Genome-Wide CRISPR/Cas9 Screening Unveils a Novel Target ATF7IP–SETDB1 Complex for Enhancing Difficult-to-Express Protein Production

• Published in: ACS synthetic biology Vol. 13; no. 2; pp. 634 - 647
• Main Authors: Kim, Su Hyun, Park, Jong-Ho, Shin, Sungwook, Shin, Seunghyeon, Chun, Dahyun, Kim, Yeon-Gu, Yoo, Jiseon, You, Weon-Kyoo, Lee, Jae Seong, Lee, Gyun Min
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.02.2024
• Subjects: CRISPR/Cas9 screen; difficult-to-express protein; histone methylation; FACS; Chinese hamster ovary cells
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.3c00646

With the emerging novel biotherapeutics that are typically difficult-to-express (DTE), improvement is required for high-yield production. To identify novel targets that can enhance DTE protein production, we performed genome-wide fluorescence-activated cell sorting (FACS)-based clustered regularly interspaced short palindromic repeats (CRISPR) knockout screening in bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cells. The screen identified the two highest-scoring genes, Atf7ip and Setdb1, which are the binding partners for H3K9me3-mediated transcriptional repression. The ATF7IP–SETDB1 complex knockout in bsAb-producing CHO cells suppressed cell growth but enhanced productivity by up to 2.7-fold. Decreased H3K9me3 levels and an increased transcriptional expression level of the transgene were also observed. Furthermore, perturbation of the ATF7IP–SETDB1 complex in monoclonal antibody (mAb)-producing CHO cells led to substantial improvements in mAb production, increasing the productivity by up to 3.9-fold without affecting the product quality. Taken together, the genome-wide FACS-based CRISPR screen identified promising targets associated with histone methylation, whose perturbation enhanced the productivity by unlocking the transgene expression.