CRISPR in Fungi: An Interview with Rebecca Shapiro

Minu: Is it difficult to perform CRISPR experiments in fungi?

Rebecca: I think with any organism, when you are developing new systems, there are always initial difficulties. I don't think that it is inherently more difficult in the case of fungi, but I do think that fungal pathogens, in particular, tend to be quite understudied compared to other pathogens. For instance, compared to bacterial pathogens or some parasitic organisms, fungi tend to not have quite as much research devoted to them. I think that is where some of the challenges come in, lesser than the technology itself.

That is why we are excited to be working on developing some of these new technologies in these organisms that don't tend to always be at the forefront of research. We are certainly not the only ones. There is now quite a strong community of technology building and CRISPR applications for studying diverse fungal pathogens, which is exciting.

Minu: Could you elaborate on one or two of your favorite lab projects using CRISPR and fungi?

Rebecca: I started here at the University of Guelph exactly a year and a half ago. We were sort of bridging the two worlds from my training, so focusing mostly fungal pathogens of humans, but also starting to delve into the world of fungal pathogens of plants, and then integrating that with CRISPR. I see our lab as being at the interface of these two things, and a lot of the projects in the group focus on more of the technology development side of things.

We have some basic CRISPR tools for doing genome editing in some of these fungal pathogens, but how can we develop new different versions of these CRISPR tools that have different types of functionality? Secondly, how can we actually apply this to study fungal pathogens?

On the technology development side of things, one of the first research projects to come out of my new lab, which I am fairly excited about, was developing a CRISPR interference (CRISPRi) system for genetic repression in Candida pathogens—common human-associated fungal pathogens. This was exciting for us because CRISPR technologies have now really been developed in a lot of different fungal pathogens, a lot of different species, but the CRISPRi repression system hadn’t yet been applied in any fungi other than Saccharomyces. So, we were really excited to get this system up and running and working in at least a few of the fungal pathogens that we work within our lab and are hoping to expand this to other organisms as well.

We are excited about this because it allows us, without having to modify the genome directly, to repress or knockdown genes. This, in turn, enables us to study the function of essential genes because we are not deleting them or otherwise obliterating their function; we are actually just tuning down the level of expression, which can be very powerful. So that's on the technology development side of it. There are also other systems that we are in the midst of developing, some of them in collaboration with other groups.

On the application front, we are interested in lots of different questions surrounding how fungi cause disease. Some of the questions that we are asking have to do with antifungal drug resistance. So we are using CRISPR systems to create large libraries of mutant strains that have different genes deleted singly or in combinations to look at genetic interactions, and looking at how some of these specific interactions affect sensitivity to antifungal drugs. Those are some of our ongoing big-picture projects.

Minu: That sounds really interesting. You are also working on gene drive-based projects in fungi, right? Could you tell us more about that project?

Rebecca: The gene drive project was something that began during my postdoc work in Jim's lab, and is definitely something that we are continuing now. Gene drives get a lot of publicity in organisms like mosquitoes, where people talk about creating this system gene drive, which acts as a sort of selfish genetic element and continually propagates itself. It will turn any heterozygous mutation into a homozygous mutation, and through the process of mating and reproduction, it will continually propagate itself infinitely essentially. So, it's a way of ensuring the existence of this particular gene or this particular mutation in a population.

So again, this is often referred to in the context of making genetically modified organisms like mosquitoes that have a certain desirable trait, and releasing them into the wild so that these gene drive traits propagate throughout the entire population and take over. We use a similar technology, but are thinking about the application quite differently, mostly because one of the pathogens we work with commonly in the lab is Candida albicans, which is the most common human fungal pathogen, and it's a diploid. So working with it in the lab comes with some intrinsic difficulties, because if you want to look at the function of a gene and knock it out, you have to knock out both copies of that gene. And if you wanted to look at two genes and how they interact, you would have to knock out both copies of gene A and then both copies of gene B, which is four rounds essentially of creating these genetic mutations, which can be really quite laborious.

The idea with the CRISPR gene drive here was to create a system where we could have a CRISPR knockout system that would self propagate. We can take Candida cells in the haploid form, transform them with CRISPR gene drives to knock out specific genes of interests, and mate them together. And while normally mating haploid with one gene deleted to a haploid with a different gene deleted, the typical outcome would be a deploy that is actually heterozygous at those two loci. But gene drive technology actually allows us to very rapidly generate a diploid cell with a homozygous deletion at both of these loci of interests.

Building this technology is allowing us to build up these large libraries of single and double gene deletions which allow us to do large scale genetic interaction analysis in some of these fungal pathogens like Candida albicans, which is something that would have been very difficult to do technology-wise prior to this.

Minu: Another interesting topic in your lab is your work on biofilms. I have mostly heard of bacterial biofilms, and how they are generally a nuisance in most of the industries. Could you talk a little bit about fungal biofilms and your work on these?

Rebecca: Fungi, similar to bacteria, form very difficult-to-treat biofilms. Actually, fungi will commonly form biofilms in conjunction with bacterial pathogens, so you will get these interesting inter-species biofilms that will have Candida fungi for instance, as well as bacteria, and very similar to bacterial biofilms, they also adhere to surfaces. They are often found associated with medical devices like catheters or stents. And again, similar to bacterial biofilms they are extremely difficult to treat. There will be some really dense networks of cells that are encapsulated in this extracellular matrix, which makes it difficult for antimicrobials to penetrate. Clinically, it is really quite a large problem.

Candida pathogens, one of the organisms we work with a lot in the lab, is notorious for forming these robustly adhesive biofilms. This was one of the topics that we have been interested in in the lab. This was also applied using our CRISPR gene drive technology, where as I mentioned, we can use the gene drive to really create these large scale libraries of genetic mutations where we can target genes, not just individually, but also in these larger combinations. This allows us to look at a lot of genetic redundancy i.e. what occurs when we knock out just this one factor, or maybe these two factors in combination, or even higher order than that? The reason that Candida is very good at forming biofilms is that it produces these adhesion proteins on its cell surface that allow it to adhere to different substrates.

We figured this was a good opportunity using our gene drive just to study what is likely a lot of redundancy between these factors. We used our CRISPR gene drive to make these libraries, these genetic contraction libraries where we delete combinations of adhesions and all possible permutations, and look at how that interferes with the ability of the fungus to form biofilms on different surfaces.

Sure enough, what we find is that there is in fact a lot of redundancy, and often just individually knocking out any one of these adhesions will allow others to compensate and will still allow for quite a robust biofilm growth. But there are many combinations of adhesion factors that when you delete them together in combination, significantly impair the ability of the fungus to form a biofilm. This gives us some interesting insights into some of these genetic redundancies and genetic interactions involved in some of this really complex pathogenesis and processes.

Minu: Thank you for elaborating on your amazing projects. I am curious to know about your future plans. Could you tell us a little bit about that?

Rebecca: Yeah, definitely. We are moving forward on all sorts of fronts. I talked about technology building, and that's something that we will continue to do. I talked about developing the CRISPR interference system, and in parallel, we were developing a CRISPR overexpression system as well as other CRISPR based platforms, so really trying to build up that technology within the world of fungal pathogens.

I also mentioned branching into different types of pathogens. Candida albicans has been our main focus, but we are also starting to work on more recently emerging Candida pathogens as well as some plant-associated pathogens. We have got projects in the lab now focused on Fusarium, which is a very common plant-associated fungal pathogen.

A lot of what we are doing now is taking some of the existing technologies that we have and building them up to a much larger scale. When we developed the CRISPR interference system, we published this work showing as a proof of principle. Now we are working to show that not only can we knock down genes, but we can also create large scale libraries that will allow us to perturb or knockdown functional genes on a much larger scale. Using this to tackle some of the questions that we have been asking in the lab about antifungal drug resistance, about biofilm growth, things in that realm.

Minu: That all sounds amazing and really exciting! I hope we get to see a lot of your work in the next few years.