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Protein Cell. 2017 Aug;8(8):601-611. doi: 10.1007/s13238-017-0418-2. Epub 2017 Jun 5.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes.

Liang P1,2,3, Sun H4, Sun Y4, Zhang X4, Xie X4, Zhang J4, Zhang Z4,5, Chen Y4, Ding C6, Xiong Y4, Ma W4, Liu D7, Huang J8,9, Songyang Z10,11,12,13.

Author information

1
Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. liangpp5@mail.sysu.edu.cn.
2
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510275, China. liangpp5@mail.sysu.edu.cn.
3
Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China. liangpp5@mail.sysu.edu.cn.
4
Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
5
Guangzhou Magigen Biotechnology Co.Ltd, Guangzhou, 510320, China.
6
Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China.
7
Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA.
8
Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. hjunjiu@mail.sysu.edu.cn.
9
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510275, China. hjunjiu@mail.sysu.edu.cn.
10
Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. songyanz@mail.sysu.edu.cn.
11
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510275, China. songyanz@mail.sysu.edu.cn.
12
Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China. songyanz@mail.sysu.edu.cn.
13
Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA. songyanz@mail.sysu.edu.cn.

Abstract

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

KEYWORDS:

base editor; high-fidelity; mouse embryos; proximal-site deamination; whole-genome sequencing

PMID:
28585179
PMCID:
PMC5546933
DOI:
10.1007/s13238-017-0418-2
[Indexed for MEDLINE]
Free PMC Article

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