citations

                    Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells.
                

Authors:

Seki A, Rutz S.

In:

Source: J Exp Med
Publication Date: (2018)
Issue: 215(3): 985-997

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Cells used in publication:

T cell, human peripheral blood unstim.: Species: human; Tissue Origin: blood
T cell, human stim.: Species: human; Tissue Origin: blood
T cell, mouse - BALB/c: Species: mouse; Tissue Origin: blood
T cell, mouse - C57BL/6: Species: mouse; Tissue Origin: blood
T cell, mouse, stim: Species: mouse; Tissue Origin: blood
CD4+, human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® 96-well Systems

Experiment

see reference-

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.

Abstract

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

Open in PubMed