Engineering Oncogenic Hotspot Mutations on SF3B1 via CRISPR-Directed PRECIS Mutagenesis

• DOI: 10.1158/2767-9764.crc-24-0145
• Publication Date: 2024-08-28T12:52:11Z
• Authors: Mike M. Fernandez; Lei Yu; Qiong Jia; Xuesong Wang; Kevyn L. Hart; Zhenyu Jia; Ren-Jang Lin; Lili Wang
• Keywords: Gene Editing; 0303 health sciences; Phosphoproteins; Leukemia, Lymphocytic, Chronic, B-Cell; Introns; 03 medical and health sciences; Mutagenesis; Cell Line, Tumor; Mutation; Humans; RNA Splicing Factors; CRISPR-Cas Systems; Research Article

Abstract SF3B1 is the most recurrently mutated RNA splicing gene in cancer. However, research of its pathogenic role has been hindered by a lack disease-relevant cell line models. Here, our study compared four genome engineering platforms to establish mutant lines: CRISPR-Cas9 editing, AAV homology-directed repair base editing (ABEmax, ABE8e), and prime (PE2, PE3, PE5max). We showed that via PE5max achieved efficient K700E across wide range lines. Our approach was further refined coupling with fluorescent reporter leverages mutation-responsive synthetic intron mark successfully edited cells. By applying this approach, called coupled intron-assisted selection (PRECIS), we introduced hotspot mutation into two chronic lymphocytic leukemia lines, HG-3 MEC-1. demonstrated PRECIS-engineered cells faithfully recapitulate known phenotypes, including altered splicing, copy number variations, cell-growth defect. Moreover, discovered can cause loss Y chromosome leukemia. results showcase PRECIS an generalizable method for genetically faithful provides new insights on cancer enables generation lines relevant cellular context. Significance: This developed reliably efficiently engineer different contexts, thereby revealing novel roles driving aberrant clonal evolution, instability.