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 and . MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.

中文翻译：

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


CRISPR 技术已经开始彻底改变 T 细胞疗法；然而，传统的 CRISPR-Cas9 基因组编辑工具的安全性、有效性和范围有限。为了应对这些挑战，我们开发了多重效应器引导阵列 (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和可扩展调节的平台。 MEGA 能够在原代人类 T 细胞中进行定量、可逆和大规模多重基因敲除，而无需靶向或切割基因组 DNA。通过将 MEGA 应用于 CAR T 细胞耗竭模型，我们有力地抑制了抑制性受体的上调，并通过组合 CRISPR 筛选发现了 T 细胞功能的配对调节因子。我们还实施了 MEGA 的药物调节，以不依赖于受体的方式控制 CAR 激活。最后，MEGA 能够多重破坏免疫调节代谢途径，以增强 CAR T 细胞适应性和抗肿瘤活性。 MEGA 提供了一个多功能合成工具包，适用于癌症免疫治疗及其他领域的应用。