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

Methods Mol Biol. 2016;1341:321-43. doi: 10.1007/7651_2015_213.


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.

Keywords: (Homozygous) knockout; CRISPR-Cas9 paired design; CRISPR/Cas9; Genome engineering; mESCs: mouse embryonic stem cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Knockout Techniques / methods*
  • Genome
  • Mice
  • Mice, Knockout
  • Mouse Embryonic Stem Cells / metabolism*
  • Plasmids / genetics
  • RNA, Guide, Kinetoplastida / genetics
  • Transfection / methods*


  • RNA, Guide