BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B

• Published in: BMC biology Vol. 16; no. 1; p. 150
• Main Authors: Coelho, Matthew A, Li, Songyuan, Pane, Luna Simona, Firth, Mike, Ciotta, Giovanni, Wrigley, Jonathan D, Cuomo, Maria Emanuela, Maresca, Marcello, Taylor, Benjamin J M
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 28-12-2018; BioMed Central; BMC
• Subjects: Animal models; Animals; APOBEC; APOBEC-1 Deaminase - genetics; Base editing; Cell culture; Cell cycle; CRISPR; CRISPR/Cas9; Cytidine Deaminase - genetics; Deoxyribonucleic acid; DNA; DNA repair; Domains; Editing; Editors; Efficiency; Enrichment; Enzymes; Fluorescence; Fluorescent reporter; Fusion protein; Gene editing; Gene Editing - methods; Genetic engineering; Genetic Engineering - methods; Genetic modification; Genetic research; Genome editing; Genomes; Humans; Innovations; Methodology; Minor Histocompatibility Antigens - genetics; Mutation; Nucleobases; Properties; Proteins; Proteins - genetics; Rats; Thymidine; CRISPR/Cas9; Fluorescent reporter; Base editing; Gene editing; APOBEC
• ISSN: 1741-7007; 1741-7007
• DOI: 10.1186/s12915-018-0617-1

Base Editing is a precise genome editing method that uses a deaminase-Cas9 fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. However, the efficient enrichment of genetically modified cells using this technique is limited by the ability to detect such events.
We have developed a Base Editing FLuorescent Activity REporter (BE-FLARE), which allows for the enrichment of cells that have undergone editing of target loci based on a fluorescence shift from BFP to GFP. We used BE-FLARE to evaluate the editing efficiency of APOBEC3A and APOBEC3B family members as alternatives deaminase domains to the rat APOBEC1 domain used in base editor 3 (BE3). We identified human APOBEC3A and APOBEC3B as highly efficient cytidine deaminases for base editing applications with unique properties.
Using BE-FLARE to report on the efficiency and precision of editing events, we outline workflows for the accelerated generation of genetically engineered cell models and the discovery of alternative base editors.