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Updated: Jul 2, 2025
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Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
216
A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary
Victor Tieu1, Elena Sotillo2, Jeremy R Bjelajac2
1Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
Cell
|February 22, 2024
Summary
Researchers developed multiplexed effector guide arrays (MEGA), a new CRISPR tool for T cell therapies. This platform precisely regulates T cell genes without altering DNA, improving CAR T cell function and anti-tumor activity.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- CRISPR-Cas9 genome editing faces limitations in safety, efficacy, and scope for T cell therapies.
- Developing advanced tools is crucial for enhancing T cell-based immunotherapies.
Purpose of the Study:
- To introduce multiplexed effector guide arrays (MEGA), a novel CRISPR-Cas13d-based platform for T cell transcriptome regulation.
- To demonstrate MEGA's capability for programmable, scalable, and reversible gene knockdown in primary human T cells.
- To explore MEGA's application in CAR T cell exhaustion, combinatorial screening, and metabolic pathway disruption.
Main Methods:
- Utilized CRISPR-Cas13d's RNA-targeting activity for gene knockdown without genomic DNA alteration.
- Developed multiplexed effector guide arrays (MEGA) for massively multiplexed gene regulation.
- Applied MEGA in a CAR T cell exhaustion model and for combinatorial CRISPR screening.
- Implemented druggable regulation of MEGA for controlled CAR activation.
- Disrupted immunoregulatory metabolic pathways using MEGA to enhance T cell fitness.
Main Results:
- Achieved quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells.
- Successfully suppressed inhibitory receptor upregulation in a CAR T cell exhaustion model.
- Identified paired regulators of T cell function via combinatorial CRISPR screening.
- Demonstrated receptor-independent control of CAR activation using druggable MEGA.
- Enhanced CAR T cell fitness and anti-tumor activity in vitro and in vivo through metabolic pathway disruption.
Conclusions:
- MEGA provides a versatile synthetic toolkit for precise T cell transcriptome regulation.
- This platform overcomes limitations of conventional CRISPR-Cas9, offering improved safety and efficacy.
- MEGA holds significant potential for advancing cancer immunotherapy and other T cell-based applications.
