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


Authors: Zhang, Y.; Vanoli, F.; LaRocque, J. R.; Krawczyk, P. M.; Jasin, M.
Article Title: Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system
Abstract: 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. © 2014 Published by Elsevier Inc.
Keywords: controlled study; nonhuman; animal cell; mouse; mammalia; homologous recombination; gene targeting; gene expression; embryo; embryonic stem cell; gene locus; bacteria (microorganisms); genetic transfection; double stranded dna break; embryonic stem cells; caspase 9; endonuclease; enzyme specificity; dna cleavage; rna sequence; expression vector; allelism; priority journal; article; cas9; double-strand break (dsb); genome editing
Journal Title: Methods
Volume: 69
Issue: 2
ISSN: 1046-2023
Publisher: Academic Press Inc., Elsevier Science  
Date Published: 2014-09-01
Start Page: 171
End Page: 178
Language: English
DOI: 10.1016/j.ymeth.2014.05.003
PROVIDER: scopus
PUBMED: 24929070
PMCID: PMC4405113
DOI/URL:
Notes: Export Date: 4 May 2015 -- Source: Scopus
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MSK Authors
  1. Maria Jasin
    213 Jasin
  2. Jeannine Larocque Kappas
    6 Kappas
  3. Yu Zhang
    8 Zhang
  4. Fabio Vanoli
    8 Vanoli