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GEN: What are the advantages of CRISPR screening?
Goldmeyer: At the outset, CRISPR biology is a little cleaner in terms of signal-to-noise ratios and specificities than are some older platforms like RNA interference. A huge advantage, however, is that with CRISPR gene knockout (CRISPRko), you can completely remove a gene function from the cell permanently instead of transiently, which enables much longer assay timepoints. Essentially, you ablate that gene function and see how the cell responds in the complete absence of a particular gene.
CRISPR also enables you to create cell backgrounds that model a disease state. Where we know that a phenotype might be the result of a particular mutation, you can use CRISPR to create that background in an amenable model and then run gene interaction screens or drug screens to determine the biology occurring in the cell. To this end, clients routinely couple cell line engineering to other projects.
There are also CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) tools that modulate gene activity rather than knock it out and allow us to directly increase or decrease the expression of a gene. By using CRISPRa and activating the gene in the form that is innately present in the cell, we get a result that is highly biologically relevant.
GEN: Horizon offers the CRISPR screening services you mentioned, that is, CRISPRko, CRISPRa, and CRISPRi. Tell us a little more about these tools.
Goldmeyer: We do indeed offer all three of those services either individually or in concert with each other. All of them can be a custom library or extended all the way up through the entire human genome, which is what we most commonly are running. You can run a CRISPR KO experiment where you systematically knock out every single gene in the genome.
Most researchers are doing one of two things. They’re applying a potential therapeutic to the cells to find genes that are responsible for the behavior of that therapeutic (genes that when removed might provide sensitivity or resistance), or they’re looking for a mechanism of action, that is, how the drug is actually working in the cells.
Patient stratification is another aspect researchers are often interested in. If you have several different cell types with which this compound might react, which cell types are most likely to be affected and in what way? As an example, think about a drug where a small patient population is completely nonresponsive, but the majority of the patient population has a positive response.
Patient stratification is a method where we can pre-isolate those groups and determine, for example, that patient group one is a good indicator and that patient group two should not get this drug. This ultimately leads to more effective patient outcomes. Functional genomic screening enables this through comparative screens in different cell models.
CRISPRi allows us to look for what happens when a gene is repressed rather than knocked out. Using the CRISPR machinery, we’re able to target near the transcriptional start site for the gene and bring in a repressor element. We can go right to that site and in a very site- and gene-specific manner prevent transcription and see how the cells respond.
Conversely, with CRISPRa, you target near the transcriptional start site and bring in elements to activate the gene and overexpress it. In effect, you bring back something that might not be expressed or is expressed at a low level in the cell.
GEN: Horizon recently expanded its cell-based CRISPR screening services. Can you talk about what that means for potential clients?
Goldmeyer: The significant expansion recently was aimed at screening in primary B cells. We’ve been able to run screens in primary T cells for some time and are expanding our capabilities into other immune cell types. Immuno-oncology is a critical field because if researchers can figure out how to leverage the human immune system to fight cancer, we can use this intrinsic system to perform a job that a drug cannot do or cannot carry out as well.
Running screens in primary T cells has applications in fields such as CAR T-cell therapy, which is an area of oncology with tremendous promise. Similarly, being able to run screens in B cells enables us to support researchers looking at how they can leverage those cell lineages to be responders to further employ the immune system for developing treatments.
We are also looking at other immune cell types—macrophages, for example. Our goal here is that we want to enable researchers who use our services to be able to work in cell lines that are most clinically relevant for what they’re trying to do. This helps ensure they can advance their research faster and with more relevant data.
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