Abstract
The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2–19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas9 nuclease (7–320, 160.7 on average). In vivo off-target deamination is further validated through target site deep sequencing. Moreover, we demonstrated that six different ABE-gRNA complexes could be examined in a single EndoV-seq assay. Our study presents the first detection method to evaluate genome-wide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas9 nuclease.
Highlights
The adenine base editor (ABE), capable of catalyzing AT to GC conversions, is an important gene editing toolbox
We demonstrated that six different ABE-gRNA complexes could be examined in a single Endonuclease V (EndoV)-seq assay
We investigated the effects of mismatch on the AT-to-GC conversion efficiency of ABE at target sites
Summary
The adenine base editor (ABE), capable of catalyzing AT to GC conversions, is an important gene editing toolbox. We systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. Of the eight gRNAs we tested with ABE, 2–19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas[9] nuclease (7–320, 160.7 on average). Our study presents the first detection method to evaluate genomewide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas[9] nuclease. The First Affiliated Hospital, Sun Yat-sen University; MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging. Medicine of Guangdong Province, School of Life Sciences and the the First Affiliated Hospital, Sun Yat-sen University, 510275 Guangzhou, China. Medicine of Guangdong Province, School of Life Sciences and the the First Affiliated Hospital, Sun Yat-sen University, 510275 Guangzhou, China. 3 State Key
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