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

Oh SA, Senger K, Madireddi S, Akhmetzyanova I, Ishizuka IE, Tarighat S, Lo JH, Shaw D, Haley B, Rutz S
Source: J Exp Med
Publication Date: (2022)
Issue: 219(5): e20211530
Research Area:
Immunotherapy / Hematology
Gene Expression
Basic Research
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® X-Unit

After 36–48 h of stimulation, T cells were pelleted, washed with PBS, and gently resuspended in P3 buffer with supplement (Lonza Bioscience) at 2 million cells per 20 µl. The following components of a single nucleofection reaction were added to a PCR tube and mixed gently: preformed RNPs (60 pmol total), HDR template (=8 µg), and T cells resuspended in P3 buffer. In some cases, poly-L-glutamic acid (Sigma-Aldrich; 150 µg) was also added to the mixture. This mixture was then transferred to 1 well of a 16-well 4D-Nucleofector cuvette (Lonza Bioscience) and pulsed with code EH-115 unless otherwise indicated. After electroporation, the 4D-Nucleofector cuvette was placed in a 37°C tissue culture incubator for 15 min to allow for cell recovery. After recovery, the cells were transferred to a 24-well tissue culture plate containing 2 ml of prewarmed PRIME-XV medium supplemented with 25 ng/ml IL-7 and 50 ng/ml IL-15 (CD8+ T cells) or 25 ng/ml IL-7, 50 ng/ml IL-15, and 400 U/ml IL-2 (CD4+ T cells).


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.