Genome editing of CCR5 by AsCpf1 renders CD4+T cells resistance to HIV-1 infection
Zhepeng Liu, Jin Liang, Shuliang Chen, Kewu Wang, Xianhao Liu, Beibei Liu, Yang Xia, Mingxiong Guo, Xiaoshi Zhang, Guihong Sun, Geng Tian
Abstract
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.