Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool.
Embryonic Stem Cell Protocols
Methods in molecular biology, March 2015
Rahel Wettstein, Maxime Bodak, Constance Ciaudo, Wettstein, Rahel, Bodak, Maxime, Ciaudo, Constance
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.
|Members of the public||2||100%|
|Readers by professional status||Count||As %|
|Student > Ph. D. Student||19||26%|
|Student > Master||14||19%|
|Student > Bachelor||7||10%|
|Readers by discipline||Count||As %|
|Agricultural and Biological Sciences||25||35%|
|Biochemistry, Genetics and Molecular Biology||24||33%|
|Medicine and Dentistry||3||4%|