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Abstract The Chinese hamster genome serves as a reference genome for the study of Chinese hamster ovary (CHO) cells, the preferred host system for biopharmaceutical production. Recent re‐sequencing of the Chinese hamster genome resulted in the RefSeq PICR meta‐assembly, a set of highly accurate scaffolds that filled over 95% of the gaps in previous assembly versions. However, these scaffolds did not reach chromosome‐scale due to the absence of long‐range scaffolding information during the meta‐assembly process. Here, long‐range scaffolding of the PICR Chinese hamster genome assembly was performed using high‐throughput chromosome conformation capture (Hi‐C). This process resulted in a new “PICRH” genome, where 97% of the genome is contained in 11 mega‐scaffolds corresponding to the Chinese hamster chromosomes (2n = 22) and the total number of scaffolds is reduced by three‐fold from 1,830 scaffolds in PICR to 647 in PICRH. Continuity was improved while preserving accuracy, leading to quality scores higher than recent builds of mouse chromosomes and comparable to human chromosomes. The PICRH genome assembly will be an indispensable tool for designing advanced genetic engineering strategies in CHO cells and enabling systematic examination of genomic and epigenomic instability through comparative analysis of CHO cell lines on a common set of chromosomal coordinates. 

Complete, accurate genome assemblies are necessary to design targets for genetic engineering strategies. Successful gene knockdowns and knockouts in Chinese hamster ovary (CHO) cells may prevent the expression of difficult‐to‐remove host cell proteins (HCPs). HCPs, if not removed, can cause problems in stability, safety, and efficacy of the biotherapeutic. A significantly improved Chinese hamster (CH) reference genome was used to identify new knockout targets with similar predicted functions and characteristics as the difficult‐to‐remove host cell lipases, LPL, PLBL2, and LPLA2. The CHO‐K1 gene and protein sequences of several of these lipases were corrected using the updated CH genome. Sequence alignments were then used to identify conserved regions that may serve as possible targets for multiple simultaneous gene knockouts. Finally, the comparison of the CHO‐K1 lipase protein sequences to their human orthologs provided insight into which lipases, if persistent in the drug product, could possibly cause immunogenic responses in patients. Topical heading: Biomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food. © 2018 American Institute of Chemical EngineersAIChE J, 64: 4247–4254, 2018