High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA
Soyoung Oh, Kate Senger, Shravan Madireddi, Ilseyar Akhmetzyanova, Isabel E. Ishizuka, Somayeh S. Tarighat, Jerry H. Lo, David Shaw, Benjamin Haley, Sascha Rutz
Abstract
Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, has the potential to uncover unique insights into their functions and enable the development of next-generation adoptive T cell therapies. Viral gene delivery into T cells, which is currently used to generate CAR T cells, has limitations in regard to targeting precision, cargo flexibility, and reagent production. Nonviral methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, but complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a convenient and scalable nonviral method that allows precise gene edits and transgene integration in primary human T cells, using plasmid donor DNA template and Cas9-RNP. This method is highly efficient for single and multiplex gene manipulation, without compromising T cell function, and is thus valuable for use in basic and translational research.