CRISPR gene editing technology has become a powerful tool to knock out or change DNA sequences and study the resulting effects. Now, researchers at the Gladstone Institute and UCSF at the University of California, San Francisco have chosen the CRISPR cas9 system to forcibly activate genes in human immune cells rather than edit them. This method, called crispra, CRISPR activation, allows them to find genes that play a role in immune cell biology more thoroughly and faster than before.
This is an exciting breakthrough that will accelerate immunotherapy research. These crispra experiments have created a new application prospect to understand which genes play an important role in each function of immune cells. In turn, this will provide us with a new understanding of how to genetically modify immune cells to become a treatment for cancer and autoimmune diseases.
Published in the journal Science, this is the first time crispra has been successfully used on a large scale in primary human cells, which are directly isolated from humans.
The scientists activated each gene in different cell genomes and tested nearly 20000 genes in parallel. This allows them to quickly understand which genes provide the most powerful lever to reprogram the rules of cell function, which may eventually lead to more powerful immunotherapy.
CRISPR cas9 genome editing system usually relies on cas9 protein commonly described as "molecular scissors" to cut DNA at the required position of the genome.
Researchers typically use CRISPR targeted scissors to selectively remove or knock out genes from various types of human immune cells, including regulatory T cells and monocytes.
Gene knockout is very helpful to understand the basic knowledge of immune cell function, but knockout alone may miss the precise positioning of some really key genes.
In particular, knocking out a gene doesn't tell you what happens if you make it more active.
Therefore, the researchers turned to crispra. In crispra, the cas9 protein changes so that it can no longer cut DNA. Instead, scientists can connect an activator, a molecular "on" switch, to the cas9 protein so that when it binds to a gene, it activates it. Alternatively, they can connect a repressor, a "off" switch, to the cas9 protein to turn off the gene, thus achieving results similar to those achieved by a typical knockout method (called crispri for CRISPR interference).
T cell is a kind of white blood cell, which is one of the key mediators of human immunity; They not only target invading pathogens, but also guide other immune cells to increase or decrease their response to invaders or cancer cells. This information transmission is achieved by producing signal molecules called cytokines. Different types of T cells produce different cytokine libraries, and different cytokines or cytokine mixtures have different effects on immune response.
Controlling T cell cytokines will provide new opportunities to reshape the entire immune response in a variety of different disease environments. But researchers do not know exactly which genes control which cytokines.
This work enables Cris researchers to be more efficient than ever in the original and new generation of Cris.
This improved efficiency of delivering crispra or crispri machines into cells is crucial for genome-wide experiments and accelerated discovery.
The team then used these methods to activate or inactivate nearly 20000 genes in human T cells isolated directly from multiple healthy volunteers. They screened for changes in cytokine production in the resulting cells and focused on hundreds of genes that act as key cytokine regulators, including some that had never been found in knockout screening before.
These studies proved the accuracy and scalability of the technology in human T cells, and researchers soon understood the rules of which genes can be turned on to regulate certain cytokine levels.
In order to treat some cancer types, clinicians are increasingly using car-t cell therapy, in which T cells are removed from patients, engineered in the laboratory to target cancer cells, and then injected. Improving the anti-cancer ability of T cells, for example, by changing the production of cytokines, can make car-t cell therapy more powerful.
Crispra technology is the basic molecular language, which can be used to design T cells and make them have very precise characteristics.
Marson's lab is now studying some of the individual genes they have screened and trying to further use crispra and crispri to find genes that control other key features in human immune cells.
In collaboration with the Gladstone UCSF Institute of genomic immunology, the Institute of innovative genomics and the UCSF life therapy program, the research team now hopes to use the new guidance manual to create synthetic gene programs that can be engineered into the next generation of cellular immunotherapy through CRISPR to treat a variety of diseases.