In this presentation, Mathew Blurton-Jones introduces the newly established CRISPR core at the University of California, Irvine's Stem Cell Research Center, detailing its mission, services, workflow, pricing, and collaborative support. The core offers genome engineering of induced pluripotent stem cells (iPSCs) and other proliferating cell types, including gene knockouts, knock‑ins, point mutations, and reporter line creation. A typical project proceeds from an initial consultation through guide design, validation, and clonal screening, typically completing within 7–8 weeks. Pricing is subsidized by the center, making it more affordable than comparable external facilities. As a concrete example, the core generated TREM2 loss‑of‑function iPSC lines to study Alzheimer’s disease, demonstrating impaired amyloid phagocytosis in vitro and altered microglial plaque response in chimeric mouse models. The talk concludes with contact information for core staff, offers of grant support letters, and preliminary data generation for feasibility studies.
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Some of you may have heard of this, but if not, this is part of the reason I'm here is to tell you a little more about a new core that's been established at UCI just a little under a year ago actually. This is through the Stem Cell Research Center. This was encouraged by Eileen Anderson when she took over the directorship of the center. You can see is one of the websites where you can look at resources and find our CRISPR core resources link there. So what do we do? Well, we can engineer induced pluripotent stem cells to modify a gene. So for instance, we can knock out a gene. We can knock in a mutation. We can make reporter lines. And we started with iPS cells because that was sort of our background. But we have also continued to— start to do this with other cell types. So if you're not working with iPS cells but have other cell types that you're interested in, as long as they're proliferating, we should be able to help you with this. I don't want to get too technical here, but I just want to very briefly explain for those of you who aren't familiar with CRISPR what it involves. Essentially, the breakthrough of CRISPR was the ability to cut DNA on both strands in a very targeted fashion. And so you can use these guide RNAs to deliver this nucleus called Cas9 You can target it to a specific gene. That makes this double-stranded break. And then there's a couple ways that the cell will try to repair it. One is called non-homologous end joining. The other is called homologous recombination. And we can take advantage of these two repair mechanisms depending on what our interests are. So if we want to make a knockout, we can just let the cell try to fix it with non-homologous end joining. And mistakes are often made that lead to stop codons and therefore a knockout. Alternatively, if you want to fix a gene or change a single amino acid, for example, we can add a template which the cell will then use on the right side through homologous recombination. So with all these steps, we sort of induce a double-strand break and then allow the cells to repair, and we kind of help them along the way depending on what we're trying to achieve. The next step then is, of course, to screen and identify the actual clones at a clonal level, single-cell level that we then expand to find cells that have been appropriately targeted. So there's, of course, more detailed steps. And we have on our website this link that shows us sort of the steps we take through. For example, in this case, for a point mutation introduction, we have conservatively said that this takes about 2 to 3 months. We're finding it's closer to 7 to 8 weeks, actually. So we're quite efficient at doing this. But it starts with a consultation, asking the researchers about the gene they're interested in. Then we'll design the guides and validate the guide's ability to cut. And then we can go sort of a full-service run-through. We can also do parts, pieces and parts, depending on the researcher's interest. Now CRISPR, some of you may know, is a fairly expensive proposition, especially when you're working with iPS cells. So we do have a price list on this, and this is has been supported by the Stem Cell Center to keep our prices low. And sort of as proof of that, one of our closest competitions here is actually the Harvard Stem Cell Bank. And if we look at the pricing, they're actually charging about twice as much, even for their own internal users. So I think we're giving UCI a fairly good deal in terms of generating these cells. So how are we doing? Well, we started just back in January 2019, and we're already making quite a lot of progress. Up at the top, you see some completed projects that we've done for various researchers here at UCI. We've got several ongoing projects that are moving forward as well, but we're pretty sure we can handle more. So if you guys are interested, feel free to reach out to us. Now, being a researcher myself, of course, I feel uncomfortable not showing a little bit of data. So I do want to show one example of how CRISPR technology is being used at UCI. So just the last couple of slides I want to show you how some work being done, and in this case, this is in partnership with UCI's Alzheimer's Disease Research Center that helps us to provide samples from patients who have or don't have Alzheimer's disease. We were interested in studying this gene called TREM2, which has been shown to be a risk factor for AD. It increases risk of Alzheimer's about threefold. We can start by trying to make a loss-of-function mutation, essentially a knockout, by using CRISPR, allowing non-homologous end joining, We can screen our clones, in this case looking at chromatograms. The top is showing the wild-type alleles. The bottom two is looking at the two different alleles to show that we have loss-of-function mutations. So we induce stop codons, premature stop codons in both alleles. And we've done this, in this case, with three different individual lines and validated at the protein level that, yes, we've made a knockout of this protein. So what can you do with it? Then you can start to do things like in vitro studies. In this case, taking microglia that have been generated from these iPS cells, feeding them with beta amyloid that we know is very important in Alzheimer's disease pathogenesis, and finding that this knockout mutation impairs their ability to phagocytose this amyloid. That gives us some indication of how TREM2 might be influencing Alzheimer's disease. We can also actually move this forward to some in vivo studies. For instance, by transplanting some of these cells into immune deficient mice, we can actually make a chimeric model to study human microglia, iPS-derived microglia in vivo. When we do that, we see some very interesting features. Normally in Alzheimer's disease, microglia will migrate to the plaques and surround them. That's what you see up above with the wild-type cell. But if we have the knockout cell, and this is again from the exact same patient, just that knockout change, the microglia no longer pay attention to the plaques. They don't migrate to them. This has a lot of effects on their gene expression. They seem to be locked in a more homeostatic state. So they're not responding to the pathology the way they should. So this is an example of using CRISPR, making an iPS line, starting to use this for both in vitro and in vivo studies to learn about that specific gene and how it might be influencing Alzheimer's disease. So with that, I just want to end with our email there. Feel free to reach out to the core members. John Paul and Lisa are really the ones running the show. They're amazing. Incredibly efficient, incredibly bright, and we're happy to work with you guys. One important thing is we're also happy to write letters of support for grant applications and even can provide some preliminary data if that's useful for feasibility. Thank you.
