High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

• Published in: The Journal of experimental medicine Vol. 219; no. 5
• Main Authors: Oh, Soyoung A, Senger, Kate, Madireddi, Shravan, Akhmetzyanova, Ilseyar, Ishizuka, Isabel E, Tarighat, Somayeh, Lo, Jerry H, Shaw, David, Haley, Benjamin, Rutz, Sascha
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 02.05.2022
• Subjects: CRISPR-Cas Systems - genetics; DNA - genetics; Gene Editing - methods; Humans; Plasmids - genetics; T-Lymphocytes; Technical Advances and Resources
• ISSN: 0022-1007; 1540-9538
• DOI: 10.1084/jem.20211530

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.