National Primate Research Center, University of Wisconsin Graduate School, Madison, Wisconsin, USA.
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.
Mol Ther Nucleic Acids. 2015 Dec 15;4:e268. doi: 10.1038/mtna.2015.42.
The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.
趋化因子（C-C 基序）受体 5（CCR5）作为 HIV-1 的共受体，对于 CCR5 嗜性病毒的细胞感染是必不可少的。功能性受体的丧失可预防 HIV 感染。在这里，我们使用 CRISPR/Cas9 系统的单和双向导 RNA（gRNA）成功靶向 GFP 标记的人类诱导多能干细胞（iPSC）中的 CCR5。使用单 gRNA 进行 CRISPER/Cas9 介导的基因编辑后，有 12.5%的细胞集落显示 CCR5 编辑，其中 22.2%的细胞集落显示双等位基因编辑，这是通过 Surveyor 核酸酶测定和直接测序确定的。使用双 gRNA 可显著提高 CCR5 编辑的效率至 27%，双等位基因基因改变的频率为 41%。为确保细胞内基因编辑的均一性，我们使用单细胞分选来建立克隆 iPSC 系。具有纯合 CCR5 突变的单细胞衍生 iPSC 系显示出多能干细胞的典型特征，并能有效地分化为造血细胞，包括巨噬细胞。尽管来自野生型和 CCR5 编辑 iPSC 的巨噬细胞均支持 CXCR4 嗜性病毒的复制，但 CCR5 编辑 iPSC 的巨噬细胞对 CCR5 嗜性病毒的挑战具有独特的抗性。这项研究表明，应用 iPSC 技术研究 CCR5 在 HIV 感染中的作用以及产生 HIV 抗性细胞用于潜在的治疗应用是可行的。
