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Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs

Part of the Methods in Molecular Biology book series (MIMB,volume 1961)

Abstract

CRISPR/Cas9 is an effective and easy-to-use tool for editing the genome of many human cancer cell lines. However, in some hard-to-transfect cell lines and primary cells, gene editing is more challenging. This protocol details an electroporation-based protocol for the delivery of Cas9 protein from Streptococcus pyogenes complexed with chemically modified sgRNAs. We have found this protocol to work very efficiently in numerous cell lines and primary cells that are difficult to transfect by conventional chemical-based transfection methods.

Key words

  • CRISPR/Cas9
  • Gene editing
  • Chemically modified sgRNAs
  • Electroporation
  • Nucleoporation
  • Gene knockout

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Acknowledgments

R.O.B. gratefully acknowledges the support from a Lundbeck Foundation Fellowship (R238-2016-3349), an AIAS-COFUND (Marie Curie) fellowship from Aarhus Institute of Advanced Studies (AIAS) co-funded by Aarhus University’s Research Foundation and the European Union’s seventh Framework Program under grant agreement no 609033, the Novo Nordisk Foundation (NNF17OC0028894), Innovation Fund Denmark (8056-00010B), the Carlsberg Foundation (CF17-0129), Slagtermester Max Wørzner og Hustru Inger Wørzners Mindelegat, The AP Møller Foundation, and the Riisfort Foundation.

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Correspondence to Rasmus O. Bak .

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Laustsen, A., Bak, R.O. (2019). Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs. In: Luo, Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9170-9_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9170-9_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9169-3

  • Online ISBN: 978-1-4939-9170-9

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