Genome-wide profiling of adenine base editor specificity by EndoV-seq
題目：通過(guò)EndoV-seq對(duì)腺嘌呤單堿基編輯器進(jìn)行全基因組范圍的特異性分析
作者及單位：
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
發(fā)表期刊及時(shí)間：
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.
腺嘌呤堿基編輯器（ABE）能夠催化A/T轉(zhuǎn)換為G/C，是一種重要的基因編輯工具箱。在這里，我們使用自身開發(fā)的EndoV-seq平臺(tái)，系統(tǒng)地評(píng)估ABE對(duì)全基因組的脫氨作用脫靶效應(yīng)。 EndoV-seq利用核酸內(nèi)切酶V在體外切割由ABE脫氨基因組DNA的含肌苷的DNA鏈。然后對(duì)處理過(guò)的DNA進(jìn)行全基因組測(cè)序，從而鑒定脫靶位點(diǎn)。在我們用ABE測(cè)試的8種gRNA中，發(fā)現(xiàn)2-19（平均8.0）個(gè)脫靶位點(diǎn)，顯著少于經(jīng)典Cas9核酸酶產(chǎn)生的脫靶位點(diǎn)（7-320，均值為160.7）。體內(nèi)的脫氨作用脫靶效應(yīng)通過(guò)靶位點(diǎn)深度測(cè)序進(jìn)一步驗(yàn)證。此外，我們證明了可以在單個(gè)EndoV-seq測(cè)定中檢查出六種不同的ABE-gRNA復(fù)合物。我們的研究提出了第一種檢測(cè)ABE全基因組脫靶效應(yīng)的檢測(cè)方法，并揭示了ABE和經(jīng)典Cas9核酸酶之間可能存在的相似性和差異。
圖表選析
Figure 2. Using EndoV-seq to profile genome-wide off-target deamination by ABE. 利用EndoV-seq通過(guò)ABE工具分析全基因組脫氨脫靶基因。
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 使用人類HBG、VEGFA3、HEK293-2細(xì)胞，或小鼠Dmd gRNA ，用Cas9（藍(lán)色）、BE3（黃色）或ABE7.10（珊瑚色）處理的基因組DNA 的全基因組切割評(píng)分（截止分?jǐn)?shù)> 2.5）。未處理的基因組DNA（灰色）作為對(duì)照。紅色箭頭表示在靶位點(diǎn)。b EndoV捕獲位點(diǎn)（ABE7.10）、雙基因組捕獲位點(diǎn)（Cas9和BE3）、脫靶位點(diǎn)（分?jǐn)?shù)> 2.5）和所列g(shù)RNA的靶上位點(diǎn)的序列標(biāo)識(shí)。靶序列和PAM序列用藍(lán)色顯示。注意：Dmd gRNA 的長(zhǎng)度為19-nt。c 對(duì)比Cas9和BE3的雙基因組捕獲位點(diǎn)與EndoV-seq捕獲的ABE7.10位點(diǎn)（ABE7.10和BE3的得分> 0.1，Cas9的得分> 2.5）的維恩圖顯示了列出的目標(biāo)位點(diǎn)。d 含有編碼ABE7.10基因的載體與HBG gRNA（靶向HBG1和HBG2）和VEGFA3 gRNA 分別共轉(zhuǎn)染進(jìn)HEK-293T細(xì)胞當(dāng)中。在轉(zhuǎn)染后48小時(shí)，提取基因組DNA用于PCR擴(kuò)增和深度測(cè)序。GFP轉(zhuǎn)染的細(xì)胞用作對(duì)照。誤差柱代表標(biāo)準(zhǔn)誤（n ?= 3）。使用雙尾非配對(duì)t檢驗(yàn)計(jì)算統(tǒng)計(jì)顯著性（*** p?<0.001）。OT為脫靶（off-target）。VEGFA3的OT10未能通過(guò)PCR擴(kuò)增。源數(shù)據(jù)作為源數(shù)據(jù)文件提供。
