Abstract 6338: CRISPR/Cas9 <i>in vitro</i> modelling of a VUS mutation found in Peruvian patients diagnosed with hereditary breast cancer

Abstract Breast cancer is the most common neoplasm in women worldwide. In Latin America, rapid economic growth has driven epidemiological and demographic transitions that have increased the breast cancer burden. In Peru, breast cancer is the leading malignancy among women, with approximately 10% of cases linked to hereditary factors, primarily mutations in the BRCA1 and BRCA2 tumor suppressor genes. Many genetic variants are classified as Variants of Uncertain Significance (VUS), as their biological and clinical impacts remain unknown. In a previous work, our group identified six novel VUS in Peruvian families with hereditary cancer, including the c.5074+28T&amp;gt;A variant found in seven families. The overriding aim of the present study was to model the c.5074+28T&amp;gt;A VUS in a healthy breast tissue cell line (SVCT) using CRISPR/Cas9 technology. Gene editing was performed using ribonucleoprotein complexes (RNPs), whereby 03 different guide RNAs were tested. RNPs were transfected into SVCT cells by electroporation. Transfection and VUS insertion were verified via flow cytometry, T7EI assay, and Sanger sequencing. Single-cell clones were expanded, cryopreserved, and analyzed by PCR and enzymatic digestion (RFLP). Positive clones were further analyzed by Sanger sequencing, where 01 clone carrying the VUS c.5074+28T&amp;gt;A was identified (SVCT-VUS, clone C59). RT-PCR analyses showed no difference between unmodified SVCT cells (ctrl) and clone C59 at the mRNA sequence level. BRCA1 gene expression was then assessed by qPCR (Taqman assay), where BRCA1 downregulation was observed on SVCT-VUS compared to unmodified SVCTs. Although RNA from patients carrying the c.5074+28T&amp;gt;A variant was not available for analysis, SVCT-VUS cells showed a comparable BRCA1 gene expression to MDA-MB-231 breast cancer cell line, which possess a reportedly reduced BRCA1 mRNA and protein expression levels. Functional studies are still needed to confirm whether c.5074+28T&amp;gt;A affects BRCA1 expression at a protein level. These findings demonstrate a proof-of-concept for a CRISPR/Cas9 cell-modeling platform to study VUS in Peruvian patients with breast cancer and other diseases. Citation Format: Solange R. Paredes-Moscosso, Claudio Villegas-Llerena, Bernardo E. Quispe-Bravo, José Buleje, Maria L. Guevara-Fujita, Ricardo Fujita. CRISPR/Cas9 in vitro modelling of a VUS mutation found in Peruvian patients diagnosed with hereditary breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6338.