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Efficient CRISPR/Cas9-mediated multiplex genome editing in CHO cells via high-level sgRNA-Cas9 complex

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Abstract

Increasing demand for recombinant therapeutic proteins has warranted the need for an efficient host cell to produce high-quality proteins, with a high yield. Chinese hamster ovary (CHO) cells appear to meet this demand, and their genetic tailoring will facilitate improvements in their productivity for recombinant proteins. Recent advances in programmable RNA-guided Cas9 nuclease (RGN) have facilitated CHO cell engineering via site-specific genome editing. One critical determinant for increasing genomeediting efficiency is attaining a balanced expression level of Cas9 nuclease and guide RNAs in the nucleus. Here, we achieved high-level expression of Cas9 nuclease and single guide RNA (sgRNA), enhancing expression levels approximately three-fold over the conventional methodology by using an iterative transfection approach. We demonstrated that high abundance of sgRNA and Cas9 nuclease induced a two-fold increase in the site-specific mutation rate on average for both single and multiple genetic targets. Sequencing results confirmed frame-shift mutations at targeted genomic loci created by error-prone NHEJassociated mutations. Moreover, we controlled the amount of sgRNA-Cas9 complex formation in vitro and delivered the complex directly to cells, resulting in the maximization of mutation frequency by the high-level of sgRNA-Cas9 complex. Importantly, mutation rates of putative off-target sites remained minimal in spite of the improved genome-editing efficiency. These results provide an efficient strategy for editing the CHO genome with the reduction of the time-consuming screening efforts aimed at isolating clones with desirable properties.

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Authors and Affiliations

  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea

    Jongoh Shin, Namil Lee, Yoseb Song, Jinhyung Park, Sun Chang Kim, Gyun Min Lee & Byung-Kwan Cho

  2. KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea

    Jongoh Shin, Namil Lee, Yoseb Song, Sun Chang Kim & Byung-Kwan Cho

  3. Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, Seoul, 100-715, Korea

    Taek Jin Kang, Sun Chang Kim & Byung-Kwan Cho

Authors
  1. Jongoh Shin
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  2. Namil Lee
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  3. Yoseb Song
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  4. Jinhyung Park
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  5. Taek Jin Kang
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  6. Sun Chang Kim
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  7. Gyun Min Lee
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  8. Byung-Kwan Cho
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Correspondence to Byung-Kwan Cho.

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Shin, J., Lee, N., Song, Y. et al. Efficient CRISPR/Cas9-mediated multiplex genome editing in CHO cells via high-level sgRNA-Cas9 complex. Biotechnol Bioproc E 20, 825–833 (2015). https://doi.org/10.1007/s12257-015-0233-7

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  • DOI: https://doi.org/10.1007/s12257-015-0233-7

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