Nucleofection-Based CRISPR/Cas Delivery in Human T Cells for Immunotherapy Applications.

Electroporation-based delivery of CRISPR/Cas systems has emerged as a powerful and versatile approach for gene editing in primary human T cells, enabling efficient, transient, and nonviral modification while minimizing genomic integration risks. This chapter focuses on the principles and practical implementation of electroporation (nucleofection) for the delivery of Cas9 ribonucleoprotein (RNP) complexes into human T cells, highlighting critical parameters that influence editing efficiency, cell viability, and scalability for research and clinical applications. We provide a comprehensive protocol for multiplex gene editing in primary human T cells using Cas9 RNP electroporation, including optimization of cell activation status, buffer composition, electroporation settings, and post-electroporation recovery. Particular emphasis is placed on strategies to achieve high-efficiency disruption of target loci such as TRAC and B2M, enabling the generation of edited T cell products with defined functional properties. As a representative application, we describe how this delivery platform can be integrated with chimeric antigen receptor (CAR) engineering to produce edited CAR-T cells, including universal "off-the-shelf" designs with reduced risks of graft-versus-host disease and immune rejection. Downstream evaluation methods, including multiparameter flow cytometry for assessing editing efficiency and immunophenotype, are also outlined.