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.
María Ortiz-Bueno, Alejandro Millán-López, Kornel Labun et al.· Methods in molecular biology· 0 citations
The Training School on the Impact of GE Delivery Tools on Target Cells was designed to provide an in-depth overview of delivery strategies for genome editing (GE) tools, a cornerstone for gene therapy and cellular engineering. The ability to efficiently and safely introduce genome editing reagents into diverse target cells remains one of the most critical challenges in advancing translational applications of CRISPR, base editing, and related technologies. This three-day program combined theoretical lectures from international experts with complementary laboratory sessions. The lectures covered delivery vectors and nanoparticles, genome editing platforms, and safety and immunological aspects, while the hands-on sessions focused on evaluating delivery efficiency and editing outcomes using different approaches. Participants had the opportunity to explore both conceptual frameworks and experimental techniques — from lipid nanoparticles and viral vectors to genome editing assessment in primary T cells. The interactive structure of the Training School fostered scientific exchange, critical thinking, and practical skill development.
Carla Fuster‐García, Manuel Rhiel, Duško Lainšček et al.· Zenodo (CERN European Organi...· 0 citations
In this course, we investigate into the complex landscape of gene editing within aclinical framework, focusing on the generation of a comprehensive map detailingcurrent delivery methodologies employed for in vivo gene editing across variousanimal models and clinical scenarios, with some insight for ex vivo studies. Thecourse includes a meticulous comparison of efficacy and specificity data, elucidatingthe diverse array of gene editing tools administered through different delivery methodswithin specific tissues and organs of interest
Yonglun Luo, Karim Benabdellah, Mariana Köber et al.· Zenodo (CERN European Organi...· 0 citations
The Training School on the Impact of GE Delivery Tools on Target Cells was designed to provide an in-depth overview of delivery strategies for genome editing (GE) tools, a cornerstone for gene therapy and cellular engineering. The ability to efficiently and safely introduce genome editing reagents into diverse target cells remains one of the most critical challenges in advancing translational applications of CRISPR, base editing, and related technologies. This three-day program combined theoretical lectures from international experts with complementary laboratory sessions. The lectures covered delivery vectors and nanoparticles, genome editing platforms, and safety and immunological aspects, while the hands-on sessions focused on evaluating delivery efficiency and editing outcomes using different approaches. Participants had the opportunity to explore both conceptual frameworks and experimental techniques — from lipid nanoparticles and viral vectors to genome editing assessment in primary T cells. The interactive structure of the Training School fostered scientific exchange, critical thinking, and practical skill development.
Carla Fuster‐García, Manuel Rhiel, Duško Lainšček et al.· Zenodo (CERN European Organi...· 0 citations
In this course, we investigate into the complex landscape of gene editing within aclinical framework, focusing on the generation of a comprehensive map detailingcurrent delivery methodologies employed for in vivo gene editing across variousanimal models and clinical scenarios, with some insight for ex vivo studies. Thecourse includes a meticulous comparison of efficacy and specificity data, elucidatingthe diverse array of gene editing tools administered through different delivery methodswithin specific tissues and organs of interest
Yonglun Luo, Karim Benabdellah, Mariana Köber et al.· Zenodo (CERN European Organi...· 0 citations
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