Engineering of HEK293T cell factory for lentiviral production by high throughput selected genes

Lentiviral vectors (LVs) are essential tools in gene therapy and bioproduction. While there have been significant advancements in downstream production and purification technologies for LVs, there is an urgent need for high-yield lentiviral production systems to meet the growing demand. This study introduces a high-throughput approach based on CRISPR-Cas9 to identify genome-wide targets for engineering HEK293T cells for improved LV production. Out of 17,501 genes, nine genes were identified, namely LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6, which are involved in viral particle packaging and formation. Single and multiplex knockout experiments demonstrated significant improvements in LV production through the knockout of three genes: GBP3, NHLRC1, and NHLRC3. The triplex knockout cell pool showed a 2.18-fold increase in LV production compared to the control group, while the pure 3KO cell line exhibited a remarkable 6.5-fold increase in LV production without compromising normal cell growth. This study provides valuable insights into the mechanisms underlying LV production and presents methods for engineering HEK293T cell factories to achieve superior lentiviral production. The identified genes offer potential targets for genetic manipulation to enhance LV production yields, addressing the current demand for high-yield lentiviral production systems.