Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

Chris J. Jung, Junli Zhang, Elizabeth Trenchard, Kevin C K Lloyd, David B. West, Barry Rosen, Pieter J. de Jong

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.

Original languageEnglish (US)
Pages (from-to)1-15
Number of pages15
JournalTransgenic Research
StateAccepted/In press - Nov 30 2016


  • Gene targeting
  • Transgenic mouse model

ASJC Scopus subject areas

  • Biotechnology
  • Animal Science and Zoology
  • Agronomy and Crop Science
  • Genetics


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