Programmable base editing of A?T to G?C in genomic DNA without DNA cleavage

題目：無需切裂DNA，對基因組DNA的A·T到G·C進行程序化堿基編輯

作者及單位：

Nicole M. Gaudelli, Alexis C. Komor, Holly A. Rees, Michael S. Packer, Ahmed H. Badran, David I. Bryson & David R. Liu

David R. Liu

• Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
• Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA
• Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

發(fā)表刊物及時間：

Nature volume551, pages464–471 (23 November 2017) Published: 25 October 2017

摘要：

The spontaneous deamination of cytosine is a major source of transitions from C?G to T?A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A?T base pairs to G?C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A?T to G?C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A?T base pairs efficiently to G?C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

胞嘧啶的自發(fā)脫氨是 C?G 到 T?A 堿基對轉(zhuǎn)化的主要來源， 這占了已知人類致病點突變的一 半。 因此后专， 有效地將目標 A?T 堿基對轉(zhuǎn)化為 G?C 的能力可以促進遺傳疾病的研究和治療划鸽。 腺嘌呤的脫氨作用產(chǎn)生肌苷输莺，肌苷被聚合酶作為鳥嘌呤處理，但目前還不知道 DNA 中有 酶能脫氨腺嘌呤裸诽。 這里我們介紹的腺嘌呤堿基編輯器(ABEs)可以介導基因組 DNA 中 A?T 到 G?C 的轉(zhuǎn)換嫂用。 我們研發(fā)出一種轉(zhuǎn)移 RNA 腺苷脫氨酶， 當它與催化受損的 CRISPRCas9 突變體融合時丈冬，可以作用于 DNA嘱函。 廣泛的定向進化和蛋白質(zhì)工程導致第 7 代 ABEs 能有效地將目標 A?T 堿基對轉(zhuǎn)化為 G?C(在人類細胞中大約 50%的效率)， 產(chǎn)品純度高(通常 至少 99.9%)埂蕊， 脫靶率低(通常不超過 0.1%)往弓。 與目前基于 Cas9 核苷酸的方法相比， ABEs 可以更有效蓄氧、更純粹地引入點突變函似，且不需要太大的非靶向基因組修飾，還可以在人類細 胞中進行疾病糾正或抑制突變喉童。 與以前的基礎編輯器一起撇寞， ABEs 可以直接、可編程地引 入所有四種過渡突變，而無需雙鏈 DNA 切割蔑担。

圖表選摘：

image.png

Figure 1: Scope and overview of base editing by an A?T to G?C base editor.

T?A到C?G堿基編輯系統(tǒng)的范圍和概述

a, Base pair changes required to correct pathogenic human SNPs in the ClinVar database39.

a 在ClinVar數(shù)據(jù)庫里牌废，需要糾正的致病性人類SNPs堿基對改變

b, The deamination of adenosine (A) forms inosine (I), which is read as guanosine (G) by polymerase enzymes. R?=?2′-deoxyribose in DNA, or ribose in RNA.

b 腺嘌呤（A）去氨基形成次黃嘌呤（I），次黃嘌呤被聚合酶鏈作用形成鳥嘌呤（G）啤握，

c, ABE-mediated A?T to G?C base editing strategy. ABEs contain a hypothetical deoxyadenosine deaminase, which is not known to exist in nature, and a catalytically impaired Cas9. They bind target DNA in a guide RNA-programmed manner, exposing a small bubble of single-stranded DNA. The hypothetical deoxyadenosine deaminase domain catalyses conversion of adenine to inosine within this bubble. Following DNA repair or replication, the original A?T base pair is replaced with a G?C base pair at the target site.

ABE介導的A?T to G?C堿基編輯系統(tǒng)流程圖鸟缕，ABEs包含一種假想的脫氧腺苷脫氨酶， 它在自然界中并不存在排抬，ABEs還 含有一種催化受損的Cas9.它們以引導RNA調(diào)控的方式結(jié)合目標DNA叁扫，暴露出一條單鏈DNA區(qū)域。在這段單鏈區(qū)域內(nèi)畜埋， 假想的脫氧腺苷脫氨酶催化腺嘌呤轉(zhuǎn)化為次黃嘌呤莫绣。隨后進行DNA修復或復制，原始的A?T堿基對就被G?C 堿基對取代 了

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Figure 4: Product purity of late-stage ABEs.

晚期ABE產(chǎn)物純度

Product distributions and indel frequencies at two representative human genomic DNA sites in HEK293T cells treated with ABE7.10 or ABE7.9 and the corresponding sgRNA, or in untreated HEK293T cells. At every position, 22,746–111,215 sequencing reads were used.

在用 ABE7.10 或 ABE7.9 處理過的 HEK293T 細胞和相應的 sgRNA 中悠鞍，或在未處理過的 HEK293T 細胞中对室，兩個具有代表性的人類基因組 DNA 位點的產(chǎn)物分布和脫靶頻率。每個 位點均使用 22,746-111,215 個測序數(shù)據(jù)讀入咖祭。

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Figure 5: Comparison of ABE7.10-mediated base editing and Cas9-mediated HDR, and application of ABE7.10 to two disease-relevant SNPs.

圖5. ABE7.10 介導堿基編輯與 Cas9 介導的 HDR 的比較掩宜，以及 ABE7.10 在兩個疾病相關 SNPs 中的應用。

a, A?T to G?C base editing efficiencies in HEK293T cells treated either with ABE7.10 or with Cas9 nuclease and an ssDNA donor template (following the CORRECT HDR method33) targeted to five human genomic DNA sites.

a 使用 ABE7.10 或 Cas9 核酸酶和 ssDNA 供體模板(按照正確的 HDR 方法 33)處理 HEK293T 細胞么翰，以 5 個人類基因組 DNA 位點為靶點牺汤， A·T 到 G·C 堿基的編輯效率。

b, Indel formation in HEK293T cells treated as described in a.

b. 按 a 中描述處理的 HEK293T 細胞后的脫靶信息浩嫌。

c, Application of ABE to install a disease-suppressing SNP, or to correct a disease-inducing SNP. Top, ABE7.10-mediated ?198T→C mutation (on the strand complementary to the one shown) in the promoter region of HBG1 and HBG2 genes in HEK293T cells. The target adenine is at protospacer positon 7. Bottom, ABE7.10-mediated reversion of the C282Y mutation in the HFE gene in LCL cells. The target adenine is at protospacer position 5.

c. 應用 ABE 來安裝抑制疾病的 SNP檐迟，或修正致病的 SNP。 上： ABE7.10-mediated-198 t→C 突變(鏈互補的如圖所示)在 HEK293T 細胞 HBG1 和 HBG2 基因的啟動子區(qū)域码耐。 靶腺嘌呤位于原間隔 7 號追迟。 下： ABE7.10 介導 LCL 細胞 HFE 基因 C282Y 突變的逆轉(zhuǎn)。靶腺嘌呤位于原間隔 5 號位 置骚腥。

翻譯小組：

王俊豪敦间、鄧峻瑋、鄭凌伶