Generation of knock-in primary human T cells using Cas9 ribonucleoproteins
Generation of knock-in primary human T cells using Cas9 ribonucleoproteins
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DOI：
10.1073/pnas.1512503112
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发表时间：
2015-08-18
影响因子：
11.1
通讯作者：
Marson, Alexander
中科院分区：
文献类型：
--
作者：
Schumann, Kathrin;Lin, Steven;Marson, Alexander
T细胞基因组工程在癌症、艾滋病、原发性免疫缺陷和自身免疫性疾病的细胞疗法中具有巨大的应用前景，但人类T细胞的基因操作一直具有挑战性。需要改进的工具来高效地“敲除”基因以及“敲入”靶向基因组修饰，以调节T细胞功能并纠正与疾病相关的突变。CRISPR/Cas9技术促进了许多细胞类型的基因组工程，但在人类T细胞中其效率有限，且尚未证明可用于靶向核苷酸替换。在此我们报道了使用Cas9 - 单向导RNA核糖核蛋白（Cas9 RNPs）在人类CD4(+) T细胞中进行高效的基因组工程。Cas9 RNPs能够消除CXCR4（艾滋病病毒进入的辅助受体）。Cas9 RNP电穿孔导致多达约40%的细胞失去CXCR4的高水平细胞表面表达，并且编辑后的细胞可通过基于低CXCR4表达的分选进行富集。重要的是，Cas9 RNPs与同源定向修复模板寡核苷酸配对，在原代T细胞中产生了高频率的靶向基因组修饰。在CXCR4和PD - 1（PDCD1，一种T细胞耗竭的调节因子，是肿瘤免疫治疗的有效靶点）中实现了靶向核苷酸替换。对靶点的深度测序证实，Cas9 RNPs产生的敲入基因组修饰效率高达约20%，占总编辑事件的多达约三分之一。这些结果确立了Cas9 RNP技术在原代人类T细胞中多种实验和治疗性基因组工程应用的地位。
T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to similar to 40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to similar to 20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.