The CRISPR-Cas9 gene editing tool has been modified to allow the activation of genes in human immune cells and study the effect the genes have on immune system function.
CRISPR is a bacterial defense mechanism that is used by bacteria to protect against attacks by phages. The system cuts the genomes of the invaders and inserts segments into the bacteria’s own genome, which allows them to detect and attack if the phages are encountered in the future. The system has been harnessed by researchers and used as a tool to make edits to plant, animal, and human DNA, either to remove, replace, or edit genes. The Cas9 protein component of the system acts like a pair of molecular scissors to cut the DNA, with the exact position of the cut dictated by an accompanying guide RNA.
Now, researchers at UCSF Gladstone Institute have modified the CRISPR-Cas9 system to activate genes rather than edit them and have used their new tool – dubbed CRISPRa – at a large scale in primary human cells to activate genes and study their effects on human immune cell biology. Further, the new tool allows researchers to discover the roles genes play more thoroughly and much faster than other techniques.
“This is an exciting breakthrough that will accelerate immunotherapy research,” says Alex Marson, director of the Gladstone-UC San Francisco Institute of Genomic Immunology and senior author of the 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.”
With CRISPRa, instead of a Cas9 protein to make the cut, a new version was developed that does not have the ability to cut the DNA. In its place is an activator that can bind to a specific gene and switch it on. There is also a companion version, dubbed CRISPRi, which can be used to switch a gene off.
The researchers developed the tool to investigate the functions of genes on immune system T cells – a type of white blood cells that produce signaling molecules called cytokines that direct other immune system cells to target intruders such as viruses. T cells can also direct the immune system to fight cancer cells.
The new CRISPR tool allows researchers to identify the genes that are responsible for controlling cytokines, which could lead to new treatments for cancer, especially improved CAR-T cell therapy. CAR_T cell therapy involves harvesting T cells from a patient, programming them in the lab to fight cancer cells, and then reintroducing them. If genes could be activated to ramp up cytokine production, it could lead to a much more powerful and effective CAR-T cell therapy.
“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,” said Zachary Steinhart, Ph.D., a postdoctoral scholar and co-first author of the paper.
The researchers have already used CRISPRa and CRISPRi to activate and inactivate around 20,000 different genes in T cells donated by healthy volunteers to identify the genes that have the greatest influence on the production of cytokines. From the 20,000 genes, the researchers have identified a few hundred that serve as cytokine regulators. “Our new data give us this incredibly rich instruction manual for T cells,” said Marson. “Now we have a basic molecular language we can use to engineer a T cell to have very precise properties.”
You can read more about the study in the paper – CRISPR activation and interference screens decode stimulation responses in primary human T cells – which was recently published in Science. DOI: 10.1126/science.abj4008