Plasmid-Based Donor Templates for Nonviral CRISPR/Cas9-Mediated Gene Knock-In in Human T Cells

Effective and precise gene editing of T lymphocytes is critical for advancing the understanding of T cell biology and the development of next-generation cellular therapies. Although methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a simple and efficient method for nonviral CRISPR/Cas9-based gene knock-in utilizing plasmid-based donor DNA templates. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Purification of human CD4⁺ or CD8⁺ T cells from blood Basic Protocol 2: Activation of purified CD4⁺ or CD8⁺ T cells using TransAct CD3/CD28 agonist-conjugated nanomatrix Basic Protocol 3: Preparation of Cas9/sgRNA RNPs Basic Protocol 4: Transfection of CAS9-RNP and knock-in template into human T cells Support Protocol 1: Purity check following magnetic T cell isolation Support Protocol 2: Dextramer staining of TCR-edited T cells Support Protocol 3: Functional characterization of TCR knock-in T cells Support Protocol 4: Detection of knock-in reporter activity in CRISPR/CAS9-edited T cells.