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Erschienen in:

2015 | Online First | Buchkapitel

Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool

verfasst von : Rahel Wettstein, Maxime Bodak, Constance Ciaudo, Ph.D.

Erschienen in: Methods in Molecular Biology™

Verlag: Springer New York

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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.
Literatur
6.
Zurück zum Zitat Ishino Y, Shinagawa H, Makino K et al (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433PubMedCentralPubMed Ishino Y, Shinagawa H, Makino K et al (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433PubMedCentralPubMed
26.
Zurück zum Zitat Koike-Yusa H, Li Y, Tan E-P et al (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32:267–273. doi:10.1038/nbt.2800 CrossRefPubMed Koike-Yusa H, Li Y, Tan E-P et al (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32:267–273. doi:10.​1038/​nbt.​2800 CrossRefPubMed
Metadaten
Titel
Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool
verfasst von
Rahel Wettstein
Maxime Bodak
Constance Ciaudo, Ph.D.
Copyright-Jahr
2015
Verlag
Springer New York
DOI
https://doi.org/10.1007/7651_2015_213