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Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA‐methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten‐eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones., Weinguny and coworkers demonstrate that overall DNA methylation patterns in Chinese hamster ovary (CHO) cells can effectively be randomized by a knock‐down of DNA methyltransferases (DNMT) using small interfering (si)RNAs. These DNA methylation changes increase the phenotypic diversity in a CHO cell population producing a recombinant protein. This newly generated diversity eventually facilitated the isolation of cells with increased production capacities and highlights the importance of epigenetic regulation and its impact on the phenotype of mammalian cell factories.

Graphical abstract By repeatedly selecting and pooling the most rapidly outgrowing clones during multiple single cell subcloning rounds, the time required for subcloning was reduced from 3 to 2 weeks and the cloning efficiency significantly increased. Transcriptome analyses of the resulting host cell lines revealed a diverse set of pathways differentially enriched in each host cell line treated, with the only shared DE pathway related to changes in extracellular matrix. This indicates that cells struggle with the lack of cell-to-cell communication in isolation during subcloning., Chinese Hamster Ovary (CHO) cells are the working horse of the pharmaceutical industry. To obtain high producing cell clones and to satisfy regulatory requirements single cell cloning is a necessary step in cell line development. However, it is also a tedious, labor intensive and expensive process. Here we show an easy way to enhance subclonability using subcloning by single cell sorting itself as the selection pressure, resulting in improved subcloning performance of three different host cell lines. These improvements in subclonability also lead to an enhanced cellular growth behavior during standard batch culture. RNA-seq was performed to shed light on the underlying mechanisms, showing that there is little overlap in differentially expressed genes or associated pathways between the cell lines, each finding their individual strategy for optimization. However, in all three cell lines pathways associated with the extracellular matrix were found to be enriched, indicating that cells struggle predominantly with their microenvironment and possibly lack of cell-to-cell contact. The observed small overlap may hint that there are multiple ways for a cell line to achieve a certain phenotype due to numerous genetic and subsequently metabolic redundancies.

Background:Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations.Methods:Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined.Results:Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents.Conclusions:The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues.Funding:Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).

Engineering of Chinese Hamster Ovary cells by manipulating microRNA (miRNA) expression levels has been shown to induce advantageous, desired phenotypes. Most of these studies so far were concerned with increasing productivity or reducing growth rate (with the implied intention of thus freeing cellular resources to also increase productivity). Here we evaluated the ability of growth correlating miRNAs to increase the growth rate of CHO-K1 cells by transient overexpression or knock down, respectively. Candidates were selected based on the correlation between growth rate and miRNA expression levels as observed in previous studies. These candidates were then up- or downregulated initially by transfection of mimics or inhibitors and subsequently by transfection of plasmids bearing the corresponding miRNAs or sponges. None of the 40 selected candidates was able to induce a better growth phenotype under these conditions. Overlap between miRNAs identified to correlate to growth in published miRNA expression studies and those identified to actively increase growth rate in a functional screen is minimal, indicating that the here selected approach of traditional overexpression/knock down engineering of miRNAs may not be a suitable strategy for the purpose of increasing growth rate.

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available. In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells. Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.

Chinese hamster ovary (CHO) cells are the most extensively used mammalian production system for biologics intended for use in humans. A critical step in the establishment of production cell lines is single cell cloning, with the objective of achieving high productivity and product quality. Despite general use, knowledge of the effects of this process is limited. Importantly, single cell cloned cells display a wide array of observed phenotypes, which so far was attributed to the instability and variability of the CHO genome. In this study we present data indicating that the emergence of diverse phenotypes during single cell cloning is associated with changes in DNA methylation patterns and transcriptomes that occur during the subcloning process. The DNA methylation pattern of each analyzed subclone, randomly picked from all outgrowing clones of the experiment, had unique changes preferentially found in regulatory regions of the genome such as enhancers, and de-enriched in actively transcribed sequences (not including the respective promoters), indicating that these changes resulted in adaptations of the relative gene expression pattern. The transcriptome of each subclone also had a significant number of individual changes. These results indicate that epigenetic regulation is a hidden, but important player in cell line development with a major role in the establishment of high performing clones with improved characteristics for bioprocessing.

Highlights • Development of a small-scale CRISPR/AsCpf1 screen in CHO. • Usage of paired gRNAs enables full deletion of coding or noncoding genomic regions. • Growth perturbing paired gRNAs identified. • Key points for considerations in future screens identified., Chinese hamster ovary (CHO) cells are the most widely used host for the expression of therapeutic proteins. Recently, significant progress has been made due to advances in genome sequence and annotation quality to unravel the black box CHO. Nevertheless, in many cases the link between genotype and phenotype in the context of suspension cultivated production cell lines is still not fully understood. While frameshift approaches targeting coding genes are frequently used, the non-coding regions of the genome have received less attention with respect to such functional annotation. Importantly, for non-coding regions frameshift knock-out strategies are not feasible. In this study, we developed a CRISPR-mediated screening approach that performs full deletions of genomic regions to enable the functional study of both the translated and untranslated genome. An in silico pipeline for the computational high-throughput design of paired guide RNAs (pgRNAs) directing CRISPR/AsCpf1 was established and used to generate a library tackling process-related genes and long non-coding RNAs. Next generation sequencing analysis of the plasmid library revealed a sufficient, but highly variable pgRNA composition. Recombinase-mediated cassette exchange was applied for pgRNA library integration rather than viral transduction to ensure single copy representation of pgRNAs per cell. After transient AsCpf1 expression, cells were cultivated over two sequential batches to identify pgRNAs which massively affected growth and survival. By comparing pgRNA abundance, depleted candidates were identified and individually validated to verify their effect., Graphical abstract Graphical workflow for a CRISPR/AsCpf1 paired gRNA (pgRNA) genomic deletion screen in Chinese hamster ovary (CHO) cells. pgRNA library ordered as a single-stranded oligonucleotide pool, followed by PCR amplification and cloning into the delivery plasmid backbone. Next, stably pgRNA expressing pre-screening cell pools were generated. By the application of the AsCpf1 endonuclease, genomic deletions were induced, and cell pools were screened for the desired phenotype. In parallel, pre-screening cell pools were treated with Cas9 resulting in no alteration of the genome. Comparison of pgRNA abundance between AsCpf1 and Cas9 treated samples after screening procedure identified phenotype modifying hits. Critical pooled CRISPR screening steps are marked by exclamation marks.Image, graphical abstract

Recent advances in omics technologies and the broad availability of big datasets have revolutionized our understanding of Chinese hamster ovary cells in their role as the most prevalent host for production of complex biopharmaceuticals. In consequence, our perception of this "workhorse of the biopharmaceutical industry" has successively shifted from that of a nicely working, but unknown recombinant protein producing black box to a biological system governed by multiple complex regulatory layers that might possibly be harnessed and manipulated at will. Despite the tremendous progress that has been made to characterize CHO cells on various omics levels, our understanding is still far from complete. The well-known inherent genetic plasticity of any immortalized and rapidly dividing cell line also characterizes CHO cells and can lead to problematic instability of recombinant protein production. While the high mutational frequency has been a focus of CHO cell research for decades, the impact of epigenetics and its role in differential gene expression has only recently been addressed. In this review we provide an overview about the current understanding of epigenetic regulation in CHO cells and discuss its significance for shaping the cell's phenotype. We also look into current state-of-the-art technology that can be applied to harness and manipulate the epigenetic network so as to nudge CHO cells towards a specific phenotype. Here, we revise current strategies on site-directed integration and random as well as targeted epigenome modifications. Finally, we address open questions that need to be investigated to exploit the full repertoire of fine-tuned control of multiplexed gene expression using epigenetic and systems biology tools.

This study was conducted to examine putative correlations between weather parameters during April-September and the amounts of nutrients, minerals and bioactive compounds in the juices of 16 apple varieties from four harvest years in Lower Austria. For most sugar-parameters, negative correlations were found with the total precipitation (r between -0.42 and -0.64). Conversely, positive correlations were observed with the mean air temperature (r between 0.32 and 0.66), the global radiation (r between 0.32 and 0.61) and the number of tropical days (r between 0.39 and 0.51). The sum of 14 polyphenols (HPLC quantitation) was positively correlated with the mean air temperature and global radiation (rs 0.44 and 0.42). Negative correlations were observed between the global radiation and potassium, magnesium and calcium contents (correlation coefficients -0.49, -0.68 and -0.69). We conclude that increased temperatures and global radiation can be correlated with enhanced sugar synthesis and polyphenol formation.

High-throughput siRNA screens were only recently applied to cell factories to identify novel engineering targets which are able to boost cells towards desired phenotypes. While siRNA libraries exist for model organisms such as mice, no CHO-specific library is publicly available, hindering the application of this technique to CHO cells. The optimization of these cells is of special interest, as they are the main host for the production of therapeutic proteins. Here, we performed a cross-species approach by applying a mouse whole-genome siRNA library to CHO cells, optimized the protocol for suspension cultured cells, as this is the industrial practice for CHO cells, and developed an in silico method to identify functioning siRNAs, which also revealed the limitations of using cross-species libraries. With this method, we were able to identify several genes that, upon knockdown, enhanced the total productivity in the primary screen. A second screen validated two of these genes, Rad21 and Chd4, whose knockdown was tested in additional CHO cell lines, confirming the induced high productivity phenotype, but also demonstrating the cell line/clone specificity of engineering effects.

Obtaining highly productive Chinese hamster ovary (CHO)-cell clones for the production of therapeutic proteins relies on multiple time-consuming selection steps. Several CHO-cell strains with high degrees of genomic and epigenetic variation are available. Each harbor potential advantages and disadvantages for any given product, particularly those considered difficult to express. A simple test system to quickly assess compatibility of cell line and product may therefore prove useful. Transient plasmid transfection falls short of the specific productivities of stable producer cells, making it unsuitable for the elucidation of high specific productivity bottlenecks. The aim of the study is to reach specific productivities approaching those of industrial production cell lines by transfection of in vitro transcribed mRNA. The system is characterized with respect to transfection efficacy (by quantitative PCR) and protein production (by flow cytometry and biolayer interferometry). Fluorescence of intracellular eGFP saturates at higher amounts of mRNA per cell, while the amount of secreted and intracellular EPO-Fc remain linearly correlated to the amount of mRNA taken up. Nevertheless, MS shows a severe reduction in N-glycosylation quality. This method allows for rapid elucidation of bottlenecks that would otherwise remain undetected until later during cell line development, giving insight into suitable strategies for preemptive targeted metabolic engineering and host cell line optimization.

Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed ‘regulation elements (RgE)’, in the 5′-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5′-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5′-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

The existence of dynamic cellular phenotypes in changing environmental conditions is of major interest for cell biologists who aim to understand the mechanism and sequence of regulation of gene expression. In the context of therapeutic protein production by Chinese Hamster Ovary (CHO) cells, a detailed temporal understanding of cell-line behavior and control is necessary to achieve a more predictable and reliable process performance. Of particular interest are data on dynamic, temporally resolved transcriptional regulation of genes in response to altered substrate availability and culture conditions. In this study, the gene transcription dynamics throughout a 9-day batch culture of CHO cells was examined by analyzing histone modifications and gene expression profiles in regular 12- and 24-hr intervals, respectively. Three levels of regulation were observed: (a) the presence or absence of DNA methylation in the promoter region provides an ON/OFF switch; (b) a temporally resolved correlation is observed between the presence of active transcription- and promoter-specific histone marks and the expression level of the respective genes; and (c) a major mechanism of gene regulation is identified by interaction of coding genes with long non-coding RNA (lncRNA), as observed in the regulation of the expression level of both neighboring coding/lnc gene pairs and of gene pairs where the lncRNA is able to form RNA-DNA-DNA triplexes. Such triplex-forming regions were predominantly found in the promoter or enhancer region of the targeted coding gene. Significantly, the coding genes with the highest degree of variation in expression during the batch culture are characterized by a larger number of possible triplex-forming interactions with differentially expressed lncRNAs. This indicates a specific role of lncRNA-triplexes in enabling rapid and large changes in transcription. A more comprehensive understanding of these regulatory mechanisms will provide an opportunity for new tools to control cellular behavior and to engineer enhanced phenotypes. Funding: IHL received support from FWF, the Austrian Science Fund, within the Doctoral Program “BioTechnology of Proteins” W1224, and HD from the “eCHO Systems” ITN PhD program funded by the Marie Sklodowska-Curie grant no. 642663. Additional grants were received from the National Bioinformatics Institute (INB), PRB2-ISCIII (PT13/0001/0044 to JG and AEC) and MINECO (PTA2014-09515 to MD)

Galactosylation as part of N-glycan processing is conducted by a set of beta-1,4-galactosyltransferases (B4GALTs), with B4GALT1 as the dominant isoenzyme for this reaction. Nevertheless, the exact contributions of this key-player as well as of the other isoenzymes involved in N-glycosylation, B4GALT2, B4GALT3 and B4GALT4, have not been studied in-depth. To increase the understanding of the protein- and site-specific activities of individual galactosyltransferases in Chinese Hamster Ovary cells, a panel of triple deletion cell lines was generated that expressed only one isoform of B4GALT each. Two model proteins were selected for this study to cover a large spectrum of possible N-glycan structures: erythropoietin and deamine-oxidase. They were expressed as Fc-fusion constructs (EPO-Fc and Fc-DAO) and their N-glycan processing status was analyzed by site-specific mass spectrometry. The sole activity of B4GALT1 resulted in a decrease of 15-21 % of fully galactosylated structures for erythropoietin, emphasizing the involvement of other isoenzymes. Interestingly, the contributions of B4GALT2 and B4GALT3 differed for the two model proteins. Unexpectedly, removal of galactosyltransferases influenced the overall process of N-glycan maturation, with the result of a higher occurrence of poorly processed oligosaccharides. In the context of high productivity cell lines, which can push N-glycan maturation towards incomplete galactosylation, galactosyltransferases are potential targets to ensure stable product quality. In view of our results, specifically engineered "designer" cell lines may be required for different proteins.

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single-cell level are still lacking. To address this need, we apply O-propargyl-puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single-cell level. In conclusion, OPP-labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.

Chinese Hamster Ovary (CHO) cells are the preferred cell line for production of biopharmaceuticals. These cells are capable to grow without serum supplementation, but drastic changes in their phenotype occur during adaptation to protein-free growth, which typically include the change to a suspension phenotype with reduced growth rate. A possible approach to understand this transformation, with the intention to counteract the reduction in growth by targeted supplementation of protein-free media, is gene expression profiling. The increasing availability of genome-scale data for CHO now facilitates quests for a better understanding of metabolic pathways and gene networks. So far, systematic large-scale expression profiling in CHO cells by microarray was limited due to lack of publicly available array designs and limitations of alternative approaches. Based on the recent release of CHO and Chinese Hamster genome sequences, including an annotated RefSeq genome, we have constructed a publicly available microarray design for effective genome-scale expression profiling. The design employed microarray probes optimized for uniformity, sensitivity, and specificity, with probe properties computed using the latest thermodynamic models. We validated the platform in an analysis of gene expression changes in response to serum-free adaptation. The observed effects on the lipid metabolism as well as on nucleotide synthesis were used to successfully select media supplements that were able to increase growth rate.

As Chinese Hamster Ovary (CHO) cells are the cell line of choice for the production of human-like recombinant proteins, there is interest in genetic optimization of host cell lines to overcome certain limitations in their growth rate and protein secretion. At the same time, a detailed understanding of these processes could be used to advantage by identification of marker transcripts that characterize states of performance. In this context, microRNAs (miRNAs) that exhibit a robust correlation to the growth rate of CHO cells were determined by analyzing miRNA expression profiles in a comprehensive collection of 46 samples including CHO-K1, CHO-S and CHO-DUKXB11, which were adapted to various culture conditions, and analyzed in different growth stages using microarrays. By applying Spearman or Pearson correlation coefficient criteria of|0.6|, miRNAs with high correlation to the overall growth, or growth rates observed in exponential, serum-free, and serum-free exponential phase were identified. An overlap of twelve miRNAs common for all sample sets was revealed, with nine positively and three negatively correlating miRNAs. The here identified panel of miRNAs can help to understand growth regulation in CHO cells and contains putative engineering targets as well as biomarkers for cell lines with advantageous growth characteristics.

Manipulation of multiple genes to engineer Chinese Hamster Ovary (CHO) cells for better performance in production processes of biopharmaceuticals has recently become more and more popular. Yet, identification of useful genes and the unequivocally assessment of their effect alone and in combination(s) on the cellular phenotype is difficult due to high variation between subclones. Here, we present development and proof-of-concept of a novel engineering strategy using multiplexable activation of artificially repressed genes (MAARGE). This strategy will allow faster screening of overexpression of multiple genes in all possible combinations. MAARGE, in its here presented installment, comprises four different genes of interest that can all be stably integrated into the genome from one plasmid in a single transfection. Three of the genes are initially repressed by a repressor element (RE) that is integrated between promoter and translation start site. We show that an elongated 5'-UTR with an additional transcription termination (poly(A)) signal most efficiently represses protein expression. Distinct guide RNA (gRNA) targets flanking the REs for each gene then allow to specifically delete the RE by CRISPR/Cas9 and thus to activate the expression of the corresponding gene(s). We show that both individual and multiplexed activation of the genes of interest in a stably transfected CHO cell line is possible. Also, upon transfection of this stable cell line with all three gRNAs together, it was possible to isolate cells that express all potential gene combinations in a single experiment.

Despite great efforts to control and modify gene expression of Chinese Hamster Ovary (CHO) cells by conventional genetic engineering approaches, i.e. overexpression or knockdown/-out, subclonal variation, induced unknown regulatory effects as well as overexpression stress are still a major hurdle for efficient cell line engineering and for unequivocal characterization of gene function. The use of epigenetic modulators - key players in CHO clonal heterogeneity - has only been marginally addressed so far. Here, we present the application of an alternative engineering strategy in CHO cells by utilizing targeted epigenetic editing tools that enable the turning-on or -off of genes without altering the genomic sequence. The present, but silent beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) gene is activated by targeting the catalytic domain (CD) of Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) via deactivated Cas9 (dCas9) to its methylated promoter. Stable upregulation in up to 60% of transfected cells is achieved over a time span of more than 80 days. No difference in growth and recombinant protein productivity is observed between activated and control cultures. Re-silencing by targeted methylation via DNA methyltransferase (DNMT) 3A-CD resulted in an up to 5.4-fold reduction of ST6GAL1 mRNA expression in ST6GAL1 expressing cells. This proof-of-concept demonstrates the feasibility of using epigenetic editing tools to efficiently modulate gene expression and provide a promising complement to conventional genetic engineering in CHO cells.

MicroRNAs are small non‐coding RNAs that play a critical role in post‐transcriptional control of gene expression. Recent publications of genomic sequencing data from the Chinese Hamster (CGR) and Chinese hamster ovary (CHO) cells provide new tools for the discovery of novel miRNAs in this important production system. Version 20 of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences for CGR/CHO. We searched for evolutionary conserved miRNAs from miRBase v20 in recently published genomic data, derived from Chinese hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several hundred miRNA sequences in the genome. For several of these, the expression in CHO cells could be verified from multiple next‐generation sequencing experiments. In addition, several hundred unexpressed miRNAs are awaiting further confirmation by testing for their transcription in different Chinese hamster tissues. Biotechnol. Bioeng. 2015;112: 1488–1493. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.