Transcriptomic Analysis Showed Upregulation of Genes Associated with P53- and Pi3k/Akt Pathway in Primary CD4+ T Cells after CRISPR/Cas9-mediated Insertion of Replication-Competent HIV-1 into Three Target Sites

The major hurdle to cure an HIV-1 infection is the latent reservoir, which consists mainly of latently HIV-1 infected CD4+ T cells and macrophages. HIV-1 integration in primary CD4+ T cells has shown to be not random and the provirus can persist for decades in infected individuals despite receiving antiretroviral therapy. To study the fate of HIV-1 proviruses at specific recurrent integration genes (RIGs), we developed a model to integrate a full-length, replication-competent HIV-1 GFP reporter virus via the CRISPR/Cas9 system into two different RIGs, namely BACH2, NFATC3 and the genomic safe harbour AAVS1 in primary CD4+ T cells. Prior transfection primary CD4+ T cells were kept for one day in culture media containing IL-15, IL-7 and IL-2. After transfection, primary CD4+ T cells were kept in culture for 9 days, sorted and RNAseq was performed in CD4+ T cells expressing the reporter. We successfully integrated HIV-1 into the three genomic locations in the same transcriptional orientations relative to the gene. Integrated full-length proviruses expressed GFP and were replication-competent. RNAseq reviled that genes associated with P53- and Pi3k/Akt Pathway were predominantly upregulated. In summary, we successfully developed a novel method to study the fate of HIV-1 at selected genomic location in primary CD4+ T cells pertinent to HIV-1 infection, hence enabling further insights into HIV-1 latency.