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Publication
Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein.
Authors Jung CJ, Zhang J, Trenchard E, Lloyd KC, West DB, Rosen B, de Jong PJ
Submitted By Kent Lloyd on 4/4/2017
Status Published
Journal Transgenic research
Year 2017
Date Published
Volume : Pages 26 : 263 - 277
PubMed Reference 27905063
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.




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