NEB Podcast #66 -
Transcript
Interviewers: Lydia Morrison, Marketing Communications Manager & Podcast Host, New England Biolabs, Inc.
Interviewee: Ben Kleinstiver, Ph.D., Assistant Professor of Pathology, Harvard Medical School
Lydia Morrison:
Welcome to the Lessons from Lab & Life Podcast, brought to you by New England Biolabs. I'm your host, Lydia Morrison, and I hope this episode offers you some new perspective. Today I'm joined by Dr. Ben Kleinstiver, whose lab is located at the Center for Genomic Medicine at Mass General Hospital. Ben joins us to talk about programmable nucleases, genome editing and the applications of this technology in the future of healthcare. Thanks so much for joining us today, Ben. Welcome to the Lessons from Lab & Life Podcast.
Dr. Ben Kleinstiver:
Thank you. It's a pleasure to join you and I'm happy to chat more about our work.
Lydia Morrison:
Yeah. Speaking of your work, could you share with our listeners what your laboratory focuses on?
Dr. Ben Kleinstiver:
Sure. Happy to. So I'll briefly introduce myself, then talk about the lab. So and this I think motivates a lot of what we do, but I'm a biochemist by training, and I've always been interested in various forms of engineering. I started my bachelor's degree in biology like many people and also in architecture. And I learned very quickly that I had to pick one. So I picked biology. But I think the themes of molecular design and architecture and all that has really carried through my career in the sense that we design tiny little proteins now and don't work on buildings, but there's still a lot of threads to pull on in terms of how we build things.
So now I have the great fortune of running a research lab at Mass Gen Hospital in the Center for Genomic Medicine, and we're also affiliated with Harvard Medical School. I run a research lab that's comprised of these really talented and motivated grad students and postdocs and technicians and junior faculty members, and just, again, very fortunate to work in such a dynamic and fun environment. The things that we work on, I think, of course I find really interesting, but they're in three sort of main areas, the first of which we define as protein engineering. And this is how we basically develop methods to engineer new proteins or tweak existing proteins.
And a big component of this is developing assays to study proteins so that we can study many, many proteins at a time and learn as we change them, how that changes their properties. A second major area of interest for us is in genome editing technology development. So this is building tools that allow us to change DNA sequences in the genomes of living cells. And this sort of falls into two buckets where we can tweak existing tools that allow us to change DNA sequences or we try to build new enzymes and technologies that allow us to edit genomes in different ways, adding new functions to the gene editing toolbox.
And then a third really major area of interest for us is developing new genetic therapies. And this relies on genome editing tools to change very specific bases in a patient's DNA sequence. This work obviously happens in the lab, but also have the great fortune of working with amazing collaborators at MGH and at the NIH and internationally on these longer term projects to develop new molecular medicines with the hope of eventually curing disease for patients one day. So together, all these three areas really sort of are interrelated in different ways that sort of inform the needs of project one, two, or three, and it's again, just an amazing dynamic environment to work in on all these different exciting areas of science.
Lydia Morrison:
Yeah, it sounds like a diverse area of research, diverse areas of research, but I can see how they're all interconnected. I'm curious, our questions today will focus mainly on the CRISPR gene editing technologies that you work on, but I'm sure that some of the other areas of your research will interplay with that in some of your answers. So my first question is around the SpRY-Cas9 enzyme that your group developed. How did that project get started?
Dr. Ben Kleinstiver:
Yeah. So this is a long story. In many ways, we and others have been looking for this type of solution for a long time. Throughout my entire career, I've worked on methods for molecular cloning, and this is basically trying to change short or long DNA sequences in what's called a plasma DNA. And this is just a circle of DNA that we use in experiments to basically propagate genetic information. And when we want to change that sequence in a plasmid, we have to do what's called molecular cloning. And for how this has been done historically and really innovated by companies like NEB is through these enzymes called restriction enzymes that can cut a DNA plasmid.
But there are some challenges here. Restriction enzymes can only cut a plasmid at a certain spot. So for a long time, we'd been looking for solutions to try and open up plasmids at really specific locations. And this is really important for lots of downstream cloning methods like ligation or isothermal assembly or Golden Gate, all these different famous molecular biology methods where having the ability to open up a plasmid has really at a very specific location makes things a lot simpler.
So I guess the sort of conception of developing SpRY for molecular biology came after we'd built this SpRY protein. Maybe I'll just quickly explain what SpRY is. So CRISPR-Cas enzymes can be programmed with a guide RNA. This is a short RNA molecule, and that tells them where in the DNA sequence to bind and edit or cut. But unfortunately, Cas9 enzymes also have to recognize a short DNA sequence called a PAM. And this is usually two or three bases long. And what this means is that Cas9 enzymes have to bind those specific sequences in a genome.
So when I started my lab, we began working on methods to remove this constraint so that we could design Cas9 proteins that could edit any base in the genome. And this work was led by Russell Walton, who was an amazingly talented technician. He's now doing his PhD in Paul Blaney's lab. And this work was published in Science in 2020, but the upshot was that we built this protein called SpRY that didn't require PAM anymore.
And the outcome of that is that this enzyme can now bind and edit any DNA sequence that we program it to with a guide RNA. So tying that back in time many years, again, I mentioned we've always done a lot of molecular cloning and trying to change DNA sequences in plasmids. Now that we had this Cas9 enzyme that didn't require a PAM anymore, and we could just program it by changing the guide RNA, we wondered if we could repurpose this enzyme as a restriction enzyme, but now being guide RNA programmed instead of needing a big toolbox of restriction enzymes in the lab.
So one night, I remember this very vividly, I was chatting with a German medical student during my postdoc, Nicolas Wyvekens, and we were wondering, how do we get around this problem? And at the time, there weren't great PAM variants of Cas9, but there were tools called Argonaute proteins, and these are DNA programmable enzymes that can be easily reprogrammed just by giving it a different DNA guide instead of an RNA guide. And we tried a few experiments, but we couldn't really get this to work using an Argonaute as a restriction enzyme.
But subsequently, many labs, including Weimann-Zao's lab and others have shown you can use Argonaute as programmable restriction enzymes, but unfortunately, they just still can't target everywhere. Now, fast-forward back a few years now that we have this SpRY protein that can edit anywhere, we wondered could we now just use this as a restriction enzyme? And this work was led by Katie Christie, a postdoc in the lab, and others like Jimmy Groh, Rachel Silverstein and Roman Dahl.
They basically asked this simple question, if we gave SpRY a different guide RNA, could we cut a DNA plasmid in a test tube at any location? And they tested hundreds of different guide RNAs and found that across almost every experiment that we did, we could really effectively open up the DNA plasmid at the guide RNA targeted location. I think it's also really important to note that this project was enabled by scientists at NEB. Meg Mabuchi worked with us to purify and provide the SpRY protein. So this was a long-standing project in many ways, but a highly collaborative project that benefited from lots of different expertise.
Lydia Morrison:
Yeah. And I think that's what it takes to discover such a powerful tool. Could you explain what a SpRYgest is and how that can be used in cloning applications?
Dr. Ben Kleinstiver:
Of course. Yeah. So now that we had this SpRY protein and we found that we could digest DNA at essentially any position in a test tube, a SpRYgest is basically using the SpRY enzyme in a DNA digest. So that's just the concatenation of the words to be a SpRYgest. So a SpRYgest is a lot like a normal restriction enzyme digest where you would basically have your DNA that you want to cut, and then you have your restriction enzyme that cuts that DNA. Instead, now we're using the SpRY protein instead of a restriction enzyme, and we have to give it a guide RNA.
Now we can control where in the plasmid we open up that plasmid because we can program SpRY to cut anywhere. And when we think about how one would clone when using restriction enzymes, how this normally works is you basically design your cloning strategy around where you have unique and convenient restriction sites. So you think about cloning based on what restriction sites are available. But now with SpRYgest, we've sort of changed the paradigm here where instead of focusing on what restriction sites are available, we just focus on what we want to clone because we know with a SpRYgest now we can open up a plasmid anywhere.
Practically how one does a SpRYgest is relatively straightforward too. Now, fortunately, you can buy the SpRY protein from NEB. The second component that you need is just a guide RNA. And this is relatively easy to generate. You design a 50 or 54 nucleotide DNA oligo, that basically changes the one part of the guide RNA. You do a short in vitro transcription reaction. This is just a reaction to generate the RNA molecule itself. You combine the guide RNA with the SpRY protein, and then you just use that to digest the DNA.
So there's maybe one or two additional steps compared to a restriction enzyme digest, but not that much more hands-on time. And I think the real benefit here is that we can cut a plasmid anywhere. And for large DNA plasmids that don't have many unique restriction sites, SpRYgest really do enable science in ways that weren't possible before. So I think SpRYgest give us a lot more flexibility and freedom to clone DNA plasmids in ways that we couldn't before when using restriction enzymes.
Lydia Morrison:
Yeah, absolutely. It's definitely reduced some of the limitations of SpRY-Cas9, but I'm curious, what are some of the limitations that still remain?
Dr. Ben Kleinstiver:
Yeah. This is a great question. And I think in many ways there have been challenges with Cas, CRISPR-Cas proteins for the last 12 or so years. Some of them have begun to be solved through protein engineering and other methods. I think sort of the main challenges that people have always honed in on are the targeting range issue. This is why we built SpRY. Targeting range just means the Cas9 requires a PAM, so it can only access parts of the genome that encode those PAMs. So by building SpRY, we've solved this limitation, but other properties include activity.
We want to edit the DNA sequence in as many cells as possible, so we want highly active but controllable proteins. Another property is safety or specificity. And this one I think is really important. When you're cutting a DNA sequence in a test tube or you're editing a chromosome in a patient, you really only want to make that genetic change or perturbation at one spot. You want to have editing at your intended on-target site, and you want to minimize unwanted editing at off-target sites. So we and others have devoted quite a bit of effort to trying to develop Cas9 proteins with improved fidelity, meaning that they have minimized off-target effects.
There are lots of other areas where Cas9 people have explored solutions to improve Cas9 properties. The immunogenicity of the bacterial protein, of course, this will encounter our human immune systems in the context of a therapeutic. So trying to minimize the immune reactivity of a Cas9 protein as much as possible. Folks have tried to minimize the size of the Cas9 protein. This is important in how we deliver these enzymes into cells or into patients by different delivery vehicles like viruses. And then on a sort of technology development front, lots of different CRISPR technologies have been advanced in the last decade or so to try and modify what we call the edit outcome.
Because normally you want one type of edit in a DNA sequence. You either want to change a base or insert a sequence or delete a sequence. But most Cas9 proteins, when they cut and break DNA, they sort of rely on DNA repair to fix that break. And we get lots of different changes that we don't necessarily want. So many groups, including David Liu’s lab at the Broad and others have developed tools called base editors and prime editors that allow us to change DNA in a much more precise way, changing single bases at a time instead of getting a heterogeneous mix of changes.
Lydia Morrison:
It sounds like even though you addressed a major limitation with SpRY-Cas9, there's lots more room for improvement in Cas9 proteins in order to sort of help them function optimally as therapeutic agents. Are there other CRISPR nucleases that you see being useful in genome editing applications?
Dr. Ben Kleinstiver:
Yeah, this is a great question. So again, since sort of the early biology discoveries of CRISPR proteins, folks have continued to explore the vast natural diversity of CRISPR and non-CRISPR nucleases that can be useful for different applications. I think it's really fortunate that Emmanuelle Charpentier and Jennifer Doudna were working on Streptococcus pyogenes as a model organism for CRISPR biology because what they discovered is the prototypical SpCas9 that works so well. Since then, many groups have tried to find other Cas9 enzymes that work as well as SpCas9, and we haven't found that yet as a field.
So I remain optimistic though. There's just such a vast biological diversity of these enzymes, either Cas9 orthologs or even these ancestral proteins called IscB or TnpB that are ancestors of Cas9 and Cas12 proteins. And amongst this diversity, I'm sure there are other great enzymes out there with interesting and differentiated properties. But instead of looking into nature to solve these problems, there's another way to try and modify CRISPR nucleases to make them more useful for genome editing.
And that's protein engineering. That's how we built an enzyme like SpRY. I think it's worth mentioning that although we found that SpRY can be really useful in lots of applications like nuclease editing and base editing, and SpRYgest, our goal actually all along was never to build SpRY. We were trying to do something else. We were trying to build lots of different enzymes that individually still maintained what we call the PAM requirement so that they scan only a small fraction of the genome, but that together, if we build enough of these proteins, then we'd have a big enough toolbox, much like you do with restriction enzymes, where you could go and grab one enzyme and use that for your specific application.
And this has been a long-standing project in the lab led by Rachel Silverstein and initiated by Russell Walton, the guy who built SpRY, where we've just tried to build hundreds or even thousands of Cas9 proteins with different properties. So I think there are lots of different ways that we can explore the natural diversity of Cas9 enzymes. And I think the potential of all these different tools in various biological applications remain very high.
Lydia Morrison:
So we touched on cloning a lot already as an application where CRISPR nucleases are useful. What are some of the other applications beyond cloning and genome editing where these nucleases might be used?
Dr. Ben Kleinstiver:
Sure, yeah. I think we've seen a lot of recent use clinically of these enzymes for therapeutics and a lot of great successes in that realm. And I think this is what a lot of people envision as sort of the ultimate translatability of genome editing is that we can get it to work in a patient and it can be safe and effective. And the admittedly early signs in a lot of these trials and now with the approved Casgevy drug is that this is well tolerated by patients and we can in principle, provide long-lasting treatments for patients.
But beyond that, I mean, there's lots of other opportunities for genome editing tools. We use them all the time to make cell lines in the lab. So to model different disease causing mutations. We can use genome editing to create a cell line that has that specific mutation to either study its biology or to optimize genome editing strategies to turn that sequence right back to the wild type, to eliminate that or correct that disease causing mutation.
During COVID and I think still now, there's been a lot of interest in leveraging programmable CRISPR enzymes in diagnostics where that would allow you to basically identify whether or not a specific sequence is in a pool of other sequences. So for instance, if you're trying to detect a COVID nucleic acid, you could use a CRISPR protein to report whether or not that specific nucleic acid is found in that population.
Beyond diagnostics, I think there's tremendous reach of genome editing in fields like epigenetics and synthetic biology where we can take CRISPR nucleases and turn off the cutting function, but now use these enzymes as a programmable DNA binding domain to park them at specific regions of the genome and have them affect a change in a transcriptional state. So I think you can basically use your imagination now in how you can apply CRISPR enzymes for lots of different applications. They're incredibly versatile enzymes and they're easily programmed to bind or edit essentially any DNA sequence at this point.
Lydia Morrison:
Yeah, it sounds like there are tons of options. Are there emerging technologies other than CRISPR-Cas that you think could be as significantly impactful in genome editing applications?
Dr. Ben Kleinstiver:
Sure. Yeah, definitely. I mean, we generally sort of categorize genome editing tools into a couple different buckets based on the types of edits that they can make. And what we've learned over the years is that CRISPR-Cas nucleases and derived proteins like base editors and prime editors and DNA polymerase-based editors, these tools are really good at making small edits to the genome, changing one or two or maybe 10 bases at a time. And I think this is where we really leverage the strengths of CRISPR enzymes.
Our goal and a goal of many other folks in the field is to be able to make predictable, precise and programmable larger changes, being able to change exon-sized fragments of the genome at a time, or being able to even insert larger kilobase sized sequences into the genome. And this is really important when we think about how to circumvent the challenge of translatability of genome editing. And one of the challenges is that for most diseases patients all have different mutations in their gene.
And this is a problem because it takes a lot of time scientifically and a lot of resources to develop new treatments for each patient because you have to develop a genome editing strategy that corrects everyone's different mutation. Instead, if we could correct exons at a time that you could use across segments of the patient population or insert a corrected gene into every patient within that disease class, now you'd have one editor that you could use across all patients.
And although I think it's possible to use CRISPR-Cas enzymes for some of these functions, other tools that have evolved naturally are much better at inserting large sequences into the genome. And these tools are called recombinases and transposases, and they're involved in propagation of genetic elements and evolution. So they've naturally evolved to shuffle large DNA sequences around. So there's a lot of interest in tools like recombinases, transposases, CRISPR associated transposases and even these recently described enzymes from Patrick Hsu's lab at the Arc Institute's called bridge recombinases, which are RNA programmable recombinases. And all of these tools allow us to think about genome editing in a different light where we can now change thousands of bases at a time instead of small segments like a single base or 10 bases.
Lydia Morrison:
Yeah, amazing. And so powerful, and this has been such an interesting conversation. Thank you so much for being here to share your expertise and your research with us. It sounds like there are so many incredible advancements being made around genome editing, and it feels like some real solutions to healthcare challenges are right around the corner.
Dr. Ben Kleinstiver:
Yeah, I agree. I think I'm biased, but I'm obviously very optimistic about the continued impact that genome editing will have on humanity. We've seen all the fancy science prizes awarded to leaders in this field, the Nobel Prize a few years ago to Jennifer Doudna and Emmanuelle Charpentier, and just a lot of recognition of the pioneers.
But genome editing has become accessible to essentially any lab now, and I think that's where the true impact of CRISPR enzymes has democratized how we think about genome editing, because now it's relatively easy to onboard these methods, but the capabilities are incredibly powerful. So in many ways how molecular biology and PCR transforms science, we've seen this huge leap in science with genome editing tool development as well. So I think although a lot of problems have been solved, I think there's a lot of interesting problems that still lie ahead. So lots of genome editing, tech dev to still be done.
Lydia Morrison:
Yeah, I can't wait to see what the next few years hold. Thanks so much for being here today, Ben.
Dr. Ben Kleinstiver:
All right. Thanks so much. It's been a pleasure.
Lydia Morrison:
Thank you for joining us for this episode of the Lessons from Lab & Life Podcast. Please check out our show's transcript for helpful links from today's conversation. And as always, we invite you to join us for our next episode when I'm joined by the incredible Dr. Rita Colwell, an internationally recognized expert on cholera and other infectious diseases. Dr. Colwell was the first woman to become director of the National Science Foundation. She's a member of the US National Academy of Sciences, and she was awarded the National Medal of Science in 2006. Dr. Colwell is also the author of "A Lab of One's Own: One Woman's Personal Journey Through Sexism in Science."
