The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics . As cell productivity directly affects the cost of production, methods have been developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity . However, there have been no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins . Here, we performed a large-scale, CHO cell-specific small interfering RNA screen to identify genes that consistently enhanced antibody production when silenced in a panel of seven CHO cell lines . Four genes, namely Cyp1a2, Atp5s, Dgki, and P3h2, were identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines . Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, resulted in a more than 90% increase in specific antibody productivity . Overall, the knockout of Cyp1a2 demonstrated the most significant improvement of antibody production, with a minimal impact on cell growth . This article is protected by copyright . All rights reserved.