Abstract
CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.
Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.
These authors contributed equally to this work
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Acknowledgments
We thank the Epigenesys network for access to its protocol database. We thank the Bourc’his laboratory and specifically Dr. Maxim Greenberg for technical assistance and discussions. We thank Dr. Tobias Beyer and the Ciaudo laboratory for the critical reading of the manuscript and for fruitful discussions.
CRISPR reagents are available to the academic community through Addgene (http://www.addgene.org/).
This work was supported by a core grant from ETH-Z (supported by Roche) and SNF (31003A_153220). M.B. is supported by a PhD fellowship from the ETH-Z foundation (ETH-21 13-1).
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Wettstein, R., Bodak, M., Ciaudo, C. (2015). Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1341. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_213
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DOI: https://doi.org/10.1007/7651_2015_213
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