An Oocyte-Specific Cas9-Expressing Mouse for Germline CRISPR/Cas9-Mediated Genome Editing

Authors

Denise G Lanza
Jianqiang Mao
Isabel Lorenzo
Lan Liao
John R Seavitt
M Cecilia Ljungberg
Elizabeth M Simpson
Francesco J DeMayo
Jason D Heaney

Language

English

Publication Date

4-1-2024

Journal

Genesis

DOI

10.1002/dvg.23589

PMID

38523431

PMCID

PMC10987075

PubMedCentral® Posted Date

4-1-2025

PubMedCentral® Full Text Version

Author MSS

Abstract

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.

Keywords

Female, Male, Mice, Animals, Gene Editing, CRISPR-Cas Systems, RNA, Guide, CRISPR-Cas Systems, Mutation, Zygote, Animals, Genetically Modified, Oocytes, Genome editing, SpCas9, germline mutations, CRISPR

Published Open-Access

yes

Recommended Citation

Lanza, Denise G; Mao, Jianqiang; Lorenzo, Isabel; et al., "An Oocyte-Specific Cas9-Expressing Mouse for Germline CRISPR/Cas9-Mediated Genome Editing" (2024). Faculty and Staff Publications. 4979.
https://digitalcommons.library.tmc.edu/baylor_docs/4979