T cells, a type of white blood cell, are one of the key mediators of immunity in the human body they not only target invading pathogens, but also direct other immune cells to increase or decrease their responses to intruders or cancer cells. Alternatively, they can attach a repressor-an "off" switch-to Cas9 to turn genes off, achieving a result similar to a typical knockout approach (called CRISPRi for CRISPR interference). Rather, scientists can attach an activator-a molecular "on" switch-to Cas9, so that when it binds to a gene, it activates it.
In CRISPRa, the Cas9 protein is altered so that it can no longer cut DNA. So the researchers turned to CRISPRa, short for CRISPR activation. In particular, knocking out a gene does not tell you what would happen if you instead made the gene more active. "Knocking out genes is great for understanding the basics of how immune cells function, but a knock-out-only approach can miss pinpointing some really critical genes," says Zachary Steinhart, Ph.D., a postdoctoral scholar in the Marson Lab and co-first author of the new paper. But his team knew they were still missing part of the story. Their results have begun to illuminate how immune cells can be engineered to be more effective against infections, inflammation, or cancer. In recent years, Marson and his colleagues have used CRISPR's targeted scissors to selectively remove (or "knock out") genes from various types of human immune cells, including regulatory T cells and monocytes. The CRISPR-Cas9 genome-editing system typically relies on Cas9 proteins, often described as "molecular scissors," to cut DNA at desired locations along the genome. This allowed them to quickly learn the rules about which genes provide the most powerful levers to reprogram cell functions in ways that could eventually lead to more powerful immunotherapies. The scientists activated each gene in the genome in different cells, enabling them to test almost 20,000 genes in parallel. The study, published in the journal Science, is the first to successfully use CRISPRa at a large scale in primary human cells, which are cells isolated directly from a person.
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In turn, this will give us new insight into how to genetically alter immune cells so they can become treatments for cancer and autoimmune diseases." "These CRISPRa experiments create a Rosetta Stone for understanding which genes are important for every function of immune cells. "This is an exciting breakthrough that will accelerate immunotherapy research," says Alex Marson, MD, Ph.D., director of the Gladstone-UCSF Institute of Genomic Immunology and senior author of the new study.
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