Related Experiment Video
Updated: Jul 10, 2026
In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function
Published on: February 27, 2019
Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells
Carli Stewart1, Claudia Manriquez Roman2, Saad S Kenderian3
1T Cell Engineering, Mayo Clinic; Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic; Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic.
Abstract:
Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.
Insights
This study presents a novel genome-wide CRISPR knockout screening method for chimeric antigen receptor T (CART) cells. This approach helps optimize gene editing strategies to improve durable responses in cancer immunotherapy.
Area of Science:
- Immunotherapy
- Cancer Research
- Gene Editing
Background:
- Chimeric antigen receptor T (CART) cell therapy offers innovative cancer treatment but faces limitations in durable response.
- Epigenetic factors in preinfusion CART cells impact therapeutic efficacy, highlighting the need for optimized gene editing strategies.
- Genome-wide CRISPR screens are valuable for understanding resistance mechanisms and refining gene editing, but challenges exist for primary cells.
Purpose of the Study:
- To describe a method for performing genome-wide CRISPR knockout screens in primary CART cells.
- To establish a proof-of-concept model for investigating CART cell exhaustion using this screening method.
- To provide a versatile platform for exploring various resistance mechanisms in different CAR constructs and tumor models.
Main Methods:
- Development of a genome-wide CRISPR knockout screening protocol applicable to primary CART cells.
- Application of the method to investigate the development of exhaustion in CD19-targeting CART cells.
- Utilizing CRISPR technology to systematically knock out genes within CART cells to identify those affecting function.
Main Results:
- Successfully established and validated a method for genome-wide CRISPR screening in primary CART cells.
- Demonstrated the utility of the method in a model system to study CART cell exhaustion.
- The developed approach is adaptable for diverse CAR constructs and cancer targets.
Conclusions:
- The described genome-wide CRISPR screening method overcomes challenges in applying such screens to primary CART cells.
- This methodology provides a powerful tool for dissecting mechanisms of resistance and optimizing CART cell therapy.
- The approach holds significant potential for advancing the development of more effective and durable CART cell-based cancer treatments.
