Related Experiment Video
Updated: Jul 2, 2025
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
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.
None:
CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
