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SELECTBIO Conferences Genome Engineering


Efficient CRISPR/Cas9 Genome Engineering using Zygotes and Embryos Derived from Cas9 Overexpressing Transgenic Mice

Ben Davies, Group Leader, University of Oxford

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site specific nucleases has allowed the production time for mouse model development to be significantly reduced and the technique is now being widely adopted as the method of choice. For laboratories establishing these techniques, the exact mode of delivery of the CRISPR/Cas9 components needs to be optimized. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, purified in vitro transcribed mRNA and recombinant protein. As an alternative to these options, we have investigated the feasibility of supplying Cas9 genetically and for this purpose have generated transgenic mice which overexpress Cas9 ubiquitously, through the use of a CAG-Cas9 transgene, targeted to the ROSA26 locus. Heterozygous and homozygous transgenic mice are indistinguishable from wild-type litter mates and show normal fertility, suggesting that ubiquitous Cas9 expression in the absence of an activating gRNA is inert. Microinjection of fertilized zygotes prepared from transgenic Cas9 overexpressing females with guide-RNAs resulted in high numbers of mutant mice and whole embryo analysis revealed that the level of mutagenesis was found to be significantly higher when Cas9 was supplied genetically relative to exogenous supply. Genetic supply of Cas9 was able to mediate loss-of-function alleles by indel mutation, point mutation changes by homology directed repair and small deletions using two gRNAs.

Add to Calendar ▼2015-10-07 00:00:002015-10-08 00:00:00Europe/LondonGenome EngineeringGenome Engineering in Hanover, GermanyHanover,