Scientists at Gladstone Institutes and UC San Francisco (UCSF) say they have co-opted the CRISPR-Cas9 system to forcibly activate genes—rather than edit them—in human immune cells. The method, known as CRISPRa, lets them discover genes that play a role in immune cell biology more thoroughly and rapidly than previously possible, according to the researchers.

“This is an exciting breakthrough that will accelerate immunotherapy research,” says Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology and senior author of the new study. “These CRISPRa experiments create a Rosetta Stone for understanding which genes are important for every function of immune cells. 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.”

The study (“CRISPR activation and interference screens decode stimulation responses in primary human T cells”), published in Science, reportedly is the first to successfully use CRISPRa at large scale in primary human cells, which are cells isolated directly from an individual person.

The team activated each gene in the genome in different cells, enabling them to test almost 20,000 genes in parallel. 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.

“Human T cell responses to antigen stimulation, including the production of cytokines, are critical for healthy immune function and can be dysregulated in autoimmunity, immunodeficiencies, and cancer. A systematic understanding of the regulators that orchestrate T cell activation with gain-of-function and loss-of-function gene perturbations would offer additional insights into disease pathways and further opportunities to engineer next-generation immunotherapies,” write the investigators.

Zachary Steinhart (left) and Ralf Schmidt (right), scientists at the Gladstone-UCSF Institute of Genomic Immunology led by Alex Marson, engineered a new CRISPR-based pipeline to test every gene in the genome and rapidly discover genes that can be “turned on” in immune cells to enhance their functions. [Michael Short/Gladstone Institutes]
“Although CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens are powerful tools for gain-of-function and loss-of-function studies in immortalized cell lines, deploying them at scale in primary cell types has been challenging. Here, we developed a CRISPRa and CRISPRi discovery platform in primary human T cells and performed genome-wide screens for functional regulators of cytokine production in response to stimulation.

“We optimized lentiviral methods to enable efficient and scalable delivery of the CRISPRa machinery into primary human T cells. This platform allowed us to perform genome-wide pooled CRISPRa screens to discover regulators of cytokine production. Pools of CRISPRa-perturbed cells were isolated by fluorescence-activated cell sorting into high and low bins based on levels of endogenous Interleukin-2 (IL-2) production in CD4+ T cells or interferon-γ (IFN-γ) production in CD8+ T cells. Hits included proximal T cell receptor (TCR) signaling pathway genes, indicating that overexpression of these components could overcome signaling “bottlenecks” and tune stimulation and cytokine production.

“Reciprocal genome-wide loss-of-function screens with CRISPRi detected hits with critical regulatory functions, including some missed by CRISPRa. By contrast, CRISPRa also identified hits that may not be required and in some cases were expressed at only low levels under the conditions of the screen. This was strongly exemplified by regulation of IFN-γ production by the nuclear factor κ B (NF-κB) signaling pathway, in which CRISPRi identified a required TCR–NF-κB signaling circuit (including MALT1 and BCL10). CRISPRa selectively detected a set of tumor necrosis factor superfamily receptors that also signal through NF-kB, including 4-1BB, CD27, CD40, and OX40.

“These receptors were not individually required for signaling in our experimental conditions but could promote IFN-γ when overexpressed. Thus, CRISPRa and CRISPRi complement each other for the comprehensive discovery of functional cytokine regulators.

“Arrayed CRISPRa perturbation validated the effects of key hits in CD4+ and CD8+ T cells. We also assessed how individual CRISPRa perturbations more broadly reprogram cytokine production beyond IL-2 and IFN-γ by measuring a panel of secreted cytokines and chemokines.

“Finally, we developed a platform for pooled CRISPRa perturbations coupled with single-cell RNA-sequencing (scRNA-seq) readout (CRISPRa Perturb-seq) in primary human T cells. We used CRISPRa Perturb-seq for deep molecular characterization of single-cell states caused by 70 genome-wide screen hits and controls to reveal how regulators of cytokine production both tune T cell activation and program cells into different stimulation-responsive states.

“Our study demonstrates a robust platform for large-scale pooled CRISPRa and CRISPRi in primary human T cells. Paired CRISPRa and CRISPRi screens enabled comprehensive functional mapping of gene networks that can modulate cytokine production. Follow-up of CRISPRa hits with arrayed phenotypic analyses and with pooled scRNA-seq approaches enabled precise functional characterization of key screen hits, revealing how key perturbations may tune T cells to therapeutically relevant states. Future CRISPRa and CRISPRi screens in primary cells could identify targets for improved next-generation cellular therapies.”

In recent years, Marson and his colleagues have used CRISPR’s targeted scissors to knock out genes from various types of human immune cells, including regulatory T cells and monocytes. Their results have begun to illuminate how immune cells can be engineered to be more effective against infections, inflammation, or cancer. But his team knew they were still missing part of the story.

“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, PhD, a postdoctoral scholar in the Marson Lab and co-first author of the new paper.

In particular, knocking out a gene does not tell you what would happen if you instead made the gene more active. So the researchers turned to CRISPRa, short for CRISPR activation. In CRISPRa, the Cas9 protein is altered so that it can no longer cut DNA. Rather, scientists can attach an activator—a molecular “on” switch—to Cas9, so that when it binds to a gene, it activates it. 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).

Mapping T cell genes

T cells 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. This messaging is achieved through the production of cytokines. Different types of T cells produce different repertoires of cytokines, and different cytokines or cytokine cocktails have different effects on the immune response.

Controlling T cell cytokines, Marson says, would offer new opportunities to reshape entire immune responses in a wide range of different disease contexts. But researchers have an incomplete understanding of exactly which genes control which cytokines.

In the new work, Marson, Steinhart, and co-first author Ralf Schmidt, MD, worked with their colleagues to adapt CRISPRa and CRISPRi to work at high efficiency in primary T cells—something never before done.

“This improved efficiency in delivering the CRISPRa or CRISPRi machinery into the cells was critical to enable genome-wide experiments and accelerate discoveries,” explains Marson.

Then, the research team used these approaches to activate or inactivate nearly 20,000 genes in human T cells isolated directly from multiple healthy volunteers. They screened the resulting cells for changes to cytokine production and homed in on hundreds of genes that serve as key cytokine regulators, including some never before identified in knock-out screens.

“Our work demonstrates the precision and scalability of this technology in human T cells,” notes Schmidt. “And we quickly learned the rules of which genes you could turn on to dial the levels of certain cytokines.”

Better T cell therapies

To treat some cancer types, clinicians are increasingly using CAR-T cell therapy, in which T cells are removed from a patient’s body, engineered in a lab to target cancer cells, and then reinfused. Boosting the ability of T cells to fight cancer—by altering their cytokine production, for instance—could make CAR-T cell therapy even more powerful.

T-Cells Work to Fight Cancer, Immunotherapy, CAR T-cell therapy, 3d renderin
Immunotherapy. CAR T-cells work to fight cancer. [Design Cells/Getty Images]
“Our new data give us this incredibly rich instruction manual for T cells,” says Marson. “Now we have a basic molecular language we can use to engineer a T cell to have very precise properties.”

Marson’s lab is now studying some of the individual genes identified in their screen, as well as working to further leverage CRISPRa and CRISPRi to discover genes that control other critical traits in human immune cells.

“Working with the Gladstone-UCSF Institute of Genomic Immunology, the Innovative Genomics Institute, and the UCSF Living Therapeutics Initiative, our team now hopes to use our new instruction manual to create synthetic gene programs that can be CRISPR-engineered into the next generation cellular immunotherapies to treat a wide-range of diseases,” says Marson.

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