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

Methods in Molecular Biology
Rahel WettsteinConstance Ciaudo

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.

Citations

Jan 20, 2017·The Journal of Cell Biology·Daniel Cirera-SalinasConstance Ciaudo
Nov 8, 2016·Journal of Zhejiang University. Science. B·Yang GuoCharlie Xiang
Aug 29, 2019·Iranian Journal of Biotechnology·Maryam MehravarMaryam Salimi
Feb 11, 2020·Frontiers in Cell and Developmental Biology·Madlen MüllerConstance Ciaudo
Aug 22, 2017·Frontiers in Molecular Neuroscience·Rahul MittalXue Z Liu
Jul 7, 2017·Transgenic Research·Hirak Kumar BarmanPallipuram Jayasankar

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