PNAS：CRISPR/Cas9改造T细胞

摘要 : 美国加州大学旧金山分校科研人员报告了一种基因组编辑策略，它能增强改造人类T细胞的效率。


美国加州大学旧金山分校科研人员报告了一种基因组编辑策略，它能增强改造人类T细胞的效率。尽管近来取得了使用CRISPR/Cas9工具的基因组编辑的进展，对人类T细胞基因组进行有效而专一的编辑仍然是一个挑战。使用由Cas9蛋白和实验室产生的单导向RNA分子组成的Cas9 核蛋白(Cas9 RNPs)，Alexander Marson 及其同事有效而专一地编辑了CXCR4的DNA序列，CXCR4为人类助手T细胞表面的一个免疫受体编码，该受体让艾滋病病毒成为可能。他们使用一种瞄准被编辑的蛋白质的细胞分类技术分离出了这种修改后的细胞，并且使用深度测序证实了这类编辑。此外，这组作者把Cas9 核蛋白(Cas9 RNPs)与一个称为同源导向修复模板的定制设计的单链DNA分子结合起来，从而选择性地取代了从健康捐献者分离出来的人类T细胞CXCR4 的核苷酸。使用一种类似的方法，这组作者证明了PD1 的靶向核苷酸取代，PD1为人类T细胞的一个免疫检查点蛋白编码，它是癌症免疫疗法的一个成熟的靶标。由于短寿命的Cas9 核蛋白(Cas9 RNPs)通常在传送到细胞后的24小时后转变，在目标是改造人类原生T细胞从而应对免疫系统疾病的策略中，它们可能比让细胞长期暴露在Cas9种的方法更安全。

原文链接：

GENEration of knock-in primary human T cells using Cas9 ribonucleoproteins

原文摘要：

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 ∼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 ∼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.

doi: 10.1073/PNAS.1512503112

作者：Kathrin Schumann

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