[Genome-wide profiling of adenine base editor specificity by EndoV-seq](Genome-wide profiling of adenine base editor specificity by EndoV-seq)
題目：通過EndoV?seq對腺嘌呤單堿基編輯器進行全基 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*
Zhou Songyang
- 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烫幕，是一種重要的基因編輯工具箱剥懒。在這里钦勘，我們使用自身 開發(fā)的EndoV?seq平臺伏嗜，系統(tǒng)地評估ABE對全基因組的脫氨作用脫靶效應(yīng)殿衰。 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鏈袱吆。然后對處理過的DNA進行全基因組測序巧颈，從而鑒定脫靶位 點。在我們用ABE測試的8種gRNA中兜粘，發(fā)現(xiàn)2?19（平均8.0）個脫靶位點申窘，顯著少于經(jīng)典Cas9核酸酶產(chǎn)生的脫 靶位點（7?320，均值為160.7）孔轴。體內(nèi)的脫氨作用脫靶效應(yīng)通過靶位點深度測序進一步驗證剃法。此外，我們證 明了可以在單個EndoV?seq測定中檢查出六種不同的ABE?gRNA復(fù)合物路鹰。我們的研究提出了第一種檢測ABE全 基因組脫靶效應(yīng)的檢測方法贷洲，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。
圖表選摘：
Introduction
The recently developed targeted base replacement strategy using deaminases holds great promise for treating human diseases caused by pathogenic single nucleotide polymorphisms (SNPs). These RNA-directed programmable base editors can carry out single base pair conversions without inducing double strand breaks (DSBs)1. Cytosine base editors (CBEs) such as base editor 3 (BE3), which catalyze C?G to T?A base pair conversion1, have been successfully used to edit target bases in zebrafish, mouse, and human2,3,4,5,6,7. Base A deamination results in I (inosine) or X (xanthosine), where base I can pair with C and be replicated as G. Adenosine base editors (ABEs) rely on the tRNA-specific adenosine deaminase (TadA) from Escherichia coli to convert A to I on the non-complementary strand, and Cas9 nickase (nCas9) to nick the complementary strand of the target site, thus achieving A?T to G?C pair conversions8. We and others have shown efficient adenine base editing by ABEs in human cells, mouse embryos, and rat embryos8,9,10,11,12,13.
引言：
新近開發(fā)的脫氨酶靶向堿基置換策略在治療致病性單核苷酸多態(tài)性(SNPs)引起的人類疾病方面具有廣闊 的前景晋柱。這些RNA導(dǎo)向的可編程堿基編輯器可以在不引起雙鏈斷裂(DSBs)的情況下進行單堿基對轉(zhuǎn)換优构。 Ctosine堿基編輯器(CBES)，如堿基編輯器3(Be 3)雁竞，催化c·g到t·a堿基對轉(zhuǎn)換1钦椭，已成功地用于斑馬 魚、小鼠和人2浓领， 3， 4势腮， 5联贩， 6， 7的靶堿基的編輯捎拯。堿基脫氨導(dǎo)致I(肌苷)或x(黃嘌呤)泪幌，其中堿基I可以與c配對盲厌，并被復(fù)制為g。腺苷堿基編輯器(ABES)依賴于來自大腸桿菌的tRNA特異性腺苷脫氨酶(Tada)將非互補鏈上的a轉(zhuǎn)換為I祸泪，而Cas9鎳酶(Ncas9)則將目標(biāo)位點的互補鏈斷裂吗浩，從而實現(xiàn)a.t到g.c對的轉(zhuǎn)換。我 們和其他人已經(jīng)證明了ABES在人類細胞没隘、小鼠胚胎和大鼠胚胎8懂扼， 9， 10右蒲， 11阀湿， 12， 13中的有效腺嘌呤 堿基編輯瑰妄。
pproximately 48% of known pathogenic SNPs may be corrected by A?T-to-G?C conversion, and >20% of these may be targetable with SpCas9-based ABEs, indicating tremendous potential for SpCas9-based ABEs in gene therapy8,14. The advent of xCas9, with its broadened PAM sequence range (5′-NGN, 5′-GAA, 5′-GAT, and 5′-CAA), promises even wider utility of ABE, as more pathogenic G?C-to-A?T SNPs may be corrected by xCas9-ABE14. However, critical questions regarding the specificity and off-target effects of ABEs remain and must be addressed before any possible ==clinical translation==（臨床轉(zhuǎn)化）15.
大約48%的已知致病SNPs可以通過a·t-to-g.c轉(zhuǎn)換進行校正陷嘴，其中>20%的SNPs可能具有基于SpCas9 Abes 的靶向性，表明基于spcase 9的abes在基因治療方面具有巨大的潛力间坐。 Xcas9的出現(xiàn)擴大了pam序列的范圍 (5‘-NGN灾挨， 5’-gaa， 5‘-gat竹宋， 5’-CAA)劳澄，這使得abe的用途更加廣泛，因為更具有致病力的g·c-a·t SNPs可能被xcase 9-abe 14校正逝撬。然而浴骂，關(guān)于ABES的特異性和非靶點效應(yīng)的關(guān)鍵問題仍然存在，必須在任何 可能的臨床轉(zhuǎn)化之前加以解決
Digenome-seq has been developed to study genome-wide off-target effects of genome editing tools, where sequencing reads of in vitro processed genomic DNA are mapped to reference genomes with chromosomal sites scored based on DNA reads with identical 5′ or 3′ ends16,17. The method has been successfully used to evaluate genome-wide off-target effects of Cas9, Cpf1, and BE316,17,18,19,20,21,22. Because the enzymes used in these previous reports cannot cleave ABEmodified DNA22, new assays for assessing ABE activities are thus necessary.
二基因組-seq已經(jīng)被開發(fā)用來研究基因組編輯工具對基因組的非目標(biāo)效應(yīng)宪潮，在這種工具中溯警，體外處理的 基因組DNA的序列讀取被映射到參考基因組，根據(jù)dna讀取的相同5‘或3’端16狡相， 17標(biāo)記染色體位點梯轻。該 方法已成功地用于評估Cas9、 Cpf 1和316尽棕、 17喳挑、 18、 19滔悉、 20伊诵、 21、 22等基因的非靶標(biāo)效應(yīng)回官。由于以 前這些報告中使用的酶無法切割A(yù)BE修飾的dna 22曹宴，因此有必要進行新的評估ABE活性的測試
In this study, we describe a method (EndoV-seq) to investigate ABE specificity genome-wide, where in vitro deaminated genomic DNA is digested with Endonuclease V (EndoV) before being subjected to whole-genome sequencing (WGS). EndoV-seq enables us to evaluate both on-target and offtarget deamination by ABE. We further validate the results through target site deep sequencing to confirm the in vivo specificity of ABE. In addition, our findings show that EndoV-seq is amenable to multiplexing and offers clues to how ABE specificity may be improved.
在本研究中，我們描述了一種 方法(endov-seq)歉提，在進行全基因組測序(WGS)之前笛坦，在體外用內(nèi)切酶v(Endov)消化脫氨基因組DNA区转。 安托夫-塞克使我們能夠評估ABE對目標(biāo)和非目標(biāo)的破壞。我們通過靶位點深度測序進一步驗證了上述結(jié) 果版扩，證實了ABE在體內(nèi)的特異性废离。此外，我們的發(fā)現(xiàn)表明礁芦， endov-seq易于復(fù)用蜻韭，并為如何提高Abe的 特異性提供了線索
圖表選析：
Fig. 1 Using EndoV-seq to evaluate on-target deamination by ABE.
圖1. 采用EndoV-seq對ABE系統(tǒng)的中靶效應(yīng)進行評估
a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage.
以hek 293-2位點的序列為例，給出了用endov-seq評價體外ABE脫靶效應(yīng)的流程圖宴偿∠嫔樱基因組DNA先與重 組的abe7.10和適當(dāng)?shù)膅RNA共同孵育，再與endov酶切窥妇，從而使DNA既被ncas9鎳酶(黑三角)和 endov(紅三角，堿基I下游的一個殘基)切割娩践。切割的DNA隨后被碎裂活翩，末端修復(fù)為全基因組測序 (WGS)，覆蓋范圍為30-40倍.
b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100?ng) were incubated with ABE7.10 (300?nM) and HEK293-2 gRNA (900?nM) for 3?h before EndoV (1U) incubation (30?min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file.
用293 t細胞基因組DNA進行PCR擴增翻伺，擴增出跨越HEK 293-2位點的區(qū)域材泄。 PCR產(chǎn)物(10 0?ng)與 abe7.10(30 0?nm)和hek 2 93-2 gRNA(90 0?nm)共同孵育3?h后(1U)孵育30?min。產(chǎn)物經(jīng)瓊脂糖凝膠電泳 鑒定吨岭。以重組Cas9作為DNA裂解的陽性對照拉宗。分子量標(biāo)記大小為堿基對。源數(shù)據(jù)作為源數(shù)據(jù)文件提 供辣辫。
c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10?μg) treated with ABE7.10 (300?nM, 8?h)?±?EndoV (8U, 3?h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G.
用APE7.10(30 0?nm旦事， 8?h)?±?endov(8u， 3?h)處理hk 293-2 gRNA靶位點的Sanger測序圖譜急灭。模擬處理 的基因組DNA作為對照姐浮。帕姆藍色。目標(biāo)基地a葬馋，紅色和突出顯示紅色箭頭卖鲤。色譜峰， a為綠色畴嘶， t為紅 色蛋逾， c為藍色， g為黑色窗悯。
d PCR products from cwere deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red.
對c基因的PCR產(chǎn)物進行了深度測序区匣。每個等位基因的頻率顯示在右 邊。帕姆藍色蟀瞧。目標(biāo)基地a沉颂，紅色。
e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
整合基因組觀察器(IGV)對 hek293-2gRNA靶區(qū)進行全基因組測序悦污。目標(biāo)基地a铸屉，紅色。帕姆藍
Fig. 2 Using EndoV-seq to profile genome-wide off-target deamination by ABE.
圖2. 利用EndoV?seq 通過ABE工具分析全基因組脫氨脫靶效應(yīng)切端。
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彻坛、VEGFA3、HEK293?2細胞踏枣，或小鼠Dmd gRNA 昌屉，用Cas9（藍色）、BE3（黃色）或 ABE7.10（珊瑚色）處理的基因組DNA 的全基因組切割評分（截止分數(shù)> 2.5）茵瀑。未處理的基因組DNA（灰 色）作為對照间驮。紅色箭頭表示在靶位點。b EndoV捕獲位點（ABE7.10）马昨、雙基因組捕獲位點（Cas9和 BE3）竞帽、脫靶位點（分數(shù)> 2.5）和所列g(shù)RNA的靶上位點的序列標(biāo)識。靶序列和PAM序列用藍色顯示鸿捧。注意： Dmd gRNA 的長度為19?nt屹篓。c 對比Cas9和BE3的雙基因組捕獲位點與EndoV?seq捕獲的ABE7.10位點 （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)位點匙奴。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HEK?293T細胞當(dāng)中堆巧。在轉(zhuǎn) 染后48小時，提取基因組DNA用于PCR擴增和深度測序泼菌。GFP轉(zhuǎn)染的細胞用作對照谍肤。誤差柱代表標(biāo)準(zhǔn)誤（n ?= 3）。使用雙尾非配對t檢驗計算統(tǒng)計顯著性（*** p?<0.001）灶轰。OT為脫靶（off?target）谣沸。VEGFA3的OT10 未能通過PCR擴增。源數(shù)據(jù)作為源數(shù)據(jù)文件提供笋颤。
Fig. 3 Using multiplex EndoV-seq to profile off-target effects of ABE.
圖3使用多元EndoV-seq來分析ABE的脫靶效應(yīng)乳附。
a Human genomic DNA (10?μg) was treated with a mixture of 300?nM ABE7.10 and six gRNAs (200?nM each) (HEK293-2, EMX1, FANCF, HBB-28 (T?>?C), RNF2 and HBG) for 8?h and then with EndoV (8?U) for 3?h. The treated gnomic DNA was subsequently sequenced with ~30–40-fold coverage and genome-wide cleavage scores calculated. Sites with cleavage score of >2.5 are plotted here. Orange, genomic DNA treated with ABE7.10 and multiplex gRNAs. Gray, untreated genomic DNA. b Genome-wide cleavage scores (cutoff?>?2.5) of untreated (gray), monoplex EndoV-seq (blue), and multiplex EndoV-seq (orange) for the indicated gRNAs are plotted. Red arrows, on-target sites. c Sequence logos (by WebLogo) of multiplex EndoV-seq captured off-target (DNA cleavage scores of >2.5) and on-target sites. d A comparison of monoplex vs. multiplex EndoV-seq captured off-target sites (DNA cleavage scores of >2.5, on-target sites not shown)
用300nM ABE7.10和6個gRNA（每個200nM）（HEK293-2，EMX1伴澄，F(xiàn)ANCF赋除，HBB-28（T> C），RNF2和HBG）的混合物處理人基因組DNA（10μg）非凌。 8小時举农，然后使用EndoV（8 U）3小時。隨后對處理過的基因組 DNA進行測序敞嗡，覆蓋率為~30-40倍颁糟，并計算全基因組切割分數(shù)航背。這里繪制了具有> 2.5的解理分數(shù)的位點。用ABE7.10和多重gRNA處理的橙色基因組DNA棱貌【撩模灰色，未經(jīng)處理的基因組DNA婚脱。 b繪制了針對指定的gRNA的未處理（灰色）今魔，monoplex EndoV-seq（藍色）和多重EndoV-seq（橙色）的全基因組切割評分（截止值> 2.5）。紅色箭頭障贸，目標(biāo)網(wǎng)站错森。 c序列標(biāo)識（通過WebLogo）多重EndoV-seq捕獲的脫靶（DNA切割分數(shù)> 2.5）和目標(biāo)位點。 d monoplex與多重EndoV-seq捕獲的脫靶位點的比較（DNA切割得分> 2.5篮洁，未顯示的靶位點）
Fig. 4 The length of gRNAs affects ABE7.10 specificity. a HBG and VEGFA3 gRNAs of different length were designed based on the 20-mer gRNA (GX19) validated in Fig. 3d. Mismatched bases are in lower case. Target bases are in red. PAM sequences are in blue. b HEK-293T cells were co-transfected with the ABE7.10 expression vector and individual HBG or VEGFA3 gRNAs from a. Genomic DNA was then extracted for target site deep sequencing. GFP-transfected cells were used as controls. The frequencies of A-to-G conversion for each gRNA at both on-target and top potential off-target (OT) sites were calculated. OT10 of VEGFA3 was failed to be amplified. Error bars represent SEM (n?=?3). Statistical significance was calculated using a two-tailed unpaired t-test. *p?<?0.05; *p?<?0.01; and ****p?<?0.001. Source data are provided as a Source Data file. c The relative activity at each site was calculated by normalizing the A-to-G conversion frequency at that site to the on-target frequency of the GX19 gRNA. The ratios are presented as heat maps where higher values correspond to higher activities
圖4. gRNAs 的長度影響 ABE7.10 特異性涩维。 a 基于圖 3d 中驗證的 20-mer gRNA (GX19)設(shè)計不 同長度的 HBG 和 VEGFA3 gRNA。 不匹配的堿基用小寫字母表示袁波。目標(biāo)堿基是紅色的激挪。 PAM 序列是藍色的。 b HEK-293T 細胞與 ABE7.10 表達載體和 a 中的單個 HBG 或 VEGFA3 gRNAs 共轉(zhuǎn)染锋叨。 提取基因組 DNA 進行目標(biāo)位點深度測序垄分。以轉(zhuǎn)染 GFP 的細胞 作為對照。 計算了各 gRNA 在靶上和靶外(OT)點的 A-to-G 轉(zhuǎn)換頻率娃磺。 VEGFA3 的 OT10 擴增失敗薄湿。 誤差條表示 SEM (n = 3)。統(tǒng)計學(xué)顯著性采用雙尾不配對 t 檢驗偷卧。 * p < 0.05;* * p < 0.01;p < 0.001豺瘤。源數(shù)據(jù)作為源數(shù)據(jù)文件提供。 c 通過將該位點的 A-to-G 轉(zhuǎn)換頻率歸一 化為 GX19 gRNA 的中靶頻率來計算各位點的相對活性听诸。比率以熱圖的形式表示坐求，其中較 高的值對應(yīng)較高的活動。
翻譯小組：
王俊豪晌梨、葉名琛桥嗤、陳志榮、黃子亮仔蝌、鄧峻瑋泛领、鄭凌伶
