Dramatic attention has been drawn to adenovirus as a vehicle for vaccine and cancer therapy, due to the capabilities of annihilating tumor cells and stimulating host immune response. However, few producer cells are perfect for adenoviral manufacture. Herein, a novel genome editing strategy by CRISPR-Cas9 technology and sgRNA library not only modulated the cellular gene expression profile systemically that enhanced subgroup B and C adenovirus yields by 3~7 folds, but also substituted naïve adenovirus sequence in HEK293 cells with a recombinant E1 transgene, drastically suppressing the production of clinically pathogenic replication-competent adenovirus. Moreover, directed evolution towards a serum-free gradient in bioreactor generated an adaptive clone MC09 with both safety and potency for scalable adenoviral manufacture in large-scale clinical applications. Molecularly, combined inhibition of carbon metabolism, apoptosis and lysosome pathways curtailed adenoviral yield in MC09 cells, as demonstrated by triple knockdown of H6PD, Lamin A/C and caspase-3. Concomitantly, overexpression of these molecules further boosted biosynthesis of adenoviral vectors, highlighting a cooperative effect among multiple pathways to amplify adenovirus production.