Abstract
Recent development in genome editing technologies has enabled site-directed deprivation of a nucleotide sequence in the chromosome in mammalian cells. Human immunodeficiency (HIV) infection causes integration of proviral DNA into the chromosome, which potentially leads to re-emergence of the virus, but conventional treatment cannot delete the proviral DNA sequence from the cells infected with HIV. In the present study, the transcription activator-like effector nucleases (TALENs) specific for the HIV p17 gene were constructed, and their activities to destroy the target sequence were evaluated. SSA assay showed a high activity of a pair of p17-specific TALENs. A human T lymphoid cell line, Jurkat, was infected with a lentivirus vector followed by transfection with the TALEN–HIV by electroporation. The target sequence was destructed in approximately 10–95% of the p17 polymerase chain reaction clones, and the efficiencies depended on the Jurkat–HIV clones. Because p17 plays essential roles for assembly and budding of HIV, and this gene has relatively low nucleotide sequence diversity, genome editing procedures targeting p17 may provide a therapeutic benefit for HIV infection.
Highlights
Human immunodeficiency virus type 1 (HIV-1) causes latent infection in CD4+ T cells and macrophages, in which HIV-1 provirus DNA is integrated into the chromosomes
Single-Strand Annealing (SSA) S and AS, and HIV p17 transcription activator-like effector nucleases (TALENs) No 2 SSA S and AS (Figure 2A), were annealed to form double strand oligonucleotides, which were subsequently inserted into pGL4-SSA reporter plasmid that had been digested by BsaI endonuclease
The HIV TALENs 1 and 2 were prepared to target HIV gag p17 gene (Figure 1) and the activities of the TALENs were assessed by the SSA assay
Summary
Human immunodeficiency virus type 1 (HIV-1) causes latent infection in CD4+ T cells and macrophages, in which HIV-1 provirus DNA is integrated into the chromosomes. Recent genome editing technologies including the zinc finger nuclease, transcription activatorlike effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas have made it possible to efficiently induce specific alteration or truncation of target nucleotide sequence in the genomic DNA of mammalian cells (Jinwei et al, 2015; Maeder and Gersbach, 2016; Mei et al, 2016). Such technologies may realize novel therapeutic procedures against various genetic diseases by replacing and modifying the genes responsible for the pathogenesis (Jang et al, 2016).
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