Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing.

IF 5.1 2区 生物学 Q2 CELL BIOLOGY
Cells Pub Date : 2025-05-11 DOI:10.3390/cells14100692
Nadezhda Alexandrovna Orlova, Maria Valerievna Sinegubova, Denis Eduardovich Kolesov, Yulia Alexandrovna Khodak, Victor Vyacheslavovich Tatarskiy, Ivan Ivanovich Vorobiev
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Abstract

Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes-glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells.

CHO 4BGD细胞的基因组和表型特征与四敲除和两个管理基因的过表达,允许代谢选择和延长饲料批量培养。
利用基因组编辑和多个辅助基因的过表达对CHO细胞进行重组是获得用于生物制药生产的更好细胞系的核心途径。通过对CHO S细胞进行随后的两轮基因组编辑,我们开发了CHO 4BGD细胞系,其中四个敲除了两个促凋亡基因bak1和bax,以及两个常见的选择标记基因-glul (GS)和dhfr,以及用于增强巨噬的基因bcl-2和beclin-1的额外拷贝。4BGD细胞的NGS测序显示,所有8个靶向等位基因都被成功破坏。8个被编辑的基因座中有2个含有大量不相关的DNA插入。进一步的数据分析表明,细胞没有脱靶DNA编辑事件,并且所有已知的CHO基因都被保留了下来。获得的细胞对诱导凋亡具有完全的抗性,适合于产生具有dhfr选择标记的稳定转染细胞系。它们也会适当地进行靶基因扩增。分泌单克隆抗体的4bgd衍生的克隆细胞系保留了长时间补料分批培养的能力。利用多重CRISPR/Cas9编辑获得多重编辑的CHO细胞,同时稳定转染编码内务基因的质粒,适用于大规模编辑CHO细胞的快速生成。
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来源期刊
Cells
Cells Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
9.90
自引率
5.00%
发文量
3472
审稿时长
16 days
期刊介绍: Cells (ISSN 2073-4409) is an international, peer-reviewed open access journal which provides an advanced forum for studies related to cell biology, molecular biology and biophysics. It publishes reviews, research articles, communications and technical notes. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. Full experimental and/or methodical details must be provided.
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