Multi-layered proteomics identifies insulin-induced upregulation of the EphA2 receptor via the ERK pathway which is dependent on low IGF1R level.

Insulin resistance impairs the cellular insulin response, and often precedes metabolic disorders, like type 2 diabetes, impacting an increasing number of people globally. Understanding the molecular mechanisms in hepatic insulin resistance is essential for early preventive treatments. To elucidate changes in insulin signal transduction associated with hepatocellular resistance, we employed a multi-layered mass spectrometry-based proteomics approach focused on insulin receptor (IR) signaling at the interactome, phosphoproteome, and proteome levels in a long-term hyperinsulinemia-induced insulin-resistant HepG2 cell line with a knockout of the insulin-like growth factor 1 receptor (IGF1R KO).

Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.

Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells.

Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells.

The Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA.

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method.

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids.

A pooled CRISPR/AsCpf1 screen using paired gRNAs to induce genomic deletions in Chinese hamster ovary cells.

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.

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells.

Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate.

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators.

Multiplex secretome engineering enhances recombinant protein production and purity.

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs.

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures.

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells.

Awakening dormant glycosyltransferases in CHO cells with CRISPRa.

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced.

Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion.

Chinese hamster ovary (CHO) cells are the preferred host for producing biopharmaceuticals. Amino acids are biologically important precursors for CHO metabolism; they serve as building blocks for proteogenesis, including synthesis of biomass and recombinant proteins, and are utilized for growth and cellular maintenance. In this work, we studied the physiological impact of disrupting a range of amino acid catabolic pathways in CHO cells.

BCAT1 and BCAT2 disruption in CHO cells has cell line-dependent effects.

In recombinant protein expression using Chinese hamster ovary (CHO) cells, chemically defined media contain essential amino acids such as branched chain amino acids (BCAAs) leucine, isoleucine and valine. Availability of amino acids is critical as these are building blocks for protein synthesis. However, breakdown of amino acids can lead to build up of toxic intermediates and metabolites that decrease cell growth, productivity and product quality.

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms.

The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host- or biopharmaceutical-specific product quality obstacles.

CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells.

The emergence of CRISPR/Cas9 system as a precise and affordable method for genome editing has prompted its rapid adoption for the targeted integration of transgenes in Chinese hamster ovary (CHO) cells. Targeted gene integration allows the generation of stable cell lines with a controlled and predictable behavior, which is an important feature for the rational design of cell factories aimed at the large-scale production of recombinant proteins. Here we present the protocol for CRISPR/Cas9-mediated integration of a gene expression cassette into a specific genomic locus in CHO cells using homology-directed DNA repair.

Mitigating Clonal Variation in Recombinant Mammalian Cell Lines.

Mammalian expression platforms are primary production systems for therapeutic proteins that require complex post-translational modifications. Current processes used for developing recombinant mammalian cell lines generate clonal cell lines with high phenotypic heterogeneity, which has puzzled researchers that use mammalian cell culture systems for a long time. Advances in mammalian genome-editing technologies and systems biotechnology have shed light on clonal variation and enabled rational cell engineering in a targeted manner.

Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference.

Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells.

Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering.

Many branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette. Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the expression cassette components.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.

Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles.

Revealing Key Determinants of Clonal Variation in Transgene Expression in Recombinant CHO Cells Using Targeted Genome Editing.

Generation of recombinant Chinese hamster ovary (rCHO) cell lines is critical for the production of therapeutic proteins. However, the high degree of phenotypic heterogeneity among generated clones, referred to as clonal variation, makes the rCHO cell line development process inefficient and unpredictable. Here, we investigated the major genomic causes of clonal variation.

Minimizing Clonal Variation during Mammalian Cell Line Engineering for Improved Systems Biology Data Generation.

Mammalian cells are widely used to express genes for basic biology studies and biopharmaceuticals. Current methods for generation of engineered cell lines introduce high genomic and phenotypic diversity, which hamper studies of gene functions and discovery of novel cellular mechanisms. Here, we minimized clonal variation by integrating a landing pad for recombinase-mediated cassette exchange site-specifically into the genome of CHO cells using CRISPR and generated subclones expressing four different recombinant proteins.

CRISPR/Cas9-Multiplexed Editing of Chinese Hamster Ovary B4Gal-T1, 2, 3, and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins.

In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9.