Podcast: Massive chemical libraries, and CRISPR-CasX

Host: Benjamin Thompson

Welcome back to the Nature Podcast. This week, we’ll be learning about a new enzyme in the CRISPR toolbox.

Host: Nick Howe

And hearing how ‘virtual chemistry’ might speed up drug discovery. I’m Nick Howe.

Host: Benjamin Thompson

And I’m Benjamin Thompson.

[Jingle]

Host: Benjamin Thompson

Listeners, I hope you’ll join me in welcoming the newest member of the team, Nick Howe, for his debut in the hosting chair. Nick, hi!

Host: Nick Howe

Hi, it’s good to be here.

Host: Benjamin Thompson

Well it’s great to have you here. And Nick, tell us, what’s your specialist subject?

Host: Nick Howe

Well, Ben, I’m into all things bees. So, my PhD was all about looking at how pesticides influence bees’ ability to tolerate cold, and also how they overwinter.

Host: Benjamin Thompson

Right, well I’m sure there’ll be lots of opportunities to talk about that in the future, but today you’re going to be telling us about something completely different – the world of massive chemical libraries – and that’s coming up in a bit. In the meantime, what do we have first on today’s show?

Host: Nick Howe

Well Ben, this week in Nature there is a paper out about a new kind of CRISPR protein, and this caught the eye of CRISPR fan Shamini Bundell. She decided to give Nature reporter Heidi Ledford a call to find out what this new protein is, how it fits into the story of CRISPR so far and where the field of gene editing might be going. Here’s Shamini.

Interviewer: Shamini Bundell

So CRISPR is a gene-editing technique that’s become really popular in the last few years and you kind of hear about it everywhere and people are always CRISPRing this and CRISPRing that and finding ways to improve CRISPR, and now there’s this new paper out from Jennifer Doudna at the University of California, Berkeley, along with various colleagues, about a new CRISPR enzyme that they’ve been studying. But before we sort of talk about what this new discovery is, could we maybe have a bit of a catch up on what CRISPR is exactly and go over some of the basics of how exactly it works as a gene-editing tool?

Interviewee: Heidi Ledford

Yeah, sure, so CRISPR systems are found in nature. They’re used by microbes to defend against invading DNA, so when a virus enters a cell and starts to make copies of its genome, if the microbe has some sequence of that viral DNA stored in its genome in what’s called a CRISPR array, then it might be able to recognise that foreign DNA and then direct an enzyme to it to slice it up and render it inactive.

Interviewer: Shamini Bundell

So, the bacteria has a little library that says if you see this DNA, that’s viral, go destroy it, and the reason everyone’s really excited about CRISPR and using it for gene editing is that particular ability to recognise bits of DNA, specific sequences.

Interviewee: Heidi Ledford

Yeah, that’s right. Well, microbiologists are excited about it because it’s just cool – it’s this microbial immune system and it’s so complex and neat – but most people are excited about it for the reason that you mentioned because researchers have figured out how to harness these systems and then to put them to work in other cells like human cells, so that gives researchers a relatively easy way to make changes to DNA at particular sites.

Interviewer: Shamini Bundell

And we’ve actually got a 3D animation on the Nature Video YouTube channel.

Interviewee: Heidi Ledford

How cool.

Interviewer: Shamini Bundell

It’s really cool. It shows a representation of the so-called CRISPR Cas9 complex, which is made up of a guide RNA and that’s the bit that recognises the sort of specific DNA sequence whatever that might be, and then there’s also the Cas9 enzyme, and that’s the thing that cuts the DNA and it’s this cutting which is the important bit for both the bacterial immune system and for scientists using it for gene editing, right?

Interviewee: Heidi Ledford

Yeah, that’s right. So, it cuts the DNA at that specific site where you tell it to cut, and then after that the DNA repair systems in the cell come in and repair it and you can rely on those systems to either make a small deletion at that site and maybe disable the gene if you wanted to knock it out, for example. You can also try to manipulate it so that the DNA repair system inserts in a little sequence that you want in there.

Interviewer: Shamini Bundell

So CRISPR Cas9 is the main system that I’ve heard about and it’s what that film was about, but it seems that Cas9 – the molecular scissors – isn’t the only cutting enzyme that’s part of CRISPR systems that we know of.

Interviewee: Heidi Ledford

So, microbes have evolved all sorts of different variations on this CRISPR system, and they’ve got lots of different enzymes that can serve as the molecular scissors, as you said. Cas9 is a nice one for researchers to use. It’s one of the first ones that they really characterised and were able to make work in a relatively simple way in lots of different kinds of cells, but it’s not perfect. It’s a bit large which can make it difficult to shuttle into cells. If you want to stick a Cas9 enzyme into a human cell, it’s better if it’s smaller. It can also make mistakes. It can make changes occasionally to sites that you didn’t necessarily want it to.

Interviewer: Shamini Bundell

So, what are our other options then?

Interviewee: Heidi Ledford

Well the paper that’s coming out is describing in more detail a relatively new – well, new to us – member of the family called CasX. That one’s a bit smaller so that makes it appealing particularly for use as a human therapy. It may also be a bit less likely to make some of these unwanted changes, which would also be an advantage from a safety standpoint.

Interviewer: Shamini Bundell

And CasX is the latest one and the one that this particular new paper is about, but it’s not the only alternative to Cas9, is it?

Interviewee: Heidi Ledford

That’s right. So, it’s sort of been periodic. There’ll be another paper that comes out that describes another enzyme and says nobody’s going to use Cas9 anymore because we’ve got this new enzyme that’s so much better, and there have been several iterations of that. I think one other one that comes to mind is one called Cas12a which was also meant to be a bit smaller than Cas9, and there was some speculation that it might be better at making some of these true edits where you can essentially rewrite a sequence rather than just deleting a little bit out. There’s another enzyme called Cas13, for example, that instead of cutting DNA, cuts RNA, so that would then allow researchers to control gene expression at a different level, so it gives them a different range of things that they could do.

Interviewer: Shamini Bundell

And so, are researchers looking for something better than Cas9? If Cas9 was just what they happened to discover first, are we searching for like the one Cas to rule them all?

Interviewee: Heidi Ledford

Yeah, we’re always looking for something better and the thing is, there’s so many of these enzymes and so many variations on this system out there, so there’ll be a whole big toolkit, I guess, of different enzymes that you can pick from. And as they become more characterised, you probably will see more labs using different enzymes. At the moment, a lot of people really prefer Cas9 because it’s the familiar one that is sort of the standard but as people become more familiar with these other enzymes, I think you’ll see more and more of them.

Interviewer: Shamini Bundell

So, it’s basically all CRISPR and you could potentially then be picking the enzyme that happens to work for the gene that you want to tweak or for the purpose that you want to do.

Interviewee: Heidi Ledford

Yeah, it’s still fairly new. It’s only been a few years since they really figured out how to use it for gene editing, and so they’re still optimising and figuring out which systems are best. In addition to looking for these naturally occurring variations, people are engineering all sorts of things. They’re engineering Cas9 enzymes, for example, that have other enzymes attached to them so that they can serve other functions and make other changes to the genome, and it’s all becoming quite sophisticated at this point. And then also some day as people continue to study these different microbial systems, they may come up with something that’s completely different, that’s not a CRISPR system, that also works well for gene editing. There may be new advantages and ways to use that as well. It’s a very quickly changing field.

Host: Nick Howe

That was Heidi Ledford chatting to Shamini Bundell about the CasX enzyme and the whole toolkit of CRISPR proteins. You can find the paper they discuss over at nature.com/nature, and you’ll find the animation of CRISPR Cas9 at youtube.com/NatureVideoChannel or by searching for the film title ‘CRISPR: Gene editing and beyond’.

Host: Benjamin Thompson

Coming up in the show, we’ll be hearing about the rule changes in the UK’s latest audit of academic research – that’s coming up in the News Chat. Before then, Anna Nagle is here with this week’s Research Highlights.

[Jingle]

Anna Nagle

The ravers here at Nature love a bit of UV light, and it turns out that squirrels like to get in on the ultraviolet action too. A chance encounter in a Wisconsin forest between a researcher’s UV flashlight and a New World flying squirrel revealed a hot pink hue in the creature’s fur. The scientists then painstakingly directed their UV light at 135 museum squirrel specimens, and found that it was only the New World flying squirrel that glowed pink. Although many plants and animals sport dazzling fluorescent patterns only visible under UV light, it’s rare in mammals, so quite why these mammals sport a hot pink pelt is a bit of a mystery. The team behind the discovery think it might be to confuse predators or possibly to help the animals find and impress each other in low light. Read that illuminating research in the Journal of Mammalogy.

[Jingle]

Anna Nagle

Lung cancer has been linked to chronic inflammation, but quite what the connection is between the two has been rather unclear. So, scientists in the US turned to mice that have been genetically modified to develop tumours similar to a common form of lung cancer in humans. The researchers found differences in both the type and abundance of lung microbes found in the tumour-ridden mice compared to those that remained cancer free. The microbes present in the cancerous mice boosted production of a type of immune cell that produces an inflammatory protein, but germ-free mice raised in a sterile environment didn’t experience this, suggesting that the microbes were playing a key role in tumour development. Check out that research in the journal Cell.

[Jingle]

Interviewer: Nick Howe

Discovering a new medical drug is a long and resource-intensive process. It can take a decade or more for a drug from the drawing board to the hospital ward, in a process that involves a huge number of researchers. But for every drug that makes it, many more fall by the wayside in a process that costs billions of dollars. Naturally, researchers are keen to streamline and accelerate the discovery process in order to save money and time. One possible way to do this is by using computer simulations to screen early drug candidates. Whilst this technology has existed for some time, this week in Nature, there’s a paper describing how size is important in the world of virtual screening, as Bryan Roth from the University of North Carolina, one of the co-authors of the paper, describes.

Interviewee: Bryan Roth

We were trying to basically see if we could enhance the process by several orders of magnitude, by speeding up the initial screening process. The initial screen typically would take months, and is usually limited to, say, 100,000 compounds to maybe 1 million compounds.

Interviewer: Nick Howe

Usually the initial screening involves physically testing thousands of compounds. In this case, rather than testing the activity of a relatively small number of compounds against a particular drug target in the lab, the team used a ‘virtual library’ – a catalogue of hundreds of millions of compounds.

Interviewee: Bryan Roth

And so, what we did here was we took advantage of a large resource of potential chemicals – these are chemicals that are theoretical in that they have not yet been synthesised but could be made by chemists using sort of common chemical reactions or typical chemical reactions, and this is using a resource called a zinc database, which was developed by my co-authors Brian Shoichet and John Irwin at UCSF. And over the last few years, they’ve been able to increase the size of the zinc database to nearly 1 billion compounds, so a large number of compounds, so more compounds than one could ever physically screen.

Interviewer: Nick Howe

By using a lot of computing power, the team were able to virtually test the interactions of these compounds against a number of drug targets in a process known as ‘automated docking’. You might think that simulating more automated docking would slow down the discovery process, but Bryan suggests that for a number of reasons, when it comes to virtual libraries, bigger is better.

Interviewee: Bryan Roth

The main advantage of a large library is you get access to a large number of different chemotypes, so these different molecules with different shapes. And it’s become clear over the past several years that if you have compounds that have unique shapes, then when they interact with the target they’re likely to have interesting biological activities that never would have been predicted before and ultimately could be therapeutic in ways that were previously unanticipated.

Interviewer: Nick Howe

By testing such a diverse array of chemical structures, including molecular shapes that are completely novel, the team were able to find interesting chemical reactions that haven’t been seen before, and they were able to do it quickly. For example, in just over a day, the researchers were able to virtually test over 138 million compounds for their ability to dock with the D4 dopamine receptor, a drug target for treatment of diseases such as Parkinson’s. The team also looked for inhibitors of a bacterial enzyme called AmpC β-lactamase, involved in antibiotic resistance. By screening 99 million molecules, they found some promising candidates. Out of these, they synthesised a very potent chemical that appears to have a completely new mechanism of action. And this is the proposed advantage of this technique – going from rapidly screening millions of compounds in the computer to then choosing a top list to synthesise and test in the lab. This approach could massively cut down on the time needed to identify early drug candidates.

Interviewee: Bryan Roth

It turns out that having this huge library really is a tremendous resource basically because you get access to this chemical space that there is no way to physically ever obtain, and it turns out that that is a huge, huge advantage, one that we had not anticipated. We hoped that we would get that, but we didn’t really anticipate just how valuable it was.

Interviewer: Nick Howe

Of course, there’s a long way from discovering a chemical interaction to developing a drug for clinical use, but the time saved could be incredibly valuable. David Gloriam from the University of Copenhagen has written a News and Views article on this topic. He thinks that although virtual screening has been around for a while, there’s a lot to appreciate in this new work.

Interviewee: David Gloriam

Well, the method of virtual screening has been refined and applied for two or three decades now, but what is really setting their approach apart is the huge compound libraries that they screen, and previously this has not been possible because of computational limitations and maybe for many groups because of licensing issues to do with the software, and also the availability of these compound databases.

Interviewer: Nick Howe

Like with any developing technique, there are some caveats that need to be considered. For docking to work, you need to have well modelled structures of targets and even then, it will only work on molecules that contain particular binding sites that allow for the activity to be changed. David thinks that there are some other challenges of a less scientific nature too. As drug discovery is an expensive process, companies often seek unique compounds that they can patent and have exclusive rights to produce. The massive library in this paper, however, is open for anyone to use.

Interviewee: David Gloriam

Because many of these molecules are available open access, then is the question of whether this would restrict rights to having invented this molecule. There are indications that if you make compounds available on the internet, this could limit the possibilities to make a stronger type of patent.

Interviewer: Nick Howe

If the structures are freely available, then the question is whether you can claim to have invented the molecule, which is a requirement for the stronger type of patent. Without the security of a powerful patent it’s reasonable to ask whether drug companies would want to invest in drug discovery like this. Both David and Bryan are confident that these challenges could be overcome. Also, as more drug targets are getting better described, this method could be applied more widely in the future. Right now, we can already start using this approach to look for novel drugs with the online ZINC15 database. You can read Bryan’s paper along with David’s News and Views article over at nature.com/nature.

Interviewer: Benjamin Thompson

Lastly this week, it’s time for the News Chat and Holly Else, one of the reporters here at Nature, joins me in the studio. Hi Holly.

Interviewee: Holly Else

Hi Ben.

Interviewer: Benjamin Thompson

We’ve got two stories this week, and the first one is about the REF, and this is a regular review of science here in the UK, and when it’s REF time you get a lot of researchers chatting on Twitter about it. Holly, for our listeners maybe outside of the UK, could you describe what the REF is?

Interviewee: Holly Else

Well, the REF is the Research Excellence Framework, and that’s a mammoth audit that the Higher Education Funding Councils do regularly to assess the quality of research that’s going on in UK universities.

Interviewer: Benjamin ThompsonWell, if this is an audit then, what sort of things is it looking at?

Interviewee: Holly Else

So, it looks at lots of different things – the environment that the researchers work in in the university, also the research papers that they’re producing and also the impact of their work, so it will look to see if there have been any changes on policy, for example, as a result of some research that’s been going on a certain institution.

Interviewer: Benjamin Thompson

And I guess the next one must be coming up relatively soon and that’s why in the news today?

Interviewee: Holly Else

Yeah, so the next one’s in 2021, and what’s happened this week is the funding councils who administer the REF have released the rules for what each university will have to do for the next exercise.

Interviewer: Benjamin Thompson

And what will they have to do and maybe how does it differ from what’s gone before?

Interviewee: Holly Else

One of the new rules is that all staff who work in research will be submitted for assessment, and this didn’t happen in the previous REF which happened in 2014, so universities could sort of select who they wanted to put forward to be assessed, and this led to sort of this frantic transfer market, a bit like football, where universities were trying to poach the best staff in particular areas in order that they could take their research and then present it to the exercise to boost their scores.

Interviewer: Benjamin Thompson

Which seems potentially disingenuous then, but maybe this new system will give more of a level playing field?

Interviewee: Holly Else

Well, potentially. It’s also sort of designed to try and keep the cost down because the last exercise was particularly expensive – it cost £246 million I think, partly because universities spent so much time and money decided who they wanted to submit to the exercise.

Interviewer: Benjamin Thompson

Right, and who’s going to be happy about this then when it happens? Are universities happy? Are researchers happy?

Interviewee: Holly Else

No one’s happy when it comes to REF. It’s a lot of work. Administrators spend their whole lives doing it. The submissions – if you’ve ever seen one – they’re just hundreds of thousands of pages long. Researchers often berate the bureaucracy of having to submit all these forms and fill in these things, but the other side of the coin is that this means that the research money in the UK is being spent wisely, some could argue.

Interviewer: Benjamin Thompson

Well, let’s move on to our second story today, Holly, and this one is about some ongoing negotiations about access to journals.

Interviewee: Holly Else

So, it’s been six months now since researchers in Sweden and most of Germany had the plug pulled on access to Elsevier journals, so that means that they are unable to view and download the most recent papers.

Interviewer: Benjamin Thompson

So why has access to these journals been lost then?

Interviewee: Holly Else

The university libraries no longer have a subscription with Elsevier because the one that they had previously has ended and no new agreement has been made in its place so therefore these countries are not paying Elsevier for their products, so Elsevier argue why should we supply them to you.

Interviewer: Benjamin Thompson

What’s the state of the negotiations at the moment then Holly? Is there a sort of particular roadblock that’s preventing these journals being available?

Interviewee: Holly Else

Yeah, so the university libraries in Sweden and Germany sort of come together to create a collective which means that they can negotiate with the publishers as one on behalf of the whole country. And what has happened is that the negotiators in these countries want to start paying less for their subscription fees because they are now increasingly publishing open access articles, and to publish open access often you have to pay a fee to the publisher because the publisher will not get any revenue from subscriptions because this article is freely available. And so, what these negotiators want to do is kind of incorporate those fees into the subscription cost, so they’re not paying twice – once to publish and then again to read – if an increasing amount of the literature is available for free. And what’s happening is Elsevier’s not happy to do that. Talks in Germany have been going on for years over this exact issue and similar in Sweden.

Interviewer: Benjamin Thompson

And so, what effect is this having on scientists, on research academics?

Interviewee: Holly Else

Well, it’s different for both countries. In Germany because the university libraries didn’t previously have a nationwide contract – they had individual contracts with each library – they’re all being cut off at different rates. So, within Germany there are still a very small amount of institutions that do still have access, and so researchers in those countries are able to get hold of the most recent papers by an interlibrary loan, for example. So, the situation perhaps there may not be as bad, but the researchers I spoke to in Sweden where all universities have no access to Elsevier are really talking about how frustrated they are, how much extra time it takes them to get hold of a copy of an article that they need. So, they might do this by ordering it from the library who would then pay for it, or they might do this by emailing the authors of the study to ask for a copy, and obviously that all takes time and in some cases, they still can’t get hold of a copy and then they have to try and find the information that they need from a different source, so maybe a different journal article. And they argue that when you’re up against a deadline for your grant application or your paper that you’re writing, sometimes you just haven’t got time to do that. So, a lot of frustration out there I think.

Interviewer: Benjamin Thompson

What about the publisher themselves – what are they saying?

Interviewee: Holly Else

They say they’re really committed to finding a solution to this problem in Germany and Sweden and, as we mentioned, the talks are still ongoing. It’s not clear what’s going to happen, but what’s happening in Sweden is the National Library of Sweden who negotiate these deals have been surveying their researchers to see how they’re getting on without Elsevier. We don’t know what they’re going to find – they’ll release the results later on this year – but I think that will be a really interesting thing to know because Sweden is the first country that’s really gone through this.

Interviewer: Benjamin Thompson

Well, thank you for joining us Holly, and listeners, for more on these stories head over to nature.com/news.

Host: Nick Howe

That’s it for this week’s show. Don’t forget you can get in touch with us on Twitter - we’re @NaturePodcast - or on email - podcast@nature.com.

Host: Benjamin Thompson

And listeners, it would be amazing if you could leave some stars or a nice review wherever you get your podcasts from. Thanks! I’m Benjamin Thompson.

Host: Nick Howe

And I’m Nick Howe. Thanks for listening. See you all next time.