A ribonucleoprotein‐based decaplex <scp>CRISPR</scp>/Cas9 knockout strategy for <scp>CHO</scp> host engineering

Carver, Joseph O.; Kern, Marie; Ko, Peggy; Greenwood‐Goodwin, Midori; Yu, Xin; Duan, Dana; Tang, Danming; Misaghi, Shahram; Auslaender, Simon; Haley, Ben; Yuk, Inn H.; Shen, Amy

doi:10.1002/btpr.3212

Cited by 4 publications

(3 citation statements)

References 29 publications

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“…New insights in genomics in general, with annotations of the CHO genome, have helped researchers successfully overcome obstacles in CHO cell engineering [169,170], but there are still data gaps for efficient sgRNA design. Advances in engineering therapeutic proteins, and modulating and editing gene expression, will enable easier production of DTE proteins with specific glycopatterns [174,175]. An increasing number of approved biosimilars in the EU and US markets [4] is contributing to the pace of innovation and development of innovative biologics and biosimilars.…”

Section: Discussionmentioning

confidence: 99%

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering

Glinšek

Bozovičar

Bratkovič

2023

IJMS

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The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.

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Section: Discussionmentioning

confidence: 99%

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering

Glinšek

Bozovičar

Bratkovič

2023

IJMS

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“… 17 , 22 , 23 Quantify relative protein expression for HCP knockout targets. 24 , 25 Supporting downstream process development Identify and track co-purifying proteins. 12 , 26 , 27 Track subset of high-risk proteins.…”

Section: Application Of Lc-ms/ms-based Methods For Hcp Identification...mentioning

confidence: 99%

Technical advancement and practical considerations of LC-MS/MS-based methods for host cell protein identification and quantitation to support process development

Guo

Kufer

et al. 2023

mAbs

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Host cell proteins (HCPs) are process-related impurities derived from the manufacturing of recombinant biotherapeutics. Residual HCP in drug products, ranging from 1 to 100 ppm (ng HCP/mg product) or even below sub-ppm level, may affect product quality, stability, efficacy, or safety. Therefore, removal of HCPs to appropriate levels is critical for the bioprocess development of biotherapeutics. Liquid chromatography-mass spectrometry (LC-MS) analysis has become an important tool to identify, quantify, and monitor the clearance of individual HCPs. This review covers the technical advancement of sample preparation strategies, new LC-MS-based techniques, and data analysis approaches to robustly and sensitively measure HCPs while overcoming the high dynamic range analytical challenges. We also discuss our strategy for LC-MS-based HCP workflows to enable fast support of process development throughout the product life cycle, and provide insights into developing specific analytical strategies leveraging LC-MS tools to control HCPs in process and mitigate their potential risks to drug quality, stability, and patient safety.

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“…A. Khan et al, 2017). Alternative strategies for simultaneous or sequential transfection of multiple synthetic gRNAs also exist for multigene knockout in culture cells (Carver et al, 2022; F. J. Khan et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

A simple, rapid, and efficient method for generating multigene‐knockout culture cells by the CRISPR/Cas9 system

et al. 2023

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We evaluated the efficacy of simultaneous multiple‐gene knockout in human culture cells. By simple co‐transfection of HeLa cells with a mixture of pX330‐based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin‐resistant cells, Cas9/single‐guide RNA (sgRNA)‐transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co‐transfection of up to seven targeting plasmids for p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA‐induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co‐transfection‐based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene‐knockout cell lines.

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A ribonucleoprotein‐based decaplex CRISPR/Cas9 knockout strategy for CHO host engineering

Cited by 4 publications

References 29 publications

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering

Technical advancement and practical considerations of LC-MS/MS-based methods for host cell protein identification and quantitation to support process development

A simple, rapid, and efficient method for generating multigene‐knockout culture cells by the CRISPR/Cas9 system

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