Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) and human CD3⁺ T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.

中文翻译：

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使用 CRISPRa 和 CRISPRi 对原代细胞中的转录进行靶向调控


CRISPR-Cas9 系统 (CRISPRa/CRISPRi) 使用核酸酶失活的 Cas9 与转录效应分子融合，可以诱导靶向转录激活或干扰。这些技术已用于癌细胞系，特别是使用慢病毒载体进行全基因组功能遗传筛选。然而，由于缺乏有效且无毒的递送方式，CRISPRa和CRISPRi尚未广泛应用于具有治疗相关性的离体培养原代细胞。在这里，我们开发了基于 RNA 或核糖核蛋白 (RNP) 电穿孔递送的 CRISPRa 和 CRISPRi 平台，并显示了原代细胞中瞬时的、可编程的基因调控，包括人 CD34 ⁺造血干细胞和祖细胞 (HSPC) 和人 CD3 ⁺ T 细胞。我们展示了使用来自不同细菌物种的多个 sgRNA 和 CRISPR 系统进行多重和正交基因调节，并且我们表明 CRISPRa 可用于操纵 HSPC 的分化轨迹。这些平台构成了瞬时控制转录的简单而有效的方法，并且很容易被合成的 sgRNA 采用和重编程为新的靶基因。我们相信这些技术将广泛应用于转录组工程以研究干细胞生物学和基因功能，并且我们预计它们将被用于开发和增强细胞疗法。