Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA TemplatesMatthew J. Johnson 1,2,3, Anthony P. DeFeo 1,2,3, Nicholas J. Slipek 1,2,3, Timothy D. Folsom 1,2,3, Tom Henley 4, Modassir S. Choudhry 4, Beau R. Webber 1,2,3,5, Branden S. Moriarity 1,2,3,5
1Department of Pediatrics, University of Minnesota, 2Masonic Cancer Center, University of Minnesota, 3Center for Genome Engineering, University of Minnesota, 4Intima Bioscience, Inc., 5Stem Cell Institute, University of Minnesota
A detailed protocol is provided for using CRISPR/Cas9 technology to achieve highly efficient targeted knock-in of large, multicistronic constructs in primary human T cells via the homology-mediated end joining (HMEJ) DNA repair pathway. T cells engineered with this cGMP-adaptable protocol maintain excellent cell expansion, cytotoxicity, and cytokine production.
