Strategies to improve engineering cancer associated SNVs with base editing Alyna Katti 1,2, Miguel Foronda 1, Eric Gardner 1, Jill Zimmerman 1,2, Bianca Diaz 1,2, Maria Paz Zafra 1, Sukanya Goswami 1, Lukas E. Dow 1,3,4 1. Sandra and Edward Meyer Cancer Center 2. Weill Cornell Graduate School 3. Department of Medicine, 4. Department of Biochemistry, Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids, and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, unlike Cas9, which shows remarkable efficacy in creating homozygous disruptive mutations following DNA double-strand breaks (DSBs), BE is relatively inefficient and identifying and isolating edited cells for analysis has proven challenging. To enable simple identification and enrichment of base editing events, we developed a “Gene On” (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We validate GO using an activatable GFP and use it to measure the kinetics, efficiency, PAM specificity, and fidelity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes, or enzymes such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. We further demonstrate the application of GO in detecting in vivo BE activity using a newly generated mouse model with inducible and reversible base editor expression that will enable generation of novel cancer models in vivo, explicitly exploring the role of missense mutations in disease. GO, together with the newly generated base editor expressing mouse model, is powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.