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Nat Commun. 2019 Jan 8;10(1):67. doi: 10.1038/s41467-018-07988-z.

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

Liang P1,2, Xie X1,3, Zhi S1, Sun H1, Zhang X1, Chen Y1, Chen Y1, Xiong Y1, Ma W1, Liu D4, Huang J5,6,7,8, Songyang Z9,10,11,12.

Author information

1
The First Affiliated Hospital, Sun Yat-sen University; MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, China.
2
Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the the First Affiliated Hospital, Sun Yat-sen University, 510275, Guangzhou, China.
3
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 3000000, Tianjin, China.
4
Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, 77030, Houston, TX, USA.
5
The First Affiliated Hospital, Sun Yat-sen University; MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, China. hjunjiu@mail.sysu.edu.cn.
6
Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the the First Affiliated Hospital, Sun Yat-sen University, 510275, Guangzhou, China. hjunjiu@mail.sysu.edu.cn.
7
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China. hjunjiu@mail.sysu.edu.cn.
8
Key Laboratory of Reproductive Medicine of Guangdong Province, the Third Affiliated Hospital of Guangzhou Medical University, 510150, Guangzhou, China. hjunjiu@mail.sysu.edu.cn.
9
The First Affiliated Hospital, Sun Yat-sen University; MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, China. songyanz@mail.sysu.edu.cn.
10
Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the the First Affiliated Hospital, Sun Yat-sen University, 510275, Guangzhou, China. songyanz@mail.sysu.edu.cn.
11
Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, 77030, Houston, TX, USA. songyanz@mail.sysu.edu.cn.
12
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China. songyanz@mail.sysu.edu.cn.

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.

PMID:
30622278
PMCID:
PMC6325126
DOI:
10.1038/s41467-018-07988-z
[Indexed for MEDLINE]
Free PMC Article

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