ABSTRACT
The advent of base editors (BEs) holds great potential for correcting pathogenic-related point mutations to treat relevant diseases. However, Cas9 nickase (nCas9)-derived BEs lead to DNA double-strand breaks, which can trigger unwanted DNA damage response (DDR). Here, we show that the original version of catalytically dead Cas12a (dCas12a)-conjugated BEs induce a basal level of DNA breaks and minimally activate DDR proteins, including H2AX, ATM, ATR, and p53. By fusing dCas12a with engineered human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A), we further develop the BEACON (base editing induced by human APOBEC3A and Cas12a without DNA break) system to achieve enhanced deamination efficiency and editing specificity. Efficient C-to-T editing is achieved by BEACON in mammalian cells at levels comparable to AncBE4max, with only low levels of DDR and minimal RNA off-target mutations. Importantly, BEACON induces in vivo base editing in mouse embryos, and targeted C-to-T conversions are detected in F0 mice.
Subject(s)
Bacterial Proteins/metabolism , CRISPR-Associated Proteins/metabolism , DNA Damage , Endodeoxyribonucleases/metabolism , Gene Editing/methods , 17-Hydroxysteroid Dehydrogenases/genetics , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Bacterial Proteins/genetics , CRISPR-Associated Proteins/genetics , Cytidine/metabolism , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA Replication , Deamination , Endodeoxyribonucleases/genetics , Female , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Phosphorylation , Proteins/genetics , Proteins/metabolism , Thymidine/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitins/metabolismABSTRACT
A variety of base editors have been developed to achieve C-to-T editing in different genomic contexts. Here, we compare a panel of five base editors on their C-to-T editing efficiencies and product purity at commonly editable sites, including some human pathogenic C-to-T mutations. We further profile the accessibilities of 20 base editors to all possible pathogenic mutations in silico. Finally, we build the BEable-GPS (Base Editable prediction of Global Pathogenic SNVs) database for users to select proper base editors to model or correct disease-related mutations. The in vivo comparison and in silico profiling catalog the availability of base editors and their broad applications in biomedical studies.
