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

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Author(s)Hamaker, Nathaniel K.
Author(s)Lee, Kelvin H.
Date Accessioned2023-08-01T15:11:23Z
Date Available2023-08-01T15:11:23Z
Publication Date2023-04-11
DescriptionThis article was originally published in Biotechnology and Bioengineering. The version of record is available at: https://doi.org/10.1002/bit.28393. © 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.
AbstractEfforts 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.
SponsorThe authors would like to thank Erica Green for assistance with the droplet digital polymerase chain reaction and William Hilliard for help with guide RNA design. They would also like to thank Mark Shaw at the University of Delaware DNA Sequencing and Genotyping Center for generating Sanger sequence reads. This study was supported by the National Science Foundation (1736123), the National Institutes of Health (T32GM008550), and the Department of Commerce, National Institute of Standards and Technology (70NANB17H002).
CitationHamaker, N. K., & Lee, K. H. (2023). High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors. Biotechnology and Bioengineering, 1– 22. https://doi.org/10.1002/bit.28393
ISSN1097-0290
URLhttps://udspace.udel.edu/handle/19716/33044
Languageen_US
PublisherBiotechnology and Bioengineering
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
Keywordscell line development
KeywordsChinese hamster ovary cell
KeywordsDNA repair
Keywordsrecombinase-mediated cassette exchange
KeywordssfOrange
Keywordssite-specific integration
TitleHigh-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors
TypeArticle
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