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 journalArticle

10 Citations (Scopus)

Abstract

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
DOIs
StateAccepted/In press - Nov 30 2016

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Gene Targeting
Zygote
gene targeting
zygote
mice
Proteins
proteins
Guide RNA
Alleles
RNA Cleavage
alleles
Gene Knockout Techniques
Homologous Recombination
homologous recombination
embryonic stem cells
Reporter Genes
reporter genes
exons
Exons

Keywords

  • CRISPR
  • Gene targeting
  • Transgenic mouse model

ASJC Scopus subject areas

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

Cite this

Jung, C. J., Zhang, J., Trenchard, E., Lloyd, K. C. K., West, D. B., Rosen, B., & de Jong, P. J. (Accepted/In press). Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein. Transgenic Research, 1-15. https://doi.org/10.1007/s11248-016-9998-5

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein. / Jung, Chris J.; Zhang, Junli; Trenchard, Elizabeth; Lloyd, Kevin C K; West, David B.; Rosen, Barry; de Jong, Pieter J.

In: Transgenic Research, 30.11.2016, p. 1-15.

Research output: Contribution to journalArticle

Jung, Chris J. ; Zhang, Junli ; Trenchard, Elizabeth ; Lloyd, Kevin C K ; West, David B. ; Rosen, Barry ; de Jong, Pieter J. / Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein. In: Transgenic Research. 2016 ; pp. 1-15.
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