Original Research · Volume 12, Issue 4 · December 2025
1 Department of Molecular Oncology, Peking University, Beijing, China
2 Institute for Genome Research, University of Tokyo, Japan
3 Cancer Research UK Cambridge Institute, University of Cambridge, UK
BRCA1 is a critical tumor suppressor gene implicated in hereditary breast and ovarian cancers. Understanding the functional consequences of BRCA1 loss in mammary epithelial cells is essential for developing targeted therapeutic strategies. While previous studies have examined BRCA1 deficiency in human cell lines, murine models offer advantages for studying tissue-specific tumor initiation.
We utilized CRISPR-Cas9 technology to generate stable BRCA1 knockout clones in murine mammary epithelial cell lines (NMuMG and EpH4). Guide RNAs targeting exons 2 and 11 of the murine Brca1 gene were designed and validated. Knockout efficiency was confirmed through Western blot, Sanger sequencing, and functional DNA repair assays. Clones were characterized for proliferation rate, migration capacity, and genomic instability over 30 passages.
We achieved >95% knockout efficiency across both cell lines. BRCA1-null cells exhibited a 2.3-fold increase in proliferation rate, impaired homologous recombination repair (p<0.001), and elevated levels of chromosomal aberrations. Migration assays revealed a 40% increase in invasive capacity compared to wild-type controls. Transcriptomic analysis identified 847 differentially expressed genes, with enrichment in DNA damage response and epithelial-mesenchymal transition pathways.
Our CRISPR-based BRCA1 knockout system provides a robust platform for studying the molecular mechanisms of BRCA1-associated tumorigenesis. The observed phenotypes recapitulate key features of BRCA1-deficient human cancers, validating this model for preclinical drug screening and mechanistic studies.