Abstract
BackgroundBase 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.ResultsWe 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.ConclusionsUsing 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.
Highlights
Introduction or correction of mutations usingCRISPR-Cas9 generally depends on DNA double-strand breaks and homology-directed repair (HDR) using an exogenous DNA repair template
We introduced a cassette for guide RNA (gRNA) expression under the human U6 promoter into the base editor 3 (BE3) expression vector to generate a single delivery construct for targeted base editing
Green Fluorescent Protein (GFP) signal was detectable after 72 h by flow cytometry only in cells transfected with the construct encoding BE3 together with the gRNA targeting BFP H66, but not a non-targeting control gRNA
Summary
CRISPR-Cas generally depends on DNA double-strand breaks and homology-directed repair (HDR) using an exogenous DNA repair template This can be a very inefficient process, dependent upon the cell type and cell cycle phase [4,5,6]. Base Editing is a precise genome editing method that uses a deaminase-Cas fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. The third iteration of the Base Editor protein (BE3) is a fusion of three enzymes: rat APOBEC1 cytidine deaminase, Cas D10A nickase, and uracil DNA glycosylase inhibitor (UGI) [1] This multi-enzyme complex can introduce high-frequency C to T mutations (or G to A on the complementary strand) through enzymatic deamination of cytidine to uracil at the targeted locus. Replication across the uracil will lead to incorporation of a thymidine at this position due to the misrecognition of uracil
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