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Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering

Part of the Methods in Molecular Biology book series (MIMB,volume 1772)

Abstract

Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

Keywords

  • CRISPR/Cas9
  • Cas9 ribonucleoproteins
  • Cas9 RNP complex
  • Gene deletion
  • T7E1 assay
  • CHO cells

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Acknowledgments

This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031957 to B.-K.C) and Basic Science Research Program (2015R1A2A2A01008006 to B.-K.C., 2015R1C1A2A01053505 to S.C.) through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (MISP).

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Correspondence to Byung-Kwan Cho .

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Shin, J., Lee, N., Cho, S., Cho, BK. (2018). Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_8

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  • DOI: https://doi.org/10.1007/978-1-4939-7795-6_8

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  • Publisher Name: Humana Press, New York, NY

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