A new paper published in the Journal of Immunology describes how researchers used a plasmid-based RNA-guided CRISPR system to successfully genetically modify primary murine T cells. The team developed a simple method to knockout genes in up to 90% of cells and to add targeted SNPs in up to 25% of the transfected primary T cells. This new method of directly engineering immune cells avoids the requirement of breeding mice which can be a time-consuming process.
The team used a plasmid-based approach for gene ablation in EL-4 cells, a mouse T cell line. Electroporation was used to transfer a commonly available plasmid expressing an sgRNA, Cas9, and GFP into the cells. The results showed that very high deletion efficiencies were achieved for CD90.2 and CD45.2 proteins. The team also used the same method to edit primary mouse CD4+ T cells and found that, similar to EL-4 cells, both surface proteins were lost in the transfected cells. The modified T cells survived once transplanted to the mouse and exhibited full functionality. They were able to multiply, migrate to immune system organs, and exhibited normal defensive behavior against an infection.
Another set of experiments showed that the researchers were able to efficiently introduce single nucleotide polymorphisms (SNPs) into T-cells which are small, precise mutations. The plasmid-based method was shown to be effective at repairing an altered FOXP3 gene, which is known to cause scurfy in mice.
"Our method allows targeted gene surgery in T-cells and opens up new perspectives for research into the immune system, and possibly for the development of new T-cell-based therapies," says Lukas Jeker, Professor of Experimental Transplantation Immunology and Nephrology at the University of Basel.
T-cell therapies have shown much promise as effective cancer therapies. The next steps for the researchers involved in this mouse T cell study is to modify their technique to work in human T cells.