Department of Molecular Biology, Genentech, South San Francisco, CA, USA.
Department of Cancer Immunology, Genentech, South San Francisco, CA, USA.
Methods Mol Biol. 2023;2618:201-217. doi: 10.1007/978-1-0716-2938-3_15.
Genetically engineered myeloid cells such as monocytes, macrophages, and dendritic cells have broad applications in basic and translational research. Their central roles in innate and adaptive immunity make them attractive as putative therapeutic cell products. However, efficient gene editing of primary myeloid cells presents unique challenges owing to their sensitivity to foreign nucleic acids and poor editing efficiencies using current methodologies (Hornung et al., Science 314:994-997, 2006; Coch et al., PLoS One 8:e71057, 2013; Bartok and Hartmann, Immunity 53:54-77, 2020; Hartmann, Adv Immunol 133:121-169, 2017; Bobadilla et al., Gene Ther 20:514-520, 2013; Schlee and Hartmann, Nat Rev Immunol 16:566-580, 2016; Leyva et al., BMC Biotechnol 11:13, 2011). This chapter describes nonviral CRISPR-mediated gene knockout in primary human and murine monocytes as well as monocyte-derived or bone marrow-derived macrophages and dendritic cells. Electroporation-mediated delivery of recombinant Cas9 complexed with synthetic guide RNAs can be applied for population-level disruption of single or multiple gene targets.
基因工程髓系细胞，如单核细胞、巨噬细胞和树突状细胞，在基础和转化研究中有广泛的应用。它们在固有和适应性免疫中的核心作用使它们成为有吸引力的潜在治疗性细胞产品。然而，由于其对异源核酸的敏感性以及当前方法编辑效率低下，对原代髓系细胞进行有效的基因编辑提出了独特的挑战 (Hornung 等人，Science 314:994-997, 2006; Coch 等人，PLoS One 8:e71057, 2013; Bartok 和 Hartmann, Immunity 53:54-77, 2020; Hartmann, Adv Immunol 133:121-169, 2017; Bobadilla 等人，Gene Ther 20:514-520, 2013; Schlee 和 Hartmann, Nat Rev Immunol 16:566-580, 2016; Leyva 等人，BMC Biotechnol 11:13, 2011)。本章描述了非病毒 CRISPR 介导的原代人源和鼠源单核细胞以及单核细胞衍生或骨髓来源的巨噬细胞和树突状细胞中的基因敲除。重组 Cas9 复合物与合成向导 RNA 的电穿孔递送可用于对单个或多个基因靶标进行群体水平的破坏。
