使用 CRISPR-Cas9 核糖核蛋白高效生成同基因原代人髓系细胞。
Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins.
机构信息
Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
出版信息
Cell Rep. 2021 May 11;35(6):109105. doi: 10.1016/j.celrep.2021.109105.
Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14 human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.
利用 CRISPR-Cas9 对原代人类细胞进行基因组工程改造，彻底改变了细胞生物学的实验和治疗方法，但人类髓系细胞在很大程度上仍然难以进行基因编辑。我们提出了一种通过电穿孔将 CRISPR-Cas9 核糖核蛋白 (RNP) 复合物直接递送至从外周血中纯化的 CD14 人单核细胞中的方法，从而实现高精度基因敲除。这些细胞可以有效地分化为单核细胞来源的巨噬细胞或树突状细胞。该过程产生的基因编辑细胞保留了髓样分化和吞噬功能的转录物和蛋白质标记。限制因子 SAMHD1 的基因缺失使 HIV-1 感染增加了 50 多倍，证明了该系统在基因型-表型研究中的强大功能。该快速、灵活和可扩展的平台可用于免疫信号、炎症、癌症免疫学、宿主-病原体相互作用等方面的人类髓系细胞的基因研究，并可能促进髓系细胞疗法的发展。
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