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

题目：通过EndoV-seq对腺嘌呤单碱基编辑器进行全基因组范围的特异性分析

作者及单位：

Puping Liang, Xiaowei Xie, Shengyao Zhi, Hongwei Sun, Xiya Zhang, Yu Chen, Yuxi Chen, Yuanyan Xiong, Wenbin Ma, Dan Liu, Junjiu Huang* & Zhou Songyang*

Junjiu Huang：
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

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

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China

Key Laboratory of Reproductive Medicine of Guangdong Province, the Third Affiliated Hospital of Guangzhou Medical University, 510150, Guangzhou, China

Yangzhou Sun：
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

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

Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, 77030, Houston, TX, USA

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China

发表期刊及时间：

Nature Communicationsvolume 10, Article number: 67 (2019)

Published: 08 January 2019

摘要：

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.

腺嘌呤碱基编辑器（ABE）能够催化A/T转换为G/C，是一种重要的基因编辑工具箱。在这里，我们使用自身开发的EndoV-seq平台，系统地评估ABE对全基因组的脱氨作用脱靶效应。 EndoV-seq利用核酸内切酶V在体外切割由ABE脱氨基因组DNA的含肌苷的DNA链。然后对处理过的DNA进行全基因组测序，从而鉴定脱靶位点。在我们用ABE测试的8种gRNA中，发现2-19（平均8.0）个脱靶位点，显着少于经典Cas9核酸酶产生的脱靶位点（7-320，均值为160.7）。体内的脱氨作用脱靶效应通过靶位点深度测序进一步验证。此外，我们证明了可以在单个EndoV-seq测定中检查出六种不同的ABE-gRNA复合物。我们的研究提出了第一种检测ABE全基因组脱靶效应的检测方法，并揭示了ABE和经典Cas9核酸酶之间可能存在的相似性和差异。

图表选析

Figure 2. Using EndoV-seq to profile genome-wide off-target deamination by ABE. 利用EndoV-seq通过ABE工具分析全基因组脱氨脱靶基因。

a Genome-wide cleavage scores (cutoff score of >2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG, VEGFA3, HEK293-2, or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of >2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of >0.1 for ABE7.10 and BE3, score of >2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM (n = 3). Statistical significance was calculated using a two-tailed unpaired t-test (***p < 0.001). OT, off-target. OT10 of VEGFA3 failed to be amplified by PCR. Source data are provided as a Source Data file.

a 使用人类HBG、VEGFA3、HEK293-2细胞，或小鼠Dmd gRNA ，用Cas9（蓝色）、BE3（黄色）或ABE7.10（珊瑚色）处理的基因组DNA 的全基因组切割评分（截止分数> 2.5）。未处理的基因组DNA（灰色）作为对照。红色箭头表示在靶位点。b EndoV捕获位点（ABE7.10）、双基因组捕获位点（Cas9和BE3）、脱靶位点（分数> 2.5）和所列gRNA的靶上位点的序列标识。靶序列和PAM序列用蓝色显示。注意：Dmd gRNA 的长度为19-nt。c 对比Cas9和BE3的双基因组捕获位点与EndoV-seq捕获的ABE7.10位点（ABE7.10和BE3的得分> 0.1，Cas9的得分> 2.5）的维恩图显示了列出的目标位点。d 含有编码ABE7.10基因的载体与HBG gRNA（靶向HBG1和HBG2）和VEGFA3 gRNA 分别共转染进HEK-293T细胞当中。在转染后48小时，提取基因组DNA用于PCR扩增和深度测序。GFP转染的细胞用作对照。误差柱代表标准误（n  = 3）。使用双尾非配对t检验计算统计显着性（*** p <0.001）。OT为脱靶（off-target）。VEGFA3的OT10未能通过PCR扩增。源数据作为源数据文件提供。