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Genome editing of CCR5 by AsCpf1 renders CD4+T cells resistance to HIV-1 infection.
Cell and Bioscience ( IF 6.1 ) Pub Date : 2020-07-08 , DOI: 10.1186/s13578-020-00444-w
Zhepeng Liu 1, 2 , Jin Liang 2 , Shuliang Chen 3 , Kewu Wang 4 , Xianhao Liu 2 , Beibei Liu 2 , Yang Xia 2 , Mingxiong Guo 5 , Xiaoshi Zhang 1 , Guihong Sun 3 , Geng Tian 2
Affiliation  

The chemokine receptor CCR5 is one of the co-receptor of HIV-1 infection. People with homozygous CCR5Δ32 deletion resist HIV-1 infection, which makes the CCR5 an important target for HIV-1 gene therapy. Although the CRISPR/Cas9 has ever been used for HIV-1 study, the newly developed CRISPR/AsCpf1 has never been utilized in HIV-1 co-receptor disruption. The CRISPR/Cpf1 system shows many advantages over CRISPR/Cas9, such as lower off-target, small size of nuclease, easy sgRNA design for multiplex gene editing, etc. Therefore, the CRISPR/Cpf1 mediated gene editing will confer a more specific and safe strategy in HIV-1 co-receptor disruption. Here, we demonstrated that CRISPR/AsCpf1 could ablate the main co-receptor of HIV-1 infection-CCR5 efficiently with two screened sgRNAs via different delivery strategies (lentivirus, adenovirus). The edited cells resisted R5-tropic HIV-1 infection but not X4-tropic HIV-1 infection compared with the control group in different cell types of HIV-1 study (TZM.bl, SupT1-R5, Primary CD4+T cells). Meanwhile, the edited cells exhibited selective advantage over unedited cells while under the pressure of R5-tropic HIV-1. Furthermore, we clarified that the predicted off-target sites of selected sgRNAs were very limited, which is much less than regular using sgRNAs for CRISPR/Cas9, and no evident off-target was observed. We also showed that the disruption of CCR5 by CRISPR/AsCpf1 took no effects on cell proliferation and apoptosis. Our study provides a basis for a possible application of CCR5-targeting gene editing by CRISPR/AsCpf1 with high specific sgRNAs against HIV-1 infection.

中文翻译:

AsCpf1 对 CCR5 的基因组编辑使 CD4+T 细胞对 HIV-1 感染产生抵抗力。

趋化因子受体 CCR5 是 HIV-1 感染的共同受体之一。CCR5Δ32纯合缺失的人抵抗HIV-1感染,这使得CCR5成为HIV-1基因治疗的重要靶点。尽管 CRISPR/Cas9 曾用于 HIV-1 研究,但新开发的 CRISPR/AsCpf1 从未用于 HIV-1 共受体破坏。与 CRISPR/Cas9 相比,CRISPR/Cpf1 系统具有许多优势,例如脱靶率低、核酸酶体积小、易于 sgRNA 设计用于多重基因编辑等。因此,CRISPR/Cpf1 介导的基因编辑将赋予更特异和更HIV-1共受体破坏的安全策略。在这里,我们证明了 CRISPR/AsCpf1 可以通过不同的递送策略(慢病毒、腺病毒)与两种筛选的 sgRNA 有效地消融 HIV-1 感染的主要共同受体-CCR5。在 HIV-1 研究的不同细胞类型(TZM.bl、SupT1-R5、原代 CD4+T 细胞)中,与对照组相比,编辑的细胞抵抗 R5-tropic HIV-1 感染,但不抵抗 X4-tropic HIV-1 感染。同时,在 R5-tropic HIV-1 的压力下,经过编辑的细胞比未经编辑的细胞表现出选择性优势。此外,我们澄清了所选 sgRNA 的预测脱靶位点非常有限,这比常规使用 CRISPR/Cas9 的 sgRNA 少得多,并且没有观察到明显的脱靶。我们还表明,CRISPR/AsCpf1 对 CCR5 的破坏对细胞增殖和凋亡没有影响。我们的研究为通过 CRISPR/AsCpf1 与针对 HIV-1 感染的高特异性 sgRNA 进行 CCR5 靶向基因编辑的可能应用提供了基础。
更新日期:2020-07-24
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