Targeted Gene Deletion Using DNA Free RNA Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.
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.
ACS synthetic biology