Accurate and complete genome sequences are essential in biotechnology to facilitate genome‐based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, ...are fragmented and replete with gap sequences and misassemblies, consistent with most short‐read‐based assemblies. Here, we completely resequenced C. griseus using single molecule real time sequencing and merged this with Illumina‐based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28‐fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up‐ and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important Chinese hamster ovary cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.
The authors completely resequenced the Chinese hamster genome using Single Molecule Real Time (SMRT) sequencing and merged the resulting SMRT‐based assembly with the publicly available draft Illumina‐based assemblies. The new assembly is more contiguous and complete than genome assemblies from either technology alone, reducing the number of scaffolds by >28‐fold, with 90% of the sequence in the 122 longest scaffolds. This new assembly will be an invaluable resource for continued basic and pharmaceutical CHO cell research.
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.
Within the epigenetic mechanisms that control gene expression in CHO cells, DNA methylation of promoters is associated with silencing of the corresponding gene. In this study, targeted demethylation of the highly methylated ST6GAL1 promoter with a repurposed CRISPR system transcriptionally activated the beforehand silent gene. The long‐lasting effect can be reversed by targeted re‐methylation of the ST6GAL1 promoter, which demonstrates the great potential of targeted epigenetic editing tools for detailed gene‐of‐function studies as well as for cell engineering approaches.
Recent transcriptome annotation using deep sequencing approaches have annotated a large number of long non-coding RNAs in zebrafish, a popular model organism for human diseases. These studies ...characterized lncRNAs in critical developmental stages as well as adult tissues. Each of the studies has uncovered a distinct set of lncRNAs, with minor overlaps. The availability of the raw RNA-Seq datasets in public domain encompassing critical developmental time-points and adult tissues provides us with a unique opportunity to understand the spatiotemporal expression patterns of lncRNAs. In the present report, we created a catalog of lncRNAs in zebrafish, derived largely from the three annotation sets, as well as manual curation of literature to compile a total of 2,267 lncRNA transcripts in zebrafish. The lncRNAs were further classified based on the genomic context and relationship with protein coding gene neighbors into 4 categories. Analysis revealed a total of 86 intronic, 309 promoter associated, 485 overlapping and 1,386 lincRNAs. We created a comprehensive resource which houses the annotation of lncRNAs as well as associated information including expression levels, promoter epigenetic marks, genomic variants and retroviral insertion mutants. The resource also hosts a genome browser where the datasets could be browsed in the genome context. To the best of our knowledge, this is the first comprehensive resource providing a unified catalog of lncRNAs in zebrafish. The resource is freely available at URL: http://genome.igib.res.in/zflncRNApedia.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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Chinese Hamster Ovary (CHO) cell lines are considered to be the preferred platform for the production of biotherapeutics, but issues related to expression instability remain ...unresolved. In this study, we investigated potential causes for an unstable phenotype by comparing cell lines that express stably to such that undergo loss in titer across 10 passages. Factors related to transgene integrity and copy number as well as the genomic profile around the integration sites were analyzed. Horizon Discovery CHO-K1 (HD-BIOP3) derived production cell lines selected for phenotypes with low, medium or high copy number, each with stable and unstable transgene expression, were sequenced to capture changes at genomic and transcriptomic levels. The exact sites of the random integration events in each cell line were also identified, followed by profiling of the genomic, transcriptomic and epigenetic patterns around them. Based on the information deduced from these random integration events, genomic loci that potentially favor reliable and stable transgene expression were reported for use as targeted transgene integration sites. By comparing stable vs unstable phenotypes across these parameters, we could establish that expression stability may be controlled at three levels: 1) Good choice of integration site, 2) Ensuring integrity of transgene and observing concatemerization pattern after integration, and 3) Checking for potential stress related cellular processes. Genome wide favorable and unfavorable genomic loci for targeted transgene integration can be browsed at https://www.borthlabchoresources.boku.ac.at/
ABSTRACT
The human mitochondrial genome has been reported to have a very high mutation rate as compared with the nuclear genome. A large number of mitochondrial mutations show significant phenotypic ...association and are involved in a broad spectrum of diseases. In recent years, there has been a remarkable progress in the understanding of mitochondrial genetics. The availability of next‐generation sequencing (NGS) technologies have not only reduced sequencing cost by orders of magnitude but has also provided us good quality mitochondrial genome sequences with high coverage, thereby enabling decoding of a number of human mitochondrial diseases. In this study, we report a computational and experimental pipeline to decipher the human mitochondrial DNA variations and examine them for their clinical correlation. As a proof of principle, we also present a clinical study of a patient with Leigh disease and confirmed maternal inheritance of the causative allele. The pipeline is made available as a user‐friendly online tool to annotate variants and find haplogroup, disease association, and heteroplasmic sites. The “mit‐o‐matic” computational pipeline represents a comprehensive cloud‐based tool for clinical evaluation of mitochondrial genomic variations from NGS datasets. The tool is freely available at http://genome.igib.res.in/mitomatic/.
Mitochondrial disorders are one of the most common genetic disorders; and are caused by genetic mutations in the mitochondrial genome. The application of Next Generation Sequencing in diagnosis of mitochondrial diseases has become popular and has given an added advantage for detecting heteroplasmic mutations. In this paper, we describe a comprehensive cloud‐based, online resource for clinical analysis and interpretation of mutations in mitochondrial genome.
• 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 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. Display omitted
Recombinant mammalian host cell lines, in particular CHO and HEK293 cells, are used for the industrial production of therapeutic proteins. Despite their well-known genomic instability, the control ...mechanisms that enable cells to respond to changes in the environmental conditions are not yet fully understood, nor do we have a good understanding of the factors that lead to phenotypic shifts in long-term cultures. A contributing factor could be inherent diversity in transcriptomes within a population. In this study, we used a full-length coverage single-cell RNA sequencing (scRNA-seq) approach to investigate and compare cell-to-cell variability and the impact of standardized and homogenous culture conditions on the diversity of individual cell transcriptomes, comparing suspension CHO-K1 and adherent HEK293FT cells. Our data showed a critical batch effect from the sequencing of four 96-well plates of CHO-K1 single cells stored for different periods of time, which was and may be therefore identified as a technical variable to consider in experimental planning. Besides, in an artificial and controlled culture environment such as used in routine cell culture technology, the gene expression pattern of a given population does not reveal any marker gene capable to disclose relevant cell population substructures, both for CHO-K1 cells and for HEK293FT cells. The variation observed is primarily driven by the cell cycle.
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•scRNA-seq of CHO-K1 suspension and HEK293FT adherent cultures shows cellular homogeneity and lack of clear subpopulations.•The minor source of heterogeneity of HEK293FT attributable to the cell cycle may be due to the limited cells examined.•53 highly variable genes shared between cell lines display the transcriptomes as close neighbours in the 2D-embedding plot.•The holding time of printed plates may affect the quality of the sequencing data and the variance in the data sets.
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.
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•Endogenous and exogenous (viral) promoters respond differently to artificially induced modifications in their promoter methylation.•Cells autologously respond to altered promoter methylation by adapting the respective histone marks.•Promoters of genes that are expressed in normal CHO cells receive marks of facultative heterochromatin that can be easily re-activated.•Promoters of silent genes in CHO and the viral CMV promoter acquire histone marks of constitutive, permanent heterochromatin.
Modulation of expression levels of endogenous or recombinant genes can be of great interest for diverse applications, such as the study of genotype-phenotype relationships for a gene of interest, or ...fine-tuning of transcription to determine physiologically relevant effects of gene expression levels. During the last decades, several synthetic biology tools were established to control gene expression in mammalian cells such as Chinese hamster ovary (CHO) cells, one of the most important cell systems for basic research as well as the production of biopharmaceuticals. Here we describe the use of triplex forming oligos (TFOs), short RNA or ssDNA molecules that can bind to the major grove of their target duplex with great specificity, to control transgene expression in CHO cells. For proof of concept, a panel of TFOs with a size of 10–20 nts were designed with the help of the on-line tool Triplexator targeting the viral cytomegalovirus (CMV) promoter/enhancer region controlling the downstream reporter gene hCD4. The effect of TFOs was tested as ssDNA oligos pre-annealed to the promoter/enhancer region in vitro as well as upon endogenous transcription of the TFO as an RNA molecule binding to their target duplex in vivo. Results showed that not only binding of the TFO, but the exact location of triplex formation within the promoter/enhancer is paramount for transcription inhibition. After relieving a binding conflict by introducing a point mutation within the CMV promoter, longer TFOs (26–30 nts) could be designed and analysed. Selected TFOs achieved a reduction in recombinant hCD4 expression of up to 85% in CHO-K1 cells.
•Triplex forming oligo sequences (TFOs) with a size of 10–20 nts were designed to target the viral cytomegalovirus (CMV) promoter/enhancer .•Binding of the TFO, but also the exact location of triplex formation within the promoter/enhancer is paramount for transcription inhibition.•Selected TFOs achieved a reduction in recombinant gene expression of up to 85% in CHO-K1 cells.
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.
For CHO cell bioprocesses, a detailed temporal understanding of transcriptional control is necessary to achieve reliable performance. Here, transcriptional dynamics throughout a batch examined by analyzing histone modifications and gene expression at closely spaced intervals revealed three levels of regulation: i) an ON/OFF switch based on promoter methylation; ii) a temporally resolved correlation to presence of active transcription‐ and promoter‐specific histone marks; iii) major fold changes in transcription mediated by RNA‐DNA‐DNA triplexes between long non‐coding RNAs and their target genes.
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