Novel CRISPR/Cas9-mediated knockout of LIG4 increases efficiency of site-specific integration in Chinese hamster ovary cell line

Wang, Chuanjie; Sun, Zhaolin; Wang, Ming; Jiang, Zhiyang; Zhang, Mengmeng; Cao, Hongxu; Luo, Longlong; Qiao, Chun‐Feng; He, X. T.; Chen, Guojiang; Li, Xinying; Liu, Jinqing; Wei, Zeliang; Shen, Beifen; Wang, Jing; Feng, Jiannan

doi:10.1007/s10529-022-03282-7

Cited by 6 publications

(4 citation statements)

References 37 publications

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“…This suggests that some DNA repair inhibitors may not be cross‐reactive with hamster orthologs or that the type of activity inhibited is dispensable for the mechanism of RI, which may be especially relevant for multifunctional proteins like Polθ, DNA‐PKcs, Ku70‐80, and ligase IV (Lig IV). Similarly, a recent report demonstrated that knock‐out of Lig IV significantly increased SSI and inhibited RI in CHO cells (Wang et al, 2022), despite observations documented here and by others that treatment with the Lig IV inhibitor SCR7 yields unproductive outcomes (Lee et al, 2016). We note that permanent inactivation of repair factors is not a prudent strategy for reducing RI since these factors contribute to genome maintenance and stability, and their removal is likely to exacerbate long‐term productivity decline in production cell lines.…”

Section: Discussionsupporting

confidence: 49%

High‐efficiency and multilocus targeted integration in CHO cells using CRISPR‐mediated donor nicking and DNA repair inhibitors

Hamaker

Lee

2023

Biotech & Bioengineering

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Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all‐in‐one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site‐specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double‐cut and double‐nick donor formats significantly improved targeting accuracy by 2.3–8.3‐fold and 19–22‐fold, respectively, compared to standard circular donors. Notably, Cas9‐mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low‐fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR‐based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.

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Section: Discussionsupporting

confidence: 49%

High‐efficiency and multilocus targeted integration in CHO cells using CRISPR‐mediated donor nicking and DNA repair inhibitors

Hamaker

Lee

2023

Biotech & Bioengineering

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“…While the Rosa26 locus has become standard as target site in human and mouse cell lines for many years, it has been shown that the homologous Rosa26 in the CHO genome can be used to exchange expression cassettes for constitutive expression [ 33 , 56 , 57 ]. In the present work, Rosa26 was found to be a suitable target site that allows for the incorporation of large expression cassettes, such as the complete regulatory units required for induced expression in CHO cells.…”

Section: Discussionmentioning

confidence: 99%

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Schloßhauer,

Tholen,

Körner

et al. 2024

Synthetic and Systems Biotechnology

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“…Notably, the SSI efficiency dramatically enhanced 9.2‐fold under the precise control of the promoter in the ROSA26 site in LIG4‐CHO cells, whereas the RI efficiency, most likely mediated by NHEJ in CHO, was hindered by LIG4 KO. Furthermore, the proliferation of CHO cells was unaffected by the ablation of LIG4 (Wang et al, 2022).…”

Section: Crispr‐cas9‐mediated Gene Ko For Cell Line Engineeringmentioning

confidence: 99%

CRISPR‐interceded CHO cell line development approaches

Amiri

Adibzadeh

Ghanbari

et al. 2023

Biotech & Bioengineering

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For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high‐quality biologics with human‐like posttranslational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long‐term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR‐Cas systems, the use of CRISPR‐Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast‐track product development process with consistent, improved product yield and quality.

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Novel CRISPR/Cas9-mediated knockout of LIG4 increases efficiency of site-specific integration in Chinese hamster ovary cell line

Cited by 6 publications

References 37 publications

High‐efficiency and multilocus targeted integration in CHO cells using CRISPR‐mediated donor nicking and DNA repair inhibitors

High‐efficiency and multilocus targeted integration in CHO cells using CRISPR‐mediated donor nicking and DNA repair inhibitors

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

CRISPR‐interceded CHO cell line development approaches

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