Genome editing of CCR5 by AsCpf1 renders CD4T cells resistance to HIV-1 infection.

Background: The chemokine receptor CCR5 is one of the co-receptor of HIV-1 infection. People with homozygous deletion resist HIV-1 infection, which makes the 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.

Results: Here, we demonstrated that CRISPR/AsCpf1 could ablate the main co-receptor of HIV-1 infection- 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 CD4T 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 by CRISPR/AsCpf1 took no effects on cell proliferation and apoptosis.

Conclusions: Our study provides a basis for a possible application of -targeting gene editing by CRISPR/AsCpf1 with high specific sgRNAs against HIV-1 infection.