Abstract P3-09-02: CRISPR-Cas9 mediated BRCA1 mutation in primary cells: Mutation efficiency and effects

Background: Germline mutations in Breast Cancer Associated (BRCA) 1 or 2 genes confer an increased risk of the development of breast and ovarian cancer. Germline mutation is followed by somatic loss of heterozygosity (LOH) resulting in biallelic inactivation. BRCA1 is involved in multiple homeostatic functions including control of chromatin organization, gene transcription, protein stability and cell division. Recent studies have demonstrated heterogeneity in LOH within and between premalignant and malignant breast tissues of BRCA1 mutation carriers. We hypothesize that LOH does not have a unitary effect on phenotype but differs by the function that is abrogated. Methods: To test our hypothesis, we adopted CRISPR-Cas9 gene editing technology. The guide RNAs for targeting the exon sequence in the RING finger, nuclear export signal (NES), nuclear localization signal (NLS) and BRCA1 C Terminus (BRCT) domain/motif of BRCA1 were designed and synthesized. MCF10A cells were transfected with a complex of guide RNA and Cas9 protein (RNP) to cause in/del mutation. The mutation was analyzed by both T7E1 assay, and an innovative and more precise method developed in our lab that utilizes linked nucleic acids (LNA) and qPCR. Proliferation and apoptosis assays were performed using the transfected cells. Organoids prepared from BRCA1 mutation carriers also were transfected with RNPs and the mutation burden determined. Results: Since single cell clones of the transfected MCF10A cells could not be selected and expanded, a pool of transfected cells was used for the analyses. T7E1 assay and qPCR analysis using LNAs demonstrated the presence of the mutations. A standard curve was created to enable the calculation of the mutation burden. IncuCyte analysis revealed increased proliferation and apoptosis, induced by irradiation, in cells with the mutation in Exon 10, where the extent of increase varied from 11% to 48% depending on the degree of mutation. In contrast, cells with the mutation in Exon 5 displayed diminished proliferation with no change in apoptosis. That mutations in exon10 and 5 have distinct biological effects when compared to the mutations in other exons is intriguing, and modification of binding proteins will be investigated. Organoids generated from BRCA1 mutation carriers (primary and nonmalignant cells) were able to be successfully transfected using the NEON electroporation system. Mutations were introduced by the CRISPR-Cas9 system and their extent quantified by our LNA-mediated qPCR method. Conclusions: CRISPR-Cas9-mediated gene editing of BRCA1 in MCF10A resulted in a change in the proliferation rate and the extent of apoptosis that is dependent on the location of the de novo mutation within the gene. The development of a novel method, LNA-mediated qPCR, provides quantitative information regarding the mutations that may be used to correlate mutation burden with biological functional change. Successful establishment of this BRCA1 tumorigenesis model has provided us with a method to test other putative tumor suppressors. Citation Format: Choi MR, Yadav S, Shidfar A, Khan SA, Clare SE. CRISPR-Cas9 mediated BRCA1 mutation in primary cells: Mutation efficiency and effects [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-02.