Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins
Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins
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DOI：
10.3791/58237
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发表时间：
2018-06-01
影响因子：
1.2
通讯作者：
Lee, Dean A.
中科院分区：
文献类型：
--
作者：
Kararoudi, Meisam Naeimi;Dolatshad, Hamid;Lee, Dean A.
CRISPR/Cas9技术正在加速许多细胞类型的基因组工程，但到目前为止，基因递送和稳定的基因修饰在原代NK细胞中一直具有挑战性。例如，使用慢病毒或逆转录病毒转导的转基因递送由于大量程序相关的NK细胞凋亡而导致基因工程化NK细胞的产量有限。我们在此描述了使用Cas9核糖核蛋白复合物（Cas9/RNP）对人原代和扩增的NK细胞进行基因组编辑的无DNA方法。该方法允许有效敲除NK细胞中的TGFBR 2和HPRT 1基因。RT-PCR数据显示基因表达水平显著降低，代表性细胞产物的细胞毒性测定表明RNP修饰的NK细胞对TGF β变得不那么敏感。经遗传修饰的细胞可以在电穿孔后通过用经辐照的mbIL 21表达饲养细胞刺激来扩增。
CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF beta. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.