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Updated: Sep 8, 2025
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy
Puneeth Guruprasad1,2,3,4, Ranjani Ramasubramanian1,2,3,4, Siena Nason2,3,4
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
Abstract:
Editing chimeric antigen receptor (CAR) T cells by using CRISPR-Cas9 has become a routine strategy to improve their antitumor function or safety profile. Xenograft tumor models in immunodeficient mice are often used to evaluate the function of CRISPR-edited human CAR T cells. These models, however, lack functional immune systems and thus fail to recapitulate barriers such as the immunosuppressive tumor microenvironment (TME) that CAR T cells will encounter in patients. Thus, genetically modifying mouse CAR T cells for use in immune-intact models is an attractive approach to explore the impact of a given gene deletion on CAR T cells within a natural TME. Here, we describe a protocol to perform CRISPR-Cas9 editing in primary mouse T cells, thereby enabling studies of gene-edited CAR T within the TME and in the presence of a functional immune system. This protocol is integrated into a standard mouse CAR T manufacturing workflow, a process that typically spans ~5-6 days. The first stage of this protocol involves isolating mouse T cells, electroporating them with a ribonucleoprotein complex and activating them by using magnetic bead stimulation. The second stage involves transducing the CAR gene and expanding these cells, and the third stage focuses on validating knockout efficiency and the functionality of gene-edited mouse CAR T cells. This procedure requires a proficiency in aseptic cell culture techniques and a basic understanding of T cell biology. We anticipate that efficient and reliable genetic modification of mouse T cells will have wide-ranging applications for cancer immunotherapies and related fields.
Insights
This study presents a CRISPR-Cas9 editing protocol for mouse T cells, enabling the evaluation of chimeric antigen receptor (CAR) T cells within a natural tumor microenvironment. This method facilitates research into cancer immunotherapies in immune-intact models.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Research
Background:
- CRISPR-Cas9 editing of chimeric antigen receptor (CAR) T cells is crucial for enhancing antitumor efficacy and safety.
- Current xenograft models using human CAR T cells in immunodeficient mice do not fully replicate the patient tumor microenvironment (TME).
- Evaluating CAR T cell function in a natural TME requires the use of gene-edited mouse CAR T cells in immune-intact models.
Purpose of the Study:
- To describe a protocol for CRISPR-Cas9 editing of primary mouse T cells.
- To enable studies of gene-edited CAR T cells within a functional immune system and natural TME.
- To facilitate research into cancer immunotherapies by providing a reliable method for mouse CAR T cell modification.
Main Methods:
- A 5-6 day protocol integrating CRISPR-Cas9 editing into standard mouse CAR T cell manufacturing.
- Isolation of mouse T cells, electroporation with ribonucleoprotein complexes, and magnetic bead stimulation.
- CAR gene transduction, cell expansion, knockout efficiency validation, and functional assessment of gene-edited mouse CAR T cells.
Main Results:
- Successful implementation of CRISPR-Cas9 editing in primary mouse T cells.
- Enabling the study of gene-edited CAR T cells within a natural tumor microenvironment and functional immune system.
- Validation of knockout efficiency and functionality of the modified CAR T cells.
Conclusions:
- The developed protocol allows for reliable genetic modification of mouse T cells using CRISPR-Cas9.
- This method supports the investigation of CAR T cell behavior and efficacy in more physiologically relevant immune-intact models.
- The protocol has broad applications for advancing cancer immunotherapies and related research fields.
