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

Biotechnology and Bioprocess Engineering volume 20, pages 825–833 (2015)Cite this article

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

Keywords

  • genome editing
  • CRISPR/Cas9
  • Chinese hamster ovary (CHO) cells
  • iterative transfection