Biotechnology and Bioprocess Engineering

, Volume 20, Issue 5, pp 825–833 | Cite as

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

  • Jongoh Shin
  • Namil Lee
  • Yoseb Song
  • Jinhyung Park
  • Taek Jin Kang
  • Sun Chang Kim
  • Gyun Min Lee
  • Byung-Kwan Cho
Research Paper

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.

Keywords

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

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Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Jongoh Shin
    • 1
    • 2
  • Namil Lee
    • 1
    • 2
  • Yoseb Song
    • 1
    • 2
  • Jinhyung Park
    • 1
  • Taek Jin Kang
    • 3
  • Sun Chang Kim
    • 1
    • 2
    • 3
  • Gyun Min Lee
    • 1
  • Byung-Kwan Cho
    • 1
    • 2
    • 3
  1. 1.Department of Biological SciencesKorea Advanced Institute of Science and TechnologyDaejeonKorea
  2. 2.KI for the BioCenturyKorea Advanced Institute of Science and TechnologyDaejeonKorea
  3. 3.Department of Chemical and Biochemical EngineeringDongguk University-SeoulSeoulKorea

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