Department of Biomedicine, Basel University Hospital and University of Basel, CH-4031 Basel, Switzerland; and Transplantation Immunology and Nephrology, Basel University Hospital, CH-4031 Basel, Switzerland.
Department of Biomedicine, Basel University Hospital and University of Basel, CH-4031 Basel, Switzerland; and Transplantation Immunology and Nephrology, Basel University Hospital, CH-4031 Basel, Switzerland
J Immunol. 2018 Apr 1;200(7):2489-2501. doi: 10.4049/jimmunol.1701121. Epub 2018 Feb 14.
Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in and repairing the clinically relevant Foxp3 mutation restored Foxp3 expression in primary T cells.
过继细胞转移是基础研究的重要方法，并且作为一种针对多种疾病（包括感染和血液癌症）的有效治疗手段而出现。对主要免疫细胞的直接遗传操作开辟了前所未有的研究机会，并可应用于增强细胞治疗产品。在本文中，我们报告了使用基于质粒的 RNA 指导的 CRISPR 系统在原代小鼠 T 细胞中进行高效基因组工程的方法。我们开发了一种简单的方法，可在多达 90%的细胞中消除基因，并在多达 25%的转染原代 T 细胞中精确靶向单核苷酸多态性。我们使用基因编辑介导的等位基因转换来定量同源定向修复，系统优化实验参数，并在原代 T 细胞中绘制天然 B 细胞表位。替代细胞表面标记物的等位基因转换可用于富集细胞，并可成功同时编辑第二个感兴趣的基因。最后，我们将该方法应用于纠正 基因中的两个致病突变。修复 X-连锁重症联合免疫缺陷病的致病原因，即 中的 2 个碱基插入，以及修复临床上相关的 Foxp3 突变，可恢复原代 T 细胞中的 Foxp3 表达。
