CRISPR/Cas9 Mediated Deletion Of MiR-146a Enhances Antiviral Responses In HIV-1 Infected Cells

Objective:The human Immunodeficiency Virus type 1(HIV-1)causes persistent infection in humans and induces miR-146a expression in infected cells.miR-146a represses the innate immune response by inhibiting the expression of TRAF6 and IRAKI genes,thus negatively controlling the NF-?B-related cytokines and interferon stimulated genes.In this study,we utilized the CRISPR/Cas9 system to abrogate miR-146a in HIV-1 infected cells,and evaluated their antiviral status by measuring changes in the expression of cytokines,type ? IFNs and IFN stimulated genes,as well as T cell exhaustion markers.Methods:To test whether CRISPR/Cas9 targeting precursor miR-146a genomic DNA loci could abrogate miR-146a expression,the four gRNAs targeting the conserved sites of human precursor miR-146a genomic sequences were designed by CRISPR DESIGN.Then we extracted genomic DNA and performed T7 endonuclease I assay,and the target region was amplified by PCR and cloned into pGEM-T vector for sequencing.And the expression of pri-miR-146,mature miR-146a,TRAF6,IRAK1,IL-1?,IL-6,IL-8,TNF-?,IFN-?,IFN-?,Gag,MxB,IFITM1 and Tetherin was measured by qPCR and Western Blot.ELISA was performed to determine the level of IL-1?,IL-6,IL-8,TNF-?,IFN-?,IFN-? and viral particles p24.Finally,Flow cytometry were uesd to evaluate the reactivation of HIV-1 provirus.Results:We reported that lentiviral CRISPR/Cas9 system was highly efficient in introducing mutations in the precursor miR-146a genomic sequences,resulting in a loss of miR-146a expression and function.miR-146a ablation led to increasing cytokines production in LPS-stimulated A549 cells.Moreover,miR-146a knockout in HIV-1 infected MT2 cells markedly increased the expression of cytokines and HIV-1 restriction factors and reversed T cell exhaustion markers,thus influencing HIV-1 replication.Finally,we further found the percentage of GFP-positive cells decreased to 22.36%as compared to control cells(44.93%),Our data showed that miR-146a knockout efficiently suppressed HIV-1 provirus reactivation.Conclusion:In conclusion,CRISPR/Cas9 mediated gene editing can ablate miR-146a expression in HIV-1 infected cells,thus breaking the miR-146a-cytokine signaling circuit,markedly increasing the expression of cytokines and ISGs but reversing the expression of T cell exhaustion markers,finally enhancing immune responses.The newly acquired antiviral state will suppress HIV-1 replication and provirus reactivation.These findings indicate that CRISPR/Cas9 mediated miR-146a gene editing may contribute a great potential for AIDS gene therapy.