title: Targeted Gene Deletion Using DNA-Free RNA-Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.
abstract: Chinese hamster ovary (CHO) cells are the preferred host for the production of a wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome engineering platform for increasing desirable phenotypes of CHO cells based upon the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9) nuclease-based genome editing. For commercial production of therapeutic proteins, CHO cells have been adapted for suspension culture in serum-free media, which is highly beneficial with respect to productivity and economics. To engineer CHO cells for rapid adaptation to a suspension culture, we exploited strand-specific RNA-seq to identify genes differentially expressed according to their adaptation trajectory in serum-free media. More than 180 million sequencing reads were generated and mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We identified significantly downregulated genes according to the adaptation trajectory and then verified their effects using the genome editing method. Growth-based screening and targeted amplicon sequencing revealed that the functional deletions of Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The availability of this strand-specific transcriptome sequencing and DNA-free RNA-guided Cas9 nuclease mediated genome editing facilitates the rational design of the CHO cell genome for efficient production of high quality therapeutic proteins.
authors: Department of Biological Sciences and KI for the BioCentury, KAIST , Daejeon 305-701, Republic of Korea.
authors: Namil
authors: N
authors: Lee
authors: Department of Biological Sciences and KI for the BioCentury, KAIST , Daejeon 305-701, Republic of Korea.
authors: JongOh
authors: J
authors: Shin
authors: Department of Biological Sciences and KI for the BioCentury, KAIST , Daejeon 305-701, Republic of Korea.
authors: Jin Hyoung
authors: JH
authors: Park
authors: Department of Biological Sciences and KI for the BioCentury, KAIST , Daejeon 305-701, Republic of Korea.
authors: Gyun Min
authors: GM
authors: Lee
authors: Department of Biological Sciences and KI for the BioCentury, KAIST , Daejeon 305-701, Republic of Korea.
authors: Suhyung
authors: S
authors: Cho
authors: Department of Biological Sciences and KI for the BioCentury, KAIST , Daejeon 305-701, Republic of Korea.|Intelligent Synthetic Biology Center, Daejeon 305-701, Republic of Korea.
authors: Byung-Kwan
authors: BK
authors: Cho
mesh_terms: D000818:Animals; D016466:CHO Cells; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D003412:Cricetulus; D017353:Gene Deletion; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D015202:Protein Engineering; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D017423:Sequence Analysis, RNA
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: Chinese hamster ovary (CHO) cells; DNA-free CRISPR/Cas9; genome-editing; high-throughput RNA-seq; suspension culture