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Methods. 2014 Sep;69(2):171-178. doi: 10.1016/j.ymeth.2014.05.003. Epub 2014 Jun 12.

Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system.

Author information

Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 USA.
Department of Human Science, Georgetown University Medical Center, 3700 Reservoir Rd. NW, Washington, DC 20057, USA.
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.
Contributed equally


Gene targeting - homologous recombination between transfected DNA and a chromosomal locus - is greatly stimulated by a DNA break in the target locus. Recently, the RNA-guided Cas9 endonuclease, involved in bacterial adaptive immunity, has been modified to function in mammalian cells. Unlike other site-specific endonucleases whose specificity resides within a protein, the specificity of Cas9-mediated DNA cleavage is determined by a guide RNA (gRNA) containing an ∼20 nucleotide locus-specific RNA sequence, representing a major advance for versatile site-specific cleavage of the genome without protein engineering. This article provides a detailed method using the Cas9 system to target expressed genes in mouse embryonic stem cells. In this method, a promoterless marker flanked by short homology arms to the target locus is transfected into cells together with Cas9 and gRNA expression vectors. Importantly, biallelic gene knockout is obtained at high frequency by only one round of targeting using a single marker.


Cas9; Double-strand break (DSB); Embryonic stem cells; Gene targeting; Genome editing; Homologous recombination

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