Background: 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 offtarget, 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-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. Conclusions: 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.
In this study, construction of conductive and biocompatible three-dimensional nickel scaffolds (NiF) with electrodeposited chitosan (CS) for tissue engineering. The scaffolds were characterized by scanning electron microscopy (SEM), mechanical testing, water absorption, retention capacity and conductive sensitivity. Three-dimensional nickel scaffolds with electrodeposited chitosan (NiFC-n) exhibited uniformly filling structure on their surfaces and the inner structure and good mechanical property. When the versatile NiFC-n sensors were attached to different deformation, they could detect a variety of motion signals. MTT assay, Cells were stained with carboxyfluoresceinsuccinimidyl ester (CFSE) assay, apoptosis experiment and cell culture experiment results showed that NiFCn had good biocompatibility. The results indicated that the NiFC2 had a low immunogenicity, and can promote cell proliferation and support cell adhesion. This work provides a safe and feasible electrodeposition method to construct conductive and biocompatible three-dimensional nickel scaffolds with electrodeposited chitosan for tissue engineering. Therefore, NiFCn had potential application as biomaterials that may contact with real time measurement of rehabilitation for tissue engineering.
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