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

IF 45.5 1区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Cell Pub Date : 2024-02-29 Epub Date: 2024-02-21 DOI:10.1016/j.cell.2024.01.035

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

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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.

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用于原代人类 T 细胞中多重转录组调控和代谢工程的多功能 CRISPR-Cas13d 平台。

CRISPR技术已开始彻底改变T细胞疗法；然而，传统的CRISPR-Cas9基因组编辑工具在安全性、有效性和范围上都受到了限制。为了应对这些挑战，我们开发了多重效应引导阵列（MEGA），这是一个利用 CRISPR-Cas13d 的 RNA 引导、RNA 靶向活性对 T 细胞转录组进行可编程、可扩展调控的平台。MEGA 能在原代人类 T 细胞中实现定量、可逆和大规模多重基因敲除，而无需靶向或切割基因组 DNA。我们将 MEGA 应用于 CAR T 细胞衰竭模型，通过组合式 CRISPR 筛选，有力地抑制了抑制性受体上调，并发现了 T 细胞功能的配对调节因子。此外，我们还对MEGA进行了药物调控，以不依赖受体的方式控制CAR的激活。最后，MEGA 还能多重破坏免疫调节代谢通路，从而提高 CAR T 细胞的体外和体内适应性和抗肿瘤活性。MEGA 为癌症免疫疗法及其他领域的应用提供了一个多功能合成工具包。

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来源期刊

Cell 生物-生化与分子生物学

CiteScore

110.00

自引率

0.80%

发文量

396

审稿时长

2 months

期刊介绍： Cells is an international, peer-reviewed, open access journal that focuses on cell biology, molecular biology, and biophysics. It is affiliated with several societies, including the Spanish Society for Biochemistry and Molecular Biology (SEBBM), Nordic Autophagy Society (NAS), Spanish Society of Hematology and Hemotherapy (SEHH), and Society for Regenerative Medicine (Russian Federation) (RPO). The journal publishes research findings of significant importance in various areas of experimental biology, such as cell biology, molecular biology, neuroscience, immunology, virology, microbiology, cancer, human genetics, systems biology, signaling, and disease mechanisms and therapeutics. The primary criterion for considering papers is whether the results contribute to significant conceptual advances or raise thought-provoking questions and hypotheses related to interesting and important biological inquiries. In addition to primary research articles presented in four formats, Cells also features review and opinion articles in its "leading edge" section, discussing recent research advancements and topics of interest to its wide readership.