Strategies and applications for CRISPRko, RNAi and beyond – SelectScience

Posted: January 9, 2021 at 2:55 am

Watch this on-demand webinar to learn more about manipulating the genes within physiologically relevant human immune cells

In this on-demand SelectScience webinar, immunology expert Dr. Verena Brucklacher-Waldert, Horizon Discovery, shares successful strategies for the manipulation of genes within physiologically relevant human immune cells. A number of case studies are presented to highlight the techniques used in a variety of applications employed for gene interrogation, including CRISPRko, RNAi and more.

Read on for highlights from our Q&A session that followed the live webinar with Ryan Donnelly, Senior Product Manager for Gene Editing at Horizon Discovery.

RD: We've spoken to a lot of customers who do what we call orthogonal studies for hit follow-up. Say you run a CRISPR knockout screen, and you get a handful of hits back, you can then see what reagents are available, maybe via traditional RNAi methods, so siRNA or shRNA. You can then run those to see if you can replicate some of the phenotypes that you saw in your knockout screen for those specific hits. The thought is, if you see a positive correlation, you now have stronger evidence that that hit was actually real, since you've now generated a similar phenotype by targeting both DNA, with your CRISPR knockout screen, as well as the RNA with those traditional kinds of RNAi methods.

In essence, you've generated a similar phenotype by targeting two different sections of the central dogma. One of the nice things about Horizon is we have readily available reagents, all genome-wide for both CRISPR knockout, CRISPR activation, siRNA and shRNAs. So, most combinations that you would be looking at for validation, we can support.

RD: There are a few. But the first one that comes to mind is when working with primary immune cells, the availability and variability of the cell type. We can extract those cells from blood samples, but there's usually a limited amount of those types of cells that we can extract from each donor. We then need to keep in mind that there can be specific variability between donors. The complexities that come along with trying to stimulate immune cells, like T cells, also need to be considered.

Another challenge that we usually see is just in the biological differences between immune cell types. We talked a lot about proliferating cell types. Some cell types lose the ability to proliferate once they've been extracted from blood.

Lastly, is a variable that we look at routinely that also comes down to cell type: cells that are in suspension versus adherent cells. This can make screening protocols quite different, depending on whether your cell types are in suspension or if they're stuck down in the bottom of a well. Those are the three main considerations when looking to conduct screening experiments within immune cells.

RD: In principle, we usually suggest a minimum of three donors, but this is all cell type dependent. Functional assays can show a high degree of variability when using cell types such as natural killer cells. But if you shift into myeloid cells, that variability in functional assays is much more limited. Another thing to keep in mind is that donor variability is not necessarily a bad thing. By mixing donors, you spread a wider vision on things like the ethnicity of those donors, sex, age, and genetics that make up the donor pool. Since they're randomized, it will give you good insight into how a variety of donors would respond and can give you higher confidence in the performance characteristics of the target that you've identified within your screen.

RD: Controls, and multiple types of controls, are absolutely critical when doing this type of work. Without them, it's really impossible to check the efficiencies in large, arrayed screens.

At Horizon, we use multiple different types of controls, and for anybody that is taking these projects on, we would recommend a similar approach. For checking the transduction efficiency, we use a combination of both lethal controls and essential genes. This will give us a nice viability readout. In essence, the more cells that die, the higher the rate of transduction efficiency.

We also incorporate non-targeting controls, and a ROSA26 guide RNA. The non-targeting control won't cut the DNA, so incurs no DNA damage. The ROSA26 guide RNA will cut, but it cuts without a functional impact to the cell. This will give us insights into the potential for DNA damage, as well as a cutting efficiency control. Lastly, we would pick a positive control based on the cell type of interest. For example, in T cells, we target CD3, as it's consistently expressed as part of the T cell receptor complex.

By doing this, we have the ability to monitor across donors, across plates, and across replicates, to look at assay performance as a whole, with the screening experiment.

Q: How does pooled screening differ from arrayed screens? And what are the advantages?

RD: The main advantage of performing a pooled screen over an arrayed screen is really the ability to scale up and analyze the whole genome with a reasonably small amount of resources. Mining the whole genome is very important when we're looking to understand new biology without any preconceived ideas. It's really a discovery approach.

Thinking about using a pool versus an arrayed screen comes down to the biological question you're attempting to answer. If it's a simple, black and white question, such as "Do my cells survive a particular stimulus?", a pooled screen is a really nice way to go. If, however, the screen needs to assess multiple different types of outcomes that use different techniques so maybe combining FACS and HTRF assays for looking at more than one parameter, arrayed screens are what you would need to use.

Learn more about modulating gene function in primary immune cells: Watch this webinar on demand here>>

SelectScience runs 10+ webinars a month across various scientific topics, discover more of our upcoming webinars>>

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Strategies and applications for CRISPRko, RNAi and beyond - SelectScience

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