CRISPR genome editing has become a powerful tool for knocking out or altering DNA sequences and studying the consequences. Now, in a new study, researchers from the Gladstone Institutes and the University of California, San Francisco used the CRISPR-Cas9 system to forcibly activate, rather than edit, genes in human immune cells. The method, called CRISPR activation (CRISPRa), allowed them to discover genes that play a role in immune cell biology more thoroughly and quickly than before. The research results were published in the journal of Science.
The authors of the paper claimed this is an exciting breakthrough that will accelerate immunotherapy research. These CRISPRa experiments provide solid clues for understanding which genes are important for each function of immune cells. This, in turn, will give researchers new insights into how immune cells can be genetically altered so that they can be therapeutics for cancer and autoimmune diseases.
This study is the first successful large-scale use of CRISPRa in primary human immune cells isolated directly from humans. The authors activated every gene in the genome in different immune cells, allowing them to test nearly 20,000 genes in parallel. This allowed them to quickly understand which rules provided the most powerful levers to reprogram cell function so that it could ultimately lead to more powerful immunotherapies.
CRISPR-Cas9 genome editing systems typically rely on Cas9 proteins called “molecular scissors” to cut DNA at ideal locations in the genome. In recent years, Marson and his colleagues have used CRISPR’s molecular scissors to selectively remove, or knock out, genes in many types of human immune cells, including regulatory T cells and monocytes. Their findings have begun to shed light on how immune cells can be genetically modified to fight infection, inflammation or cancer more effectively. Still, his team knew they were missing part of the story.
Knocking out genes is great for understanding the basic function of immune cells, but knockout-only approaches can miss some really critical genes. In particular, knocking out a gene doesn’t explain what happens when making that gene more active. In CRISPRa, the Cas9 protein is genetically engineered to no longer cut DNA. Instead, scientists could attach an activator — a molecular “switch” to Cas9, so that when it binds to a gene, it activates that gene. Alternatively, they could attach an inhibitor — an “off” switch — to Cas9 to turn off a gene, achieving results similar to a typical gene knockout approach called CRISPR interference.
As a type of immune cell, T cells are one of the key mediators of human immunity; they not only target invading pathogens, but also direct other immune cells to increase or decrease their response to invaders or cancer cells. This information transfer is accomplished through the production of signaling molecules called cytokines. Different types of T cells produce different combinations of cytokines, and different cytokines or combinations of cytokines have different effects on the immune response.
The researchers say that controlling cytokine production by T cells will provide new opportunities to reshape the entire immune response in a variety of different disease settings. But the understanding of exactly which genes control which cytokine production is incomplete. The authors then used these methods to activate or suppress nearly 20,000 genes in human T cells isolated directly from multiple healthy volunteers. They screened the resulting cells for changes in cytokine production and targeted hundreds of genes that function as key cytokine regulators, including some that had never been identified in knockout screens before.
To treat certain types of cancer, clinicians are increasingly using CAR-T cell therapy, in which T cells are taken from a patient, genetically modified in the lab to target cancer cells, and then These genetically engineered T cells were reinfused into the same patients. Boosting the cancer-fighting ability of T cells by altering their cytokine production could make CAR-T cell therapy even more powerful.
“Our new data provide us with an extremely rich guidebook for T cells,” said the researchers. “We now have a basic molecular language with which we can genetically engineer T cells to be very precise. characteristics.”
The Marson lab is now studying some of the genes they screened and working to further use CRISPRa and CRISPRi to discover genes that control other key traits in human immune cells.
“In collaboration with the Gladstone-UCSF Institute for Genomic Immunology, the Institute for Innovative Genomics, and the UCSF Living Therapeutics Program, our team now hopes to use our new instruction manual to build synthetic genes,” said Marson. programs that can be engineered through CRISPR genetic engineering into next-generation cellular immunotherapies to treat a range of diseases.”
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