Ma Linyuan, Jang Lydia, Chen Jian, Song Jun, Yang Dongshan, Zhang Jifeng, Chen Y Eugene, Xu Jie
Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine, University of Michigan Medical Center.
Celetrix LLC.
J Vis Exp. 2019 Jun 20(148). doi: 10.3791/59512.
Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.
以CRISPR相关蛋白9（Cas9）为代表的基因编辑核酸酶正成为生物医学研究中的主流工具。通过转染将CRISPR/Cas9元件成功递送至靶细胞是高效基因编辑的先决条件。本方案表明，管电穿孔（TE）机器介导的CRISPR/Cas9核糖核蛋白（RNP）与单链寡脱氧核苷酸（ssODN）供体模板一起递送至不同类型的哺乳动物细胞，可导致强大而精确的基因编辑事件。首先，应用TE将CRISPR/Cas9 RNP和ssODN递送至兔成纤维细胞中的白细胞介素2受体亚基γ（IL2RG）基因和蝶呤还原酶（SPR）基因，以诱导致病突变。通过细菌TA克隆测序确定，精确突变率达到3.57%-20%。然后，将相同策略应用于人类诱导多能干细胞中的几个临床相关基因，包括表皮生长因子受体（EGFR）、心肌肌球蛋白结合蛋白C（Mybpc3）和血红蛋白亚基β（HBB）。同样，通过深度测序（DeepSeq）确定，实现了高度精确的突变率（11.65%-37.92%）。目前的工作表明，CRISPR/Cas9 RNP的管电穿孔代表了一种用于哺乳动物细胞基因编辑的高效转染方案。
