Naeimi Kararoudi Meisam, Dolatshad Hamid, Trikha Prashant, Hussain Syed-Rehan A, Elmas Ezgi, Foltz Jennifer A, Moseman Jena E, Thakkar Aarohi, Nakkula Robin J, Lamb Margaret, Chakravarti Nitin, McLaughlin K John, Lee Dean A
Center for Childhood Cancer and Blood Disease, Nationwide Children's Hospital.
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford.
J Vis Exp. 2018 Jun 14(136):58237. doi: 10.3791/58237.
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 TGFβ. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.
CRISPR/Cas9技术正在加速多种细胞类型中的基因组工程，但到目前为止，基因递送和稳定的基因修饰在原代自然杀伤（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进行转基因递送，由于与操作相关的大量NK细胞凋亡，导致基因工程化NK细胞的产量有限。我们在此描述一种使用Cas9核糖核蛋白复合物（Cas9/RNPs）对人原代和扩增的NK细胞进行基因组编辑的无DNA方法。该方法能够有效敲除NK细胞中的TGFBR2和HPRT1基因。逆转录聚合酶链反应（RT-PCR）数据显示基因表达水平显著降低，对一种代表性细胞产物的细胞毒性分析表明，经核糖核蛋白（RNP）修饰的NK细胞对转化生长因子β（TGFβ）的敏感性降低。基因修饰的细胞在电穿孔后可通过用表达mbIL21的辐照饲养细胞刺激进行扩增。
