Abstract
Targeted mutagenesis is required to evaluate the function of DNA segments across the genome. In recent years the CRISPR/Cas9 technology has been widely used for functional genome studies and is partially replacing classical homologous recombination methods in different aspects. CRISPR/Cas9-derived tools indeed allow the production of a wide-range of engineered mutations: from point mutations to large chromosomal rearrangements such as deletions, duplications and inversions. Here we present a protocol to engineer Embryonic Stem Cells (ESC) with desired mutations using transfection of custom-made CRISPR/Cas9 vectors. These methods allow the in vivo modeling of congenital mutations and the functional interrogation of DNA sequences.
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Acknowledgments
We thank all members of the Mundlos lab in particular Katerina Kraft for sharing their experiences and fruitful discussions about the protocol. We also thank Heiner Schrewe and Lars Wittler for helping us to establish the ESC culture.
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Andrey, G., Spielmann, M. (2017). CRISPR/Cas9 Genome Editing in Embryonic Stem Cells. In: Ørom, U. (eds) Enhancer RNAs. Methods in Molecular Biology, vol 1468. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4035-6_15
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DOI: https://doi.org/10.1007/978-1-4939-4035-6_15
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-4033-2
Online ISBN: 978-1-4939-4035-6
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