNA and RNA, and we ran biochemical cleavage assays against target DNA. I ran the first one with a large collection of these hybrid guides. When I got the data back, I sent a cheeky email and a summary to Andy for him to review – because I thought the results looked great! That night, Andy responded with excited expletives. Suffice to say, he thought it looked great too!

A lot of our initial designs had comparable activity to the normal all-RNA guide. It was a eureka moment. We called these hybrid molecules CRISPR hybrid RNA-DNA – chRDNA for short – pronounced “Chardonnay” (remember I had previously worked in the wine industry!).

As we worked more with these hybrid guides, we also stumbled across some unique properties they had over the all-RNA system. With CRISPR systems, you program the guide RNA to direct Cas9 to a DNA target sequence, but there are some liabilities. The Cas9 protein can bind to and cleave at target DNA sequences that look similar to the intended target site – in other words, off-target sites. Because of this, using CRISPR systems to edit a human cell can pose a risk. You don’t always know what off-target sites might be hit and how this will impact cellular function.

To maximize CRISPR genome editing impact, and ensure its safe use, we wanted to find ways to mitigate off-target effects – and this is where chRDNAs began to truly shine. Through a combination of both DNA and RNA bases, chRDNAs have a very discriminant activity against off-targets. Depending on where we put the DNA bases within chRDNA, we could tune the specificity of the system. In other words, we could build bespoke chRDNAs for each target site.

How is Caribou using the chRDNA technology now?
 

The complexity of the projects has changed over time, from research of CRISPR tools to development of allogeneic CAR-based cell therapies. For our first clinical program, CB-010, for treatment of B cell non-Hodgkin lymphoma, there are three edits (two gene knockouts and one gene knock-in). In our second program, CB-011, for treatment of multiple myeloma, we make four edits. Our newest program, CB-012, for treatment of acute myeloid leukaemia (AML), involves five edits. As we make more edits, we need a system to help accomplish that with maximum efficiency and safety, such as the chRDNAs.

One of the first patients in our non-Hodgkin lymphoma trial had eight prior lines of treatment before being put on our clinical trial. With a single dose of CB-010, our off-the-shelf CAR-T cell therapy, he has been cancer free for two years. It’s incredible to see the impact that these therapies can have for patients. Out of 16 patients we treated in the dose escalation portion of our CB-010 ANTLER phase 1 trial, 44 percent are cancer free out to six months and beyond. It’s very humbling to see how our science has directly impacted patients. I joined Caribou because I thought CRISPR proteins were interesting and today it has evolved far from what I imagined. 

How has your role at Caribou developed over the years?
 

Today, I'm an associate director and I lead a small team of highly motivated, very bright researchers. My role is about passing the baton on and relying on my team to come up with new ideas that will drive further innovation in CRISPR systems and how we use them.

In addition to learning to be a manager and a leader, I’ve had to develop a broader understanding of the biotech space and how a biotech company is run. Sometimes I have to get involved with our business development team and speak to potential partners about our technology, and I’ve also had opportunities to get involved with our clinical team, talking with clinicians or nurse practitioners about cell therapies. I’ve also had to interact with our legal team about IP and patents. It has all been very interesting.

Running a team now has also made me reflect on my time at Davis in terms of my early mentors and how I can mentor others. It’s important to find the best way to motivate people and to maximize their scientific creativity.

What keeps you excited about the future of CRISPR?
 

It’s been really shocking and surprising to see the rapid implementation of CRISPR across all areas of science. Everything has moved very fast; we’re now using CRISPR for cell therapies and for genome editing plants and eukaryotes. It’s almost ubiquitous and is already having medical impact with the first approval of a CRISPR-based therapy in the UK coming after just over a decade after the seminal work on CRISPR-Cas9 genome editing by Jennifer Doudna and Emmanuelle Charpentier and their colleagues. It’s amazing to take a step back and appreciate how much has changed now that we can make genetic manipulations.

I will never forget when we interviewed a scientist out of his postdoc. He gave a presentation about his work where he had been trying to tag a mouse neuronal protein with a fluorescent reporter. He spent almost a year of his postdoc trying different approaches, but he finished his presentation by saying that the work could have probably been done in two months now that CRISPR-Cas9 was a tool at researchers’ disposal. It really encapsulates how much CRISPR has changed things for the scientific community.

And yet it’s still early days. There are many new ways that we can push what we’re able to do with CRISPR systems and how our understanding and deployment of them evolves.

What are the big challenges facing the field?
 

One of the biggest challenges for any CRISPR or genome editing company is the speed of advancement. Every week – almost every day – a new paper is released showing how CRISPR is being applied in novel ways. It’s a great reminder about innovation and the push to try something new. We scientists often sit down and think about all of the ways that something won’t work (and why), as opposed to just getting into the lab and trying it out.

In retrospect when I look back on the early work I was doing on chRDNAs, if I’d have been further in my career and had known more about CRISPR systems, I could have come up with a hundred reasons as to why using DNA and RNA in CRISPR guide sequences would not be tolerated. But we went into the lab and we tried things – and it led to a huge breakthrough for Caribou. And that’s something I try to press upon my team. To truly move the needle, you need to come up with a hypothesis, throw some effort behind it, and test it in the lab.

What are your hopes for the future of the field?
 

I hope that the innovation around CRISPR systems continues and that this isn’t where we plateau from a technology perspective. I hope we continue to realize how CRISPR systems can continue to have a broad impact and I hope we continue to find new ways to re-engineer them to push the paradigm of what is possible with cell therapies – and in other areas where there are unmet needs.

I don’t see things slowing down any time soon, but whatever lies ahead we must keep in mind the ethical implications. We cannot shy away from the tough conversations in the scientific community; we need to continually ask ourselves that big question:
“Just because we can do something, should we do it?”