Chromosome‐scale scaffolds for the Chinese hamster reference genome assembly to facilitate the study of the CHO epigenome

Hilliard, William; MacDonald, Madolyn L.; Lee, Kelvin H.

doi:10.1002/bit.27432

Cited by 35 publications

(40 citation statements)

References 39 publications

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“…further exemplifies to the CHO community the power of Hi-C mediated genome scaffolding, are consistent with those published for the recent PICRH Chinese hamster assembly(Hilliard et al, 2020) and in line with what would be expected given the haploid chromosome number of the CHOK1SV 10E9 cell line. The ≈10% of sequence not included within the final chromosome-scale assembly remain pertinent for continued improvements to the assembly.…”

supporting

confidence: 87%

“…Similar levels of alignment consistency were also witnessed in comparison to the Chinese Hamster PICR reference assembly(Rupp et al, 2018; data not shown). Finally, in terms of both the overall cis/trans ratio and the distribution of close cis and far cisinteractions, data aligned to the CHOK1SV 10E9 LACHESIS assembly was comparable to high-quality human and mouse Hi-C datasets(Nagano et al, 2015;Figure 2c).The integration of data, such as long-read PacBio sequencing, along with a comparative analysis to the PICRH Chinese hamster assembly(Hilliard et al, 2020), should help refine the CHOK1SV 10E9 cell line assembly further. Importantly, however, our assembly can be used to mediate the hierarchical identification of higher order 3D chromatin structures within the CHOK1SV 10E9 cell line and could be used as a framework to aid all facets of CHO cell line genomics research where a chromosome-scale assembly is of paramount importance.…”

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confidence: 87%

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High‐resolution three‐dimensional chromatin profiling of the Chinese hamster ovary cell genome

Bevan

Schoenfelder

Young

et al. 2020

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cell lines are the pillars of a multibillion‐dollar biopharmaceutical industry producing recombinant therapeutic proteins. The effects of local chromatin organization and epigenetic repression within these cell lines result in unpredictable and unstable transgene expression following random integration. Limited knowledge of the CHO genome and its higher order chromatin organization has thus far impeded functional genomics approaches required to tackle these issues. Here, we present an integrative three‐dimensional (3D) map of genome organization within the CHOK1SV® 10E9 cell line in conjunction with an improved, less fragmented CHOK1SV 10E9 genome assembly. Using our high‐resolution chromatin conformation datasets, we have assigned ≈90% of sequence to a chromosome‐scale genome assembly. Our genome‐wide 3D map identifies higher order chromatin structures such as topologically associated domains, incorporates our chromatin accessibility data to enhance the identification of active cis‐regulatory elements, and importantly links these cis‐regulatory elements to target promoters in a 3D promoter interactome. We demonstrate the power of our improved functional annotation by evaluating the 3D landscape of a transgene integration site and two phenotypically different cell lines. Our work opens up further novel genome engineering targets, has the potential to inform vital improvements for industrial biotherapeutic production, and represents a significant advancement for CHO cell line development.

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supporting

confidence: 87%

mentioning

confidence: 87%

High‐resolution three‐dimensional chromatin profiling of the Chinese hamster ovary cell genome

Bevan

Schoenfelder

Young

et al. 2020

Biotech & Bioengineering

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“…FDR less than 0.05 and absolute logFC greater than one were used as cutoffs to classify genes as differentially expressed. After differential expression analysis, gene coordinates in the PICR assembly were converted to chromosomal coordinates using annotations lifted over from PICR to the PICRH assembly as described previously (Hilliard et al, 2020).…”

Section: Methodsmentioning

confidence: 99%

“…Reproducibility and whole chromosome similarity between the Hi-C interaction frequency matrices of host and producer cell lines was assessed by calculating the stratum-adjusted correlation coefficients (SCC) for Chinese hamster chromosomes 1 through 10 and X at 100-kb resolution using HiCRep (Yang et al, 2017). SCC was also used to score host and producer similarity to the Chinese hamster interaction frequency matrix described previously (Hilliard et al, 2020) at 1-Mb resolution. SCC was calculated for interaction frequency matrices that were generated using a version of the PICRH genome assembly with a fully intact chromosome 1 (concatemer of chr1_0 and chr1_1).…”

Section: Hi-c Reproducibility and Whole Chromosome Similarity Scoringmentioning

confidence: 99%

Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis

Hilliard

Lee

2020

Biotech & Bioengineering

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The Chinese hamster ovary (CHO) cell lines that are used to produce commercial quantities of therapeutic proteins commonly exhibit a decrease in productivity over time in culture, a phenomenon termed production instability. Random integration of the transgenes encoding the protein of interest into locations in the CHO genome that are vulnerable to genetic and epigenetic instability often causes production instability through copy number loss and silencing of expression. Several recent publications have shown that these cell line development challenges can be overcome by using site‐specific integration (SSI) technology to insert the transgenes at genomic loci, often called “hotspots,” that are transcriptionally permissive and have enhanced stability relative to the rest of the genome. However, extensive characterization of the CHO epigenome is needed to identify hotspots that maintain their desirable epigenetic properties in an industrial bioprocess environment and maximize transcription from a single integrated transgene copy. To this end, the epigenomes and transcriptomes of two distantly related cell lines, an industrially relevant monoclonal antibody‐producing cell line and its parental CHO‐K1 host, were characterized using high throughput chromosome conformation capture and RNAseq to analyze changes in the epigenome that occur during cell line development and associated changes in system‐wide gene expression. In total, 10.9% of the CHO genome contained transcriptionally permissive three‐dimensional chromatin structures with enhanced genetic and epigenetic stability relative to the rest of the genome. These safe harbor regions also showed good agreement with published CHO epigenome data, demonstrating that this method was suitable for finding genomic regions with epigenetic markers of active and stable gene expression. These regions significantly reduce the genomic search space when looking for CHO hotspots with widespread applicability and can guide future studies with the goal of maximizing the potential of SSI technology in industrial production CHO cell lines.

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“…Steady improvements in cell line development, media formulation, and bioprocessing now enable production yields exceeding 10 g/L from a fed-batch culture. Emerging resources, including the CHO and hamster genome sequence [3][4][5][6][7][8] and genome editing tools [9][10][11][12][13][14] now allow researchers to rely less on largely empirical, "trial-and-error" approaches to CHO cell line development, and move towards a more rational engineering approach, in pursuit of novel CHO lines with tailored, superior attributes [15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

Restoration of deficient DNA Repair Genes Mitigates Genome Instability and Increases Productivity of Chinese Hamster Ovary Cells

Spahn

Zhang

et al. 2021

Preprint

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Chinese Hamster Ovary (CHO) cells are the primary host used for manufacturing of therapeutic proteins. However, production instability of high-titer cell lines is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein titer. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide polymorphisms (SNPs) in DNA repair genes. Comparison with other mammalian cells confirmed DNA repair is compromised in CHO. Restoration of key DNA repair genes by SNP reversal or expression of intact cDNAs improved DNA repair and genome stability. Moreover, the restoration of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show for the first time that correction of key DNA repair genes yields considerable improvements in stability and protein expression in CHO, and provide new opportunities for cell line development and a more efficient and sustainable production of therapeutic proteins.

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Chromosome‐scale scaffolds for the Chinese hamster reference genome assembly to facilitate the study of the CHO epigenome

Cited by 35 publications

References 39 publications

High‐resolution three‐dimensional chromatin profiling of the Chinese hamster ovary cell genome

High‐resolution three‐dimensional chromatin profiling of the Chinese hamster ovary cell genome

Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis

Restoration of deficient DNA Repair Genes Mitigates Genome Instability and Increases Productivity of Chinese Hamster Ovary Cells

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