Article
Biotechnology & Applied Microbiology
Sana Pourtabatabaei, Samaneh Ghanbari, Narges Damavandi, Elham Bayat, Mozhgan Raigani, Sirous Zeinali, Fatemeh Davami
Summary: Chinese hamster ovary (CHO) cells are widely used for manufacturing therapeutic proteins, and in this study, stable cell lines were successfully generated with targeted integration of reporter genes using the CRISPR/Cas9 platform. The study showed that higher efficiency and expression homogeneity were observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.
JOURNAL OF BIOTECHNOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Sung Wook Shin, Dongwoo Kim, Jae Seong Lee
Summary: Chinese hamster ovary (CHO) cells are the most valuable expression host for commercial biotherapeutic production, with recent trends focused on site-specific integration over random integration. The DCD system enables highly efficient CRISPR/Cas9-mediated targeted integration in CHO cells, and the multi-component system shows an increased knock-in efficiency compared to the previous system. Increasing sgRNA and DCD components relative to Cas9 positively correlates with knock-in efficiency, and the system can be widely used in transgene targeted integration strategies in CHO cell line development.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Article
Biotechnology & Applied Microbiology
Klaus Leitner, Krishna Motheramgari, Nicole Borth, Nicolas Marx
Summary: In this study, a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline was developed to simultaneously obtain information on integration sites, the composition of the integrated sequence, and its DNA methylation status in CHO cells from the same sequencing run. The data generated by nCats provides sensitive, detailed, and correct information on transgene integration sites and expression vector structure, which can only be partially obtained by traditional Targeted Locus Amplification-seq data. Moreover, nCats allows for the analysis of DNA methylation status from the same raw data without prior DNA amplification.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Honggi Min, Seul Mi Kim, Dongwoo Kim, Solhwi Lee, Sumin Lee, Jae Seong Lee
Summary: In this study, a hybrid cell line development platform was developed to increase the productivity of targeted integrants through the amplification of transgene copies. The results showed that targeted integrants exhibited a 3.6-fold increase in EGFP expression and increased copy numbers of DHFR and EGFP when exposed to 200 nM MTX. A single-step MTX amplification increased the specific monoclonal antibody productivity by 2.8-fold. The study provides a new strategy for increasing the productivity of CHO cell lines.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)
Article
Biochemical Research Methods
Jin Myeong Kwak, Youngsik Lee, Sung Wook Shin, Jae Seong Lee
Summary: A cell cycle enrichment method using a lower concentration of hydroxyurea (HU) has been developed to increase HDR-mediated KI efficiency in CHO cells, resulting in a 1.2-1.5 fold improvement in KI efficiency compared to control conditions.
Review
Biotechnology & Applied Microbiology
Shahin Amiri, Setare Adibzadeh, Samaneh Ghanbari, Behnaz Rahmani, Mohammad H. Kheirandish, Aref Farokhi-Fard, Mansoureh S. Dastjerdeh, Fatemeh Davami
Summary: Chinese hamster ovary (CHO) cells are the most commonly used host cell system for industrial production of recombinant protein biopharmaceuticals. The use of CRISPR-Cas technology along with donor design strategies has allowed scientists to efficiently modify CHO cell line genomes and achieve consistent and high-quality protein expression. This review summarizes the recent advancements in CHO cell line development, including different cell engineering approaches and targeted integration strategies, which can improve the product development process and yield.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Multidisciplinary Sciences
Femila Manoj, Laura W. Tai, Katelyn Sun Mi Wang, Thomas E. Kuhlman
Summary: The GENEWRITE system combines the site-specific targetable activity of Cas endonucleases with the reverse transcriptase activity of the ORF2p protein, enabling the insertion of large genetic constructs at precise locations. Despite off-target effects, successful insertion of large genetic payloads has been achieved in E. coli through proper payload design and co-expression of helper proteins.
SCIENTIFIC REPORTS
(2021)
Article
Biotechnology & Applied Microbiology
Dongwoo Kim, Jae Seong Lee
Summary: By modifying the Cas9 expression vector, the fusion of the ZF-binding protein with the donor plasmid can expedite its translocation into the nucleus and co-localization with the DNA double-strand break site. This modification leads to an increased efficiency of homology-mediated knock-in.
BIOTECHNOLOGY AND BIOPROCESS ENGINEERING
(2023)
Article
Genetics & Heredity
Dennis Webster, Alla Bondareva, Staci Solin, Taylor Goldsmith, Lin Su, Nathalia de Lima e Martins Lara, Daniel F. Carlson, Ina Dobrinski
Summary: Pigs, sharing physiological and genetic characteristics with humans, have been used as appropriate preclinical models for studying human diseases and developing innovative treatments. Researchers have demonstrated that CRISPR/Cas9-mediated gene editing in porcine spermatogonia is more efficient than previous methods, enabling precise replication of human disease alleles.
FRONTIERS IN GENETICS
(2021)
Article
Biotechnology & Applied Microbiology
Jose Bonafont, Angeles Mencia, Esteban Chacon-Solano, Wai Srifa, Sriram Vaidyanathan, Rosa Romano, Marta Garcia, Rosario Hervas-Salcedo, Laura Ugalde, Blanca Duarte, Matthew H. Porteus, Marcela Del Rio, Fernando Larcher, Rodolfo Murillas
Summary: This study presents a gene-editing approach using CRISPR-Cas9 system to achieve gene correction in different cell types, showing therapeutic potential for RDEB.
Article
Biochemistry & Molecular Biology
Guoling Li, Xiaohui Yang, Xinxin Luo, Zhenfang Wu, Huaqiang Yang
Summary: The use of small molecules to synchronize the cell cycle in S and G2/M phases enhances the efficiency of CRISPR/Cas9-mediated homology-directed repair (HDR) in animal cells and embryos. The study reveals the common molecular mechanisms that connect cell cycle progression and HDR activity.
CELL AND BIOSCIENCE
(2023)
Article
Biotechnology & Applied Microbiology
Tatiana Dovgan, Vahid Golghalyani, Fabio Zurlo, Diane Hatton, Viv Lindo, Richard Turner, Claire Harris, Tingting Cui
Summary: This study demonstrates that for cell lines with high levels of copurifying HCPs, modest downregulation of the difficult to remove HCP Cathepsin D through stable RNA interference or CRISPR-Cas9 technology can greatly reduce levels of copurifying HCP, leading to improved product quality and reducing fragmentation of the drug product to negligible levels in degradation studies. It also shows the potential of cell engineering to target other undesired HCPs and alleviate downstream purification burden.
BIOTECHNOLOGY AND BIOENGINEERING
(2021)
Article
Cell Biology
Chenxin Wang, Sen Fang, Yangcan Chen, Na Tang, Guanyi Jiao, Yanping Hu, Jing Li, Qingtong Shan, Xin Wang, Guihai Feng, Qi Zhou, Wei Li
Summary: Due to the difficulties in manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes is inefficient. We developed a robust knock-in strategy called PAINT 3.0, which uses micro-homologues to boost targeted integration in different cells, achieving editing efficiencies up to 80% and minimizing off-target integration. PAINT 3.0 enables high-efficiency genome targeting and functional CAR-T cell production, making it a powerful tool for gene editing and potential for clinical applications.
Article
Biology
Dorottya Anna Simon, Andras Talas, Peter Istvan Kulcsar, Zsuzsanna Biczok, Sarah Laura Krausz, Gyorgy Varady, Ervin Welker
Summary: Prime editing is a gene engineering tool that allows editing of the genome by introducing insertions, deletions, and substitutions. However, the efficiency of prime editing has been low, typically achieving editing rates of only 10-30%. To address this issue, researchers have developed PEAR, a fluorescent tool that can sensitively detect prime editing activity, improving the accuracy and applicability of editing.
Article
Biology
Jordan M. Welker, Wesley A. Wierson, Maira P. Almeida, Carla M. Mann, Melanie E. Torrie, Zhitao Ming, Stephen C. Ekker, Karl J. Clark, Drena L. Dobbs, Jeffrey J. Essner, Maura McGrail
Summary: Efficient precision gene targeting in zebrafish is achieved through the use of the GeneWeld protocol and pGTag vector series, which provide a streamlined and cost-effective approach. Integration of DNA reporter cassettes is driven by short homology sequences, leading to high germline transmission rates for precision-targeted alleles.
Review
Biotechnology & Applied Microbiology
Tae Kwang Ha, Dongil Kim, Che Lin Kim, Lise Marie Grav, Gyun Min Lee
Summary: This review summarizes the factors that influence the quality of therapeutic proteins in recombinant CHO cell culture, categorizing them into culture environments, chemical additives, and host cell proteins. Understanding these factors will facilitate the development of high yield, high-quality therapeutic protein production processes.
BIOTECHNOLOGY ADVANCES
(2022)
Article
Biotechnology & Applied Microbiology
Karen Julie la Cour Karottki, Hooman Hefzi, Songyuan Li, Lasse Ebdrup Pedersen, Philipp N. Spahn, Chintan Joshi, David Ruckerbauer, Juan A. Hernandez Bort, Alex Thomas, Jae Seong Lee, Nicole Borth, Gyun Min Lee, Helene Faustrup Kildegaard, Nathan E. Lewis
Summary: Media and feed optimization have improved mammalian biopharmaceutical production, but genome editing offers a new avenue for enhancing cell metabolism. A comprehensive pooled CRISPR screen identified a glutamine response network in CHO cells, and knockout of a poorly characterized lipase gene significantly increased cell growth in glutamine-free media. The screen provides a targeted platform to study genes involved in metabolic traits and elucidate novel regulators of metabolism.
METABOLIC ENGINEERING
(2021)
Article
Biotechnology & Applied Microbiology
Su Hyun Kim, Minhye Baek, Sungje Park, Seunghyeon Shin, Jae Seong Lee, Gyun Min Lee
Summary: Controlled expression of Blimp1beta improves the production capacity of rCHO cells by regulating secretory machinery, enhancing the production of rhBMP-4 and mAbs.
METABOLIC ENGINEERING
(2022)
Article
Biotechnology & Applied Microbiology
Minji Kyeong, Jae Seong Lee
Summary: A fluorescence-based UPR monitoring system was developed for CHO cells by integrating mGFP into endogenous BiP using CRISPR/Cas9. The monitoring system can track cell stress levels based on ER stress changes, providing a tool for monitoring high productivity and quality in protein production.
METABOLIC ENGINEERING
(2022)
Article
Biotechnology & Applied Microbiology
Seunghyeon Shin, Su Hyun Kim, Jong-Ho Park, Jae Seong Lee, Gyun Min Lee
Summary: This study established a high-throughput CRISPR/Cas9 library screening platform to identify target genes associated with hyperosmotic stress, and enhanced cell resistance to stress and recombinant protein production by knockout of these genes.
METABOLIC ENGINEERING
(2022)
Article
Chemistry, Analytical
Andreas Madsen, Oscar Mejias-Gomez, Lasse E. Pedersen, Kerstin Skovgaard, Peter Kristensen, Steffen Goletz
Summary: Simultaneous targeting of different antigens with bispecific antibodies (bsAbs) has high therapeutic potential but presents challenges in analysis. Flow-induced dispersion analysis (FIDA) allows in-depth characterization of bsAbs under near-native conditions, determining equilibrium dissociation constants and analyzing binding cooperativity. FIDA is a versatile tool with low material consumption and high matrix tolerability for format selection and characterization of complex protein therapeutics.
ANALYTICAL CHEMISTRY
(2022)
Article
Biotechnology & Applied Microbiology
Tae Kwang Ha, Andreu Odena, Karen Julie la Cour Karottki, Che Lin Kim, Hooman Hefzi, Gyun Min Lee, Helene Faustrup Kildegaard, Lars K. Nielsen, Lise Marie Grav, Nathan E. Lewis
Summary: The use of a dual selection method can greatly improve the efficiency of Chinese hamster ovary (CHO) cell lines in producing biotherapeutic proteins, although it may lead to reduced growth rate and viability. By knocking out the Gs/Aspg genes, a novel dual selectable system was successfully constructed, which showed significantly improved productivity and titer compared to the standard Gs system under the same selection conditions. With further optimization of culture medium and selection conditions, this approach is expected to be an effective addition to industrial workflows for producing recombinant biotherapeutics.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Rebecca M. Lennen, Hyun Gyu Lim, Kristian Jensen, Elsayed T. Mohammed, Patrick V. Phaneuf, Myung Hyun Noh, Sailesh Malla, Rosa A. Borner, Ksenia Chekina, Emre Ozdemir, Ida Bonde, Anna Koza, Jerome Maury, Lasse E. Pedersen, Lars Y. Schoning, Nikolaus Sonnenschein, Bernhard O. Palsson, Alex T. Nielsen, Morten O. A. Sommer, Markus J. Herrgard, Adam M. Feist
Summary: This study used an automated platform to evolve Escherichia coli to grow optimally in the presence of 11 industrial chemicals and uncovered the tolerance mechanisms. The results showed that cells are tolerized through frequent mutation of membrane transporters, cell wall-associated proteins, transcription and translation machineries, and stress signaling proteins. Osmotic stress plays a significant role in tolerance, and pre-tolerized starting isolates can greatly enhance the subsequent production of chemicals.
METABOLIC ENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Johan B. Rojek, Yogesh Basavaraju, Saranya Nallapareddy, Dubhe B. B. Ocana, Roland Baumgartner, Sanne Schoffelen, Lise M. Grav, Steffen Goletz, Lasse E. Pedersen
Summary: This study demonstrates the application of Mad7 in CHO cells for gene knockout, expanding the versatility of the CRISPR toolbox for cell line engineering and research.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Review
Medicine, Research & Experimental
Monica L. Fernandez-Quintero, Anne Ljungars, Franz Waibl, Victor Greiff, Jan Terje Andersen, Torelif T. T. Gjolberg, Timothy P. P. Jenkins, Bjorn Gunnar Voldborg, Lise Marie Grav, Sandeep Kumar, Guy Georges, Hubert Kettenberger, Klaus R. R. Liedl, Peter M. M. Tessier, John McCafferty, Andreas H. H. Laustsen
Summary: Beyond potency, a good developability profile is essential for a biological drug. Early selection and screening for these attributes can save resources and avoid costly failures. This review highlights key developability properties, such as the source, biophysical and pharmacokinetic properties, and recombinant expression, and discusses methods for identifying molecules with liabilities and optimizing drug leads. The article also compares developability parameters for injectable and orally delivered biologics and provides an outlook on emerging trends in biologics development.
Article
Medicine, Research & Experimental
Oscar Mejias-Gomez, Andreas V. Madsen, Kerstin Skovgaard, Lasse E. Pedersen, J. Preben Morth, Timothy P. Jenkins, Peter Kristensen, Steffen Goletz
Summary: This study analyzes human antibody sequences to understand the factors determining antibody diversity and specificity. The study reveals the importance of specific residue positions and their influence on antibody characteristics. These findings have significant implications for the development and optimization of antibody therapeutics.
Article
Biochemical Research Methods
Oscar Mejias-Gomez, Andreas V. Madsen, Lasse E. Pedersen, Peter Kristensen, Steffen Goletz
Summary: This study presents a system based on split beta-lactamase complementation, which can remove OFF-frame clones and increase functional diversity more quickly and efficiently, suitable for constructing randomized libraries. The system can eliminate OFF-frame clones containing stop codons or frameshifts in starting mixtures, enriching the IN-frame clones to about 70%.
Article
Biotechnology & Applied Microbiology
Vratislav Stovicek, Laura Dato, Henrik Almqvist, Marie Schopping, Ksenia Chekina, Lasse Ebdrup Pedersen, Anna Koza, Diogo Figueira, Freddy Tjosas, Bruno Sommer Ferreira, Jochen Forster, Gunnar Liden, Irina Borodina
Summary: The study successfully engineered an industrial strain of the yeast Saccharomyces cerevisiae for efficient utilization of xylose in lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. Whole genome sequencing revealed genetic determinants of increased tolerance to SSL, leading to the development of strains for producing dicarboxylic acids. The engineered strains showed high productivity of malic and succinic acid from fermentable carbon content of SSL, providing potential gene targets for improving tolerance to inhibitory industrial feedstocks.
BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS
(2022)
Article
Chemistry, Analytical
Andreas Madsen, Oscar Mejias-Gomez, Lasse E. Pedersen, Kerstin Skovgaard, Peter Kristensen, Steffen Goletz
Summary: FIDA technology enables in-depth characterization of bsAbs with diverse molecular architectures and valencies, analyzing their bispecific binding functionality without being affected by molecular format, making it a versatile tool for selecting and characterizing complex protein therapeutics.
ANALYTICAL CHEMISTRY
(2022)