Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects
Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects
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DOI：
10.1126/sciadv.aba1773
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发表时间：
2020-07-01
期刊：
影响因子：
13.6
通讯作者：
Gao, Xue
中科院分区：
文献类型：
--
作者：
Lee, Sangsin;Ding, Ning;Gao, Xue
胞嘧啶碱基编辑器（CBE）使得能够在靶向基因座处进行有效的胞苷至胸苷（C-to-T）取代而没有双链断裂。然而，目前的CBE编辑其活性窗口内的所有C，产生不希望的旁观者突变。在最具挑战性的情况下，当旁观者C与靶向c相邻时，现有的碱基编辑器无法区分它们并编辑两个C。为了提高CBE的精确度，我们鉴定并工程化了人APOBEC 3G（A3 G）脱氨酶;当与Cas9切口酶融合时，所得A3 G-BE表现出对人细胞中5 '-CC-3'基序中的第二个C的选择性编辑。我们的A3 G-BE可以以高精度安装单个疾病相关的C到T替换。与BE 4 max相比，完全修饰的等位基因的百分比对于疾病校正超过6000倍，对于疾病建模超过600倍。基于双细胞胚胎注射方法和RNA测序分析，我们的A3 G-BE显示出最小的基因组和转录组范围的脱靶效应，实现了高靶向保真度。
Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted c, existing base editors fail to discriminate them and edit both Cs.To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5'-CC-3' motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.