Francesco DeMayo

Summary Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene‐edited animals creates unique issues including ill‐defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte‐specific, Gdf9 promoter driven, Cas9 transgene (Gdf9‐Cas9) targeted as a single copy into the Hprt1 locus. The X‐linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9‐Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9‐Cas9 does not mediate genome editing, confirming that Gdf9‐Cas9 is not expressed in embryos. Finally, we demonstrate that off‐target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9‐Cas9 transgene is a viable alternative to exogenous Cas9.