CHO microRNA engineering is growing up: Recent successes and future challenges

Jadhav, Vaibhav; Hackl, Matthias; Druz, Aliaksandr; Shridhar, Smriti; Chung, C.‐S.; Heffner, Kelley; Kreil, David P.; Betenbaugh, Mike; Shiloach, Joseph; Barron, Niall; Grillari, Johannes; Borth, Nicole

doi:10.1016/j.biotechadv.2013.07.007

Cited by 78 publications

(68 citation statements)

References 208 publications

(159 reference statements)

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“…In addition, Bcl-2-like protein 10 (Bcl2l10), a signaling molecule involved in apoptosis, and stress-associated endoplasmic reticulum protein 1 (Serp1) are both in PICR but not IPCR. MicroRNAs targeting these two proteins in CHO cells have been developed [Jadhav et al, 2013]. …”

Section: Resultsmentioning

confidence: 99%

A reference genome of the Chinese hamster based on a hybrid assembly strategy

Rupp

MacDonald

et al. 2018

Biotech & Bioengineering

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119

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Accurate and complete genome sequences are essential in biotechnology to facilitate genome-based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, are fragmented and replete with gap sequences and misassemblies, consistent with most short-read based assemblies. Here, we completely resequenced C. griseus using Single Molecule Real Time (SMRT) sequencing and merged this with Illumina-based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28-fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up- and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important CHO cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.

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Section: Resultsmentioning

confidence: 99%

A reference genome of the Chinese hamster based on a hybrid assembly strategy

Rupp

MacDonald

et al. 2018

Biotech & Bioengineering

Self Cite

119

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“…By virtue of its simple, fast, and highly efficient multigene knockout capability, this platform can offer distinct advantages over current targeted methods as well as traditional methods for CHO cell line engineering including overexpression or introduction of new genes, RNA interference-mediated knockdown or regulation of gene expression [20,21]. knockout CHO cell line generation using ZFNs [12], the present platform is fast and efficient in regard to the one-step generation of multiple gene knockout CHO cell lines.…”

Section: Accepted Articlementioning

confidence: 99%

One‐step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

Grav

Lee

Gerling

et al. 2015

Biotechnology Journal

116

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The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.

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“…In fact, Clarke et al 2011 found a set of 287 genes for the prediction of cellular productivity in the model they established. However, as this study was based on an early microarray chip that contained only ~3500 expressed CHO genes out of the estimated 15 000 transcripts present in a cell at any time [9], it is likely that many more mRNAs and other factors like non-coding RNAs (ncRNAs) play a role in determining productivity [10].…”

Section: Introductionmentioning

confidence: 99%

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

Harreither

Hackl

Pichler

et al. 2015

Biotechnology Journal

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Over the last three decades, product yields from CHO cells have increased dramatically, yet specific productivity (qP) remains a limiting factor. In a previous study, using repeated cell-sorting, we have established different host cell subclones that show superior transient qP over their respective parental cell lines (CHO-K1, CHO-S). The transcriptome of the resulting six cell lines in different biological states (untransfected, mock transfected, plasmid transfected) was first explored by hierarchical clustering and indicated that gene activity associated with increased qP did not stem from a certain cellular state but seemed to be inherent for a high qP host line. We then performed a novel gene regression analysis identifying drivers for an increase in qP. Genes significantly implicated were first systematically tested for enrichment of GO terms using a Bayesian approach incorporating the hierarchical structure of the GO term tree. Results indicated that specific cellular components such as nucleus, ER, and Golgi are relevant for cellular productivity. This was complemented by targeted GSA that tested functionally homogeneous, manually curated subsets of KEGG pathways known to be involved in transcription, translation, and protein processing. Significantly implicated pathways included mRNA surveillance, proteasome, protein processing in the ER and SNARE interactions in vesicular transport.

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CHO microRNA engineering is growing up: Recent successes and future challenges

Cited by 78 publications

References 208 publications

A reference genome of the Chinese hamster based on a hybrid assembly strategy

A reference genome of the Chinese hamster based on a hybrid assembly strategy

One‐step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

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