A compendium of stable hotspots in the CHO genome

Hilliard, William; Lee, Kelvin H.

doi:10.1002/bit.28390

Cited by 9 publications

(3 citation statements)

References 34 publications

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“…The optimized methods enable the generation of CHO cell clones with high-efficiency transgene integration at a single target locus as well as multiplex SSI at two loci simultaneously. Our lab has already begun to implement the DND approach for other SSI projects, which has proven to be robust and reliable (Hilliard & Lee, 2023). The genome editing tools and methods presented here could be readily adapted to support mammalian synthetic biology and advanced biomanufacturing applications.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…One of such approach is targeted integration; however, identifying a CHO cell genomic loci capable of supporting high-level protein expression is still a bottleneck. To address this need, Hilliard and Lee (2023), implemented the "Thousands of Reporters Integrated in Parallel" high-throughput screening method, identifying several hotspot candidates in the CHO genome exhibiting high transgene messenger RNA expression. In another study, Leitner et al(2023) presented a fast and robust method-the nanopore Cas9-targeted sequencing (nCats) pipeline-to characterize cell clones and isolate the most promising ones.…”

Section: Improving Cho Cell Line Performance Through Cell and Bioproc...mentioning

confidence: 99%

Advanced cell technologies: Making protein, cell, and gene therapies a reality

Geada,

Roldão,

Betenbaugh

et al. 2023

Biotech & Bioengineering

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Advanced cell technologies: Making protein, cell, and gene therapies a reality Researchers and engineers came together in Lisbon at the 27th Meeting of the European Society for Animal Cell Technology (ESACT 2022), to discuss the latest advances in technologies associated with protein-based biologics production, new modalities and cell, gene, and tissue therapies. Main contributions focused on how the capabilities of production platforms can be enhanced, and how to leverage them to generate new products. Some of the advances that were presented are discussed below, including those related to cell line development, metabolic engineering, analytics, chinese hamster ovary and insect cells platforms engineering, vesicle and viral vector production, and gene and cell therapy, along with some concluding remarks on the future of this important field.

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“…Consequently, massive parallel phenotypic perturbation screenings that are coupled to next-generation sequencing readouts in bulk or at single-cell level become feasible 25 . Recently, genetically-barcoded knock-in libraries have been used for deciphering optimal targeted integration loci in CHO antibody producer cells 26 as well as first genome-wide pooled CRISPR KO screenings to improve cellular bioproduction properties 27 .…”

Section: Introductionmentioning

confidence: 99%

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production

Bauer,

Oberist,

Poth

et al. 2024

Sci Rep

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Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.

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A compendium of stable hotspots in the CHO genome

Cited by 9 publications

References 34 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

Advanced cell technologies: Making protein, cell, and gene therapies a reality

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production

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