Genome-wide profiling of adenine base editor specificity by EndoV-seq

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.

CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors

“…6 and Supplementary Table 3). As a result, we found no noticeable off-target sites likewise to the previous ABE-based gene editing studies 24,25,26 , suggesting potential clinical utility.…”

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…EndoV‐seq has been involved in utilizing EndoV (deoxyinosine 3′ endonuclease) in vitro for nicking the DNA strand containing the inosine which is deaminated by ABE. The processed samples are then subjected to WGS for identification of off‐target sites . However, the specificity of BE and off‐target assessment needs an endonuclease for recognition of base I, the deaminated product of base A. EndoV from Thermotoga maritimais is a repair enzyme that has the ability to recognize the deoxyinosines and hydrolyze the second phosphodiester bond 3′ of the inosine base that results in nicked DNA …”

Improving the specificity of adenine base editor using high-fidelity Cas9

“…According to recent reports, the off-target sites of ABE is fewer in vitro analysis by whole-genome sequencing [22], and in vivo, the same results were observed in rice [1] and mouse embryos [28]. So we only chose the top three or four off-target sites with relatively strong off-target activity to test the specificity of ABEs.…”

“…The on-target editing activity of e-ABE7.10 is comparable to ABE7.10, but three others have varying degrees of reduction compared to ABE7.10. Therefore, we chose e-ABE7.10 for further testing on the other known sites (HBG2, HPRT and VEGFA3 [22,23]). As shown in Fig.…”

“…3A), indicating ABEs have tremendous potential in gene therapy [2]. However, ABEs can tolerate mismatch between sgRNA and target sequence, inducing off-target effects [22], which limit its application especially in clinical. The using of high-fidelity Cas9 variants in ABE have not been reported, except sniper-Cas9.…”