De novo assembly and annotation of the CHOZN® GS^−/− genome supports high‐throughput genome‐scale screening

Abstract

Chinese hamster ovary (CHO) cells have been used as the industry standard for the production of therapeutic monoclonal antibodies for several decades. Despite significant improvements in commercial‐scale production processes and media, the CHO cell has remained largely unchanged. Due to the cost and complexity of whole‐genome sequencing and gene‐editing it has been difficult to obtain the tools necessary to improve the CHO cell line. With the advent of next‐generation sequencing and the discovery of the CRISPR/Cas9 system it has become more cost effective to sequence and manipulate the CHO genome. Here, we provide a comprehensive de novo assembly and annotation of the CHO‐K1 based CHOZN® GS−/− genome. Using this platform, we designed, built, and confirmed the functionality of a whole genome CRISPR guide RNA library that will allow the bioprocessing community to design a more robust CHO cell line leading to the production of life saving medications in a more cost‐effective manner.

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene

“…To overcome this problem, we performed Oxford Nanopore Technology (ONT) long-read whole genome sequencing (WGS) using genomic DNA extracted from Pro-5, Lec5 and Lec9 CHO cells and performed de novo genome assembly for each cell line. This led to full-length assemblies that were of similar quality to those previously published (26) ( Table 1 , Table S2 ). In Pro-5 cells, DHRSX was detected towards the end of a contig of 385 kbp (contig 8156; Fig.…”

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells

“…This growing demand requires that either the biomanufacturing community invest significant financial resources into increasing production capacity of manufacturing plants, or that the scientific community develop new technologies capable of increasing therapeutic protein production. While CHO cells are the cell line of choice in producing therapeutic proteins, they have remained largely unchanged since the genetic engineering of glutamine synthetase as a selection system [18,27]. Several high-throughput screens have been performed using small molecule libraries to enhance the performance of CHO cells; however, process economics often precludes incorporating these small molecules into commercial-scale therapeutic protein production settings [3,8,9].…”

“…The performance of all genes was assessed with plots of the residual standard deviation of every gene to their average log-count with a robustly fitted trend line of the residuals. Differential expression analysis was then performed using a published CHO genome assembly [18] consisting of 20,414 annotated genes, of which 13,323 had ≥2 mapped reads in this analysis, to analyze for differences between conditions, and the results were filtered for only those genes with Benjamini-Hochberg false discovery rate-adjusted p-values less than or equal to 0.05. Reactome pathway analysis was performed via the CHOmics web-based platform [19].…”

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

“…Two proprietary CHO cell lines (Sigma-Aldrich) were used in this study: an IgG 1 producer derived from CHOZN®GS -/- 34 host and the non-producing host cell line CHOZN®GS -/- 34 . The glutamate synthase knockout host cell line (CHOZN®GS -/- ) is the parental cell line for the IgG 1 producer.…”