Their publication in the Journal of Experimental Medicine is an advanced optimization for gene knock out in primary human and mouse T cells. The final protocol achieved 85-98% gene knock out for several tested receptors. With a single transfection of RNP CRISPR/Cas9 complexes you can develop next generation chimeric antigen receptor (CAR) T cells for treatment of various cancers. You can also use this protocol for deletion of endogenous TCRs and HLA class I for off the shelf CAR T cells.
The group has tested and optimized many parameters, which are important for successful genome editing. Not only the selection of RNPs and CRISPR complexes, but also cell culture conditions and many different transfection conditions. Finally, they also titrated the best ratio of the different components for genetic modification of the cells.
Very important at this approach is that the protocol does work well for unstimulated T cells. For many viral approaches, you have to stimulate the cells for successful transduction with retro- or adenoviruses. In this non-viral approach, no cytokine cocktails are needed to stimulate the cells, after genome editing, the controls and modified cells show no impact on cell functionality. A great and very systematic article with excellent primary data and set up. I can only recommend to read this work from the research group in San Francisco.