Histone deacetylase inhibitors (iHDACs) have been extensively studied as enhancers of therapeutic protein production in recombinant Chinese hamster ovary (CHO) (rCHO) cell cultures. However, the addition of iHDACs reduces the viable cell concentration (VCC) in rCHO cell cultures, thereby reducing their potential to enhance therapeutic protein production. To mitigate the negative effects of iHDACs on VCC, screening using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based single-gene knockout (KO) library in rCHO cells was performed in the presence of CI994, a member of iHDACs, and 10 potential KO genes that enhanced the VCC of CI994-treated rCHO cells were identified. Among these, Bcor was validated as a promising KO target that improved VCC without negatively affecting the specific productivity in the presence of CI994. Bcor KO increased the VCC and therapeutic protein concentrations in both batch and fed-batch cultures in the presence of CI994. Taken together, these findings highlight the potential of the whole-genome CRISPR/Cas9-based single-gene KO cell library to identify KO target genes for the development of iHDAC-resistant rCHO cells for enhanced therapeutic protein production.,Histone deacetylase inhibitors, such as CI994, enhance therapeutic protein production, but hinder cell proliferation in recombinant CHO cell cultures. Here, knockout target genes conferring CI994 resistance were identified via virus-free genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 knockout cell library. These target genes were subsequently validated through knockout experiments.image,