无需切裂DNA，对基因组DNA的A·T到G·C进行程序化碱基编辑

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

题目：无需切裂DNA，对基因组DNA的A·T到G·C进行程序化碱基编辑

作者及单位：

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

发表刊物及时间：

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.

胞嘧啶的自发脱氨是 C•G 到 T•A 碱基对转化的主要来源， 这占了已知人类致病点突变的一 半。 因此， 有效地将目标 A•T 碱基对转化为 G•C 的能力可以促进遗传疾病的研究和治疗。 腺嘌呤的脱氨作用产生肌苷，肌苷被聚合酶作为鸟嘌呤处理，但目前还不知道 DNA 中有 酶能脱氨腺嘌呤。 这里我们介绍的腺嘌呤碱基编辑器(ABEs)可以介导基因组 DNA 中 A•T 到 G•C 的转换。 我们研发出一种转移 RNA 腺苷脱氨酶， 当它与催化受损的 CRISPRCas9 突变体融合时，可以作用于 DNA。 广泛的定向进化和蛋白质工程导致第 7 代 ABEs 能有效地将目标 A•T 碱基对转化为 G•C(在人类细胞中大约 50%的效率)， 产品纯度高(通常 至少 99.9%)， 脱靶率低(通常不超过 0.1%)。 与目前基于 Cas9 核苷酸的方法相比， ABEs 可以更有效、更纯粹地引入点突变，且不需要太大的非靶向基因组修饰，还可以在人类细 胞中进行疾病纠正或抑制突变。 与以前的基础编辑器一起， ABEs 可以直接、可编程地引 入所有四种过渡突变，而无需双链 DNA 切割。

图表选摘：

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

T•A到C•G碱基编辑系统的范围和概述

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

a 在ClinVar数据库里，需要纠正的致病性人类SNPs碱基对改变

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）去氨基形成次黄嘌呤（I），次黄嘌呤被聚合酶链作用形成鸟嘌呤（G），

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介导的A•T to G•C碱基编辑系统流程图，ABEs包含一种假想的脱氧腺苷脱氨酶， 它在自然界中并不存在，ABEs还 含有一种催化受损的Cas9.它们以引导RNA调控的方式结合目标DNA，暴露出一条单链DNA区域。在这段单链区域内， 假想的脱氧腺苷脱氨酶催化腺嘌呤转化为次黄嘌呤。随后进行DNA修复或复制，原始的A•T碱基对就被G•C 碱基对取代 了

image.png

Figure 4: Product purity of late-stage ABEs.

晚期ABE产物纯度

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 处理过的 HEK293T 细胞和相应的 sgRNA 中，或在未处理过的 HEK293T 细胞中，两个具有代表性的人类基因组 DNA 位点的产物分布和脱靶频率。每个 位点均使用 22,746-111,215 个测序数据读入。

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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.

图5. ABE7.10 介导碱基编辑与 Cas9 介导的 HDR 的比较，以及 ABE7.10 在两个疾病相关 SNPs 中的应用。

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 供体模板(按照正确的 HDR 方法 33)处理 HEK293T 细胞，以 5 个人类基因组 DNA 位点为靶点， A·T 到 G·C 碱基的编辑效率。

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

b. 按 a 中描述处理的 HEK293T 细胞后的脱靶信息。

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. 应用 ABE 来安装抑制疾病的 SNP，或修正致病的 SNP。 上： ABE7.10-mediated-198 t→C 突变(链互补的如图所示)在 HEK293T 细胞 HBG1 和 HBG2 基因的启动子区域。 靶腺嘌呤位于原间隔 7 号。 下： ABE7.10 介导 LCL 细胞 HFE 基因 C282Y 突变的逆转。靶腺嘌呤位于原间隔 5 号位 置。

翻译小组：

王俊豪、邓峻玮、郑凌伶