? CORE 1 The Viral Vector Core (Core 1) will provide customized lentiviral or retroviral vectors to all PPG projects. Lentiviral or retroviral vector particles are fundamental reagents necessary for the efficient genetic engineering of HTLV-1 infected cells. The vector particles will be used to overexpress or reduce expression of host, viral, or novel fusion genes. Reduced expression will be achieved by delivery of either shRNA or CRISPR/Cas9. The vectors for reduced expression will be matched with vectors for complementation with wild type and functional mutant genes with silent mutations to prevent shRNA or CRISPR recognition. Lentiviral vector particles will also provide fluorescent and/or selection genes. Homology directed repair induced by a CRISPR/Cas9 vector will modify the HTLV-1 genome sequence. Integrase defective lentiviral vectors will be used to quantify the off- target effects of all CRISPR/Cas9 vectors. The Core will work with individual PPG labs to develop CRISPR/Cas9 reagents and methodologies for modification of HTLV-1 infected cells. The Core will also develop a novel vector that limits Cas9 expression both spatially and temporally to transcriptionally active HTLV-1 infected cells and reduce potential off-target editing.
Specific aims of the core are:
Aim 1 to provide custom retroviral vectors, Aim 2 to design, validate, and optimize CRISPR/Cas9 vectors, Aim 3 to develop an inducible, self-limiting HTLV-1 CRISPR/Cas9 vector. The lentiviral or retroviral vector particles produced by the Core will service Projects 1-3 of this PPG.
? CORE 1 The Viral Vector Core will provide custom high quality lentiviral or retroviral vector particles to PPG labs for overexpression or reduced expression of host, viral, or novel function genes. Reduced expression vectors will deliver either shRNA or CRISPR/Cas9 to cells. Finally, a novel lentiviral CRISPR/Cas9 vector will be developed to limit Cas9 expression spatially and temporally.
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