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
Biotechnology & Applied Microbiology
Dongil Kim, Su Hyun Kim, Chansik Yoon, Gyun Min Lee
Summary: The use of CRISPR/Cas9-based single-gene knockout cell library was able to identify KO target genes conferring resistance to iHDACs, leading to improved therapeutic protein production.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biochemical Research Methods
Patrick G. Schweickert, Ning Wang, Stephanie L. Sandefur, Michael E. Lloyd, Stephen F. Konieczny, Christopher. C. Frye, Zhuo Cheng
Summary: The study demonstrates the successful application of CRISPR/Cas12a for generating CHO KO cell lines with improved product quality, with efficiency dependent on the targeting RNA used; small screens of clonally derived cell lines can be used to generate CHO KO cell lines, and a novel bulk culture analysis approach is proposed for assessing CRISPR RNA efficiency and determining ideal screen sizes; Cas12a can be directly integrated into the cell line generation process with no negative impact on titer or screen size.
BIOTECHNOLOGY JOURNAL
(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
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
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.
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
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
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
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
Pavlos Kotidis, Roberto Donini, Johnny Arnsdorf, Anders Holmgaard Hansen, Bjorn Gunnar Rude Voldborg, Austin W. T. Chiang, Stuart M. Haslam, Michael Betenbaugh, Ioscani Jimenez del Val, Nathan E. Lewis, Frederick Krambeck, Cleo Kontoravdi
Summary: Researchers constructed a comprehensive glycosylation reaction network, CHOGlycoNET, based on 200 glycan datasets from CHO cell lines. They used machine learning and dimensionality reduction techniques to explore the distribution of mannosidase and glycosyltransferase enzymes in the Golgi apparatus and identify key reactions. CHOGlycoNET can accelerate glycomodel development and predict the effect of glycoengineering strategies.
METABOLIC ENGINEERING
(2023)
Article
Multidisciplinary Sciences
Daniel M. Sapozhnikov, Moshe Szyf
Summary: PROTECTOR is a novel approach that uses a nuclease-dead Cas protein to bind to off-target sites and interfere with Cas activity, reducing off-target mutation rates without compromising on-target activity. It can be used in combination with high-fidelity Cas enzymes to further decrease off-target editing, offering an ability to protect off-target sites with identical sequences to target sites.
SCIENTIFIC REPORTS
(2023)
Article
Cell Biology
Luxing Ge, Jian Kang, Xiangchen Dong, Deji Luan, Guanghua Su, Guangpeng Li, Yong Zhang, Fusheng Quan
Summary: This study successfully utilized CRISPR/Cas9 technology to achieve gene mutation and insertion in the bovine genome, confirming the effectiveness of this gene editing strategy. Mutant myoblasts exhibited higher proliferation, myogenic differentiation, and adipogenic transdifferentiation abilities, providing breeding material and technical strategy for high-quality bovine breeding.
JOURNAL OF CELLULAR PHYSIOLOGY
(2021)
Editorial Material
Biochemistry & Molecular Biology
Brett M. Sansbury, Eric B. Kmiec
Summary: The expectations for selectively modifying the human genome have never been higher in the last five years. The development of site-specific nucleases is centerstage in effective molecular therapies, with a focus on correcting genetic mutations that cause cancer and inherited diseases. Homology directed repair, a process for reversing mutations and restoring normal protein function, is a key technique that is being researched for human gene editing.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Review
Biotechnology & Applied Microbiology
Tingting Xu, Jihong Zhang, Tianyun Wang, Xiaoyin Wang
Summary: This review focuses on the aggregation of recombinant therapeutic antibodies (RTAs), discussing factors causing aggregation during RTA production and strategies for overcoming RTA aggregation.
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
(2022)
Article
Biochemistry & Molecular Biology
Hassan Vahidnezhad, Leila Youssefian, Amir Hossein Saeidian, Andrew Touati, Sara Pajouhanfar, Taghi Baghdadi, Azam Ahmadi Shadmehri, Cecilia Giunta, Marius Kraenzlin, Delfien Syx, Fransiska Malfait, Cristina Has, Su M. Lwin, Razieh Karamzadeh, Lu Liu, Alyson Guy, Mohammad Hamid, Ariana Kariminejad, Sirous Zeinali, John A. McGrath, Jouni Uitto
Article
Chemistry, Analytical
Shakiba Zeinali, Mehrdad Khalilzadeh, Habib Bagheri
ANALYTICA CHIMICA ACTA
(2019)
Review
Medicine, Research & Experimental
Asghar Fallah, Ali Sadeghinia, Houman Kahroba, Amin Samadi, Hamid Reza Heidari, Behzad Bradaran, Sirous Zeinali, Ommoleila Molavi
BIOMEDICINE & PHARMACOTHERAPY
(2019)
Editorial Material
Immunology
Leila Youssefian, Hassan Vahidnezhad, Mehdi Yousefi, Amir Hossein Saeidian, Arghavan Azizpour, Andrew Touati, Neda Nikbakht, Kambiz Kamyab-Hesari, Mohammad Mahdi Adib-Sereshki, Sirous Zeinali, Behzad Mansoori, Ali Jazayeri, Razieh Karamzadeh, Paolo Fortina, Emmanuelle Jouanguy, Jean-Laurent Casanova, Jouni Uitto
CLINICAL INFECTIOUS DISEASES
(2019)
Article
Biotechnology & Applied Microbiology
Maryam Abbasalipour, Mohammad Ali Khosravi, Sirous Zeinali, Hossein Khanahmad, Morteza Karimipoor, Kayhan Azadmanesh
BIOMED RESEARCH INTERNATIONAL
(2019)
Article
Gastroenterology & Hepatology
Leila Youssefian, Hassan Vahidnezhad, Amir Hossein Saeidian, Sara Pajouhanfar, Soheila Sotoudeh, Parvin Mansouri, Davoud Amirkashani, Sirous Zeinali, Michael A. Levine, Ketty Peris, Roberto Colombo, Jouni Uitoo
JOURNAL OF HEPATOLOGY
(2019)
Article
Endocrinology & Metabolism
Maryam Abiri, Hassan Saei, Maryam Eghbali, Razieh Karamzadeh, Tina Shirzadeh, Zohreh Sharifi, Sirous Zeinali
METABOLIC BRAIN DISEASE
(2019)
Article
Genetics & Heredity
F. Zafarghandi Motlagh, M. S. Fallah, H. Bagherian, T. Shirzadeh, S. Ghasri, S. Dabbagh, M. Jamali, Z. Salehi, M. Abiri, S. Zeinali
ORPHANET JOURNAL OF RARE DISEASES
(2019)
Article
Pharmacology & Pharmacy
Mohammad Ali Khosravi, Maryam Abbasalipour, Jean-Paul Concordet, Johannes Vom Berg, Sirous Zeinali, Arash Arashkia, Kayhan Azadmanesh, Thorsten Buch, Morteza Karimipoor
EUROPEAN JOURNAL OF PHARMACOLOGY
(2019)
Article
Endocrinology & Metabolism
Mehdi Shafaat, Mehrdad Hashemi, Ahmad Majd, Maryam Abiri, Sirous Zeinali
METABOLIC BRAIN DISEASE
(2019)
Article
Biochemistry & Molecular Biology
Narges Damavandi, Sirous Zeinali
Summary: Genetic polymorphisms of GST M1, GST T1, TNF-α, and IL-6 are associated with non-alcoholic fatty liver disease (NAFLD) in the Iranian population, providing a potential tool for prevention and care of NAFLD.
MOLECULAR BIOLOGY REPORTS
(2021)
Article
Infectious Diseases
Mahan Ghafari, Bardia Hejazi, Arman Karshenas, Stefan Dascalu, Alireza Kadvidar, Mohammad A. Khosravi, Maryam Abbasalipour, Majid Heydari, Sirous Zeinali, Luca Ferretti, Alice Ledda, Aris Katzourakis
Summary: This study utilized genome analysis and all-cause mortality data to reveal the start date and transmission dynamics of the COVID-19 epidemic in Iran, estimating the doubling time and actual death toll. Results showed a significant level of under-reporting, highlighted the high risk of virus introduction by travelers, and underscored the importance of non-pharmaceutical interventions in epidemic control.
Article
Cell Biology
Parisa Mashayekhi, Mehrdad Noruzinia, Sirous Zeinali, Sepideh Khodaverdi