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.

Since the establishment of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, powerful strategies for engineering of CHO cell lines have emerged. Nevertheless, there is still room to expand the scope of the CRISPR tool box for further applications to improve CHO cell factories. Here, the authors demonstrate activity of the alternative CRISPR endonuclease Cpf1 in CHO-K1 for the first time and that it can be used in parallel to CRISPR/Cas9 without any interference. Both, Cas9 and Cpf1, can be effectively used for multi-gene engineering with a strategy based on paired single guide RNAs (sgRNAs) for full gene deletions. This strategy also enables the targeting of regulatory regions, which would not respond to the conventional frameshift mutations, as shown by removing the α-1,6-Fucosyltransferase 8 (FUT8) promoter resulting in a functional knock-out. FUT8 also served as model to verify that deletion efficiency is size-independent (2-150 kb). To test the suitability for multi-gene approaches in combination with gene deletion, clones harboring triple deletions in β-1,4-Galactosyltransferase (B4GALT) isozymes are identified using solely conventional PCR/qPCR. In addition, two bicistronic transcription strategies are implemented to enable unequivocal pairing of sgRNAs: a CHO-derived tRNA linker that works for both, Cas9 and Cpf1, as well as paired sgRNAs in an array format, which can be used with Cpf1 due to its RNA processing ability. These strategies broaden the range of application of CRISPR for novel gene editing approaches in CHO cells and also enable the efficient realization of a genome-wide deletion library.

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.In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools.

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine‐tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5′‐ and 3′‐end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.

No abstract

MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 KOs are significantly increased to 190-311 mg L −1 compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L −1 , pointing towards the potential of miRNA KO for cell line engineering.

No abstract

No abstract