A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells

Victor Tieu; Elena Sotillo; Jeremy R Bjelajac; Crystal Chen; Meena Malipatlolla; Justin A Guerrero; Peng Xu; Patrick J Quinn; Chris Fisher; Dorota Klysz; Crystal L Mackall; Lei S Qi

doi:10.1016/j.cell.2024.01.035

A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells

Cell. 2024 Feb 29;187(5):1278-1295.e20. doi: 10.1016/j.cell.2024.01.035. Epub 2024 Feb 21.

Authors

Affiliations

¹ Department 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.
² Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA.
⁴ Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: cmackall@stanford.edu.
⁵ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Sarafan ChEM-H, Stanford University, Stanford, CA 94305, USA; Chan Zuckerberg Biohub, San Francisco, San Francisco, CA 94080, USA. Electronic address: stanley.qi@stanford.edu.

PMID: 38387457
PMCID: PMC10965243 (available on 2025-02-28)
DOI: 10.1016/j.cell.2024.01.035

Abstract

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.

Keywords: CAR T cells; CRISPR; Cas13; RNA targeting; T cell exhaustion; cancer immunotherapy; cell therapy; gene editing; metabolic engineering; synthetic biology.

MeSH terms

Gene Expression Profiling
Humans
Metabolic Engineering* / methods
RNA
T-Lymphocytes*
Transcriptome

Substances

RNA

Abstract

MeSH terms

Substances

Grants and funding