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Updated: Dec 31, 2025
In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection
Published on: February 10, 2023
In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer
Stefano Annunziato1,2, Catrin Lutz1,2, Linda Henneman3
1Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Abstract:
Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.
Insights
This study introduces a new mouse model for precise cancer gene editing. It enables the efficient creation of missense mutations in vivo, advancing cancer driver research.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Genetically engineered mouse models (GEMMs) are crucial for cancer research.
- CRISPR gene disruption is fast but limited for missense mutations.
- A flexible platform is needed for precise in vivo mutation engineering.
Purpose of the Study:
- To develop a novel knock-in mouse model for precise somatic missense mutation engineering.
- To test the utility of a cytidine base editor system for in vivo gene editing.
- To assess the impact of defined allelic variants on mammary tumorigenesis.
Main Methods:
- Generated knock-in mice with Cre-conditional cytidine base editor expression.
- Utilized intraductal delivery of sgRNA-encoding vectors for somatic engineering.
- Assessed point mutation efficiency and effects on mammary tumorigenesis in situ.
Main Results:
- Achieved high-efficiency installation of point mutations in endogenous genes.
- Successfully recapitulated oncogenic nonsense mutations.
- Demonstrated proof of concept in a triple-negative breast cancer model.
Conclusions:
- The developed system enables precise somatic engineering of missense mutations in vivo.
- This flexible base editing platform can be extended to various tissues and cancer types.
- The model facilitates the study of defined allelic variants in cancer development.
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