Harnessing A3G for efficient and selective C-to-T conversion at C-rich sequences.
Harnessing A3G for efficient and selective C-to-T conversion at C-rich sequences.
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DOI：
10.1186/s12915-020-00879-0
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发表时间：
2021-02-18
期刊：
影响因子：
5.4
通讯作者：
Huang X
中科院分区：
文献类型：
--
作者：
Yu W;Li J;Huang S;Li X;Li P;Li G;Liang A;Chi T;Huang X
通过使用催化受损的CAS蛋白将胞苷脱氨酶招募到目标C上，实现了位点特异性的C&gT；T DNA碱基编辑；目标C通常位于由引导RNA指定的5-nT编辑窗口内。包括RAT APOBEC1(Ra1)与nCas9融合的典型胞苷碱基编辑器Be3可以不分青红皂白地对编辑窗口内的多个C进行脱氨基处理，并在转录组上产生大量的非靶标编辑。DNA非靶标编辑的一个有效对策是用APOBEC蛋白取代Ra1，APOBEC蛋白在特定基序的背景下选择性地编辑C，如靶向TC的eA3A-BE3所示。然而，类似的编辑选择其他主题还没有被描述。特别是，在富含C的序列中靶向特定的C一直是具有挑战性的。在这里，我们试图面对这一挑战，并克服在Be3中看到的RNA脱靶效应。通过用优化的全人A3G基因(OA3G)取代Ra1，我们开发了oA3G-BE3，它选择性地针对CC和CCC，并且也没有对转录组的全局脱靶效应。此外，我们创建了oA3G-BE4max，这是oA3G-BE3的升级版本，具有强大的目标编辑功能。最后，我们证明了oA3G-BE4max在基因组上具有可以忽略的Cas9独立的脱靶效应。OA3G-BE4max可以高效、选择性地编辑C(C)C，是对eA3A-编辑者的补充，拓宽了Motif选择性编辑者的集体编辑范围，填补了基础编辑工具箱的空白。
Site-specific C>T DNA base editing has been achieved by recruiting cytidine deaminases to the target C using catalytically impaired Cas proteins; the target C is typically located within 5-nt editing window specified by the guide RNAs. The prototypical cytidine base editor BE3, comprising rat APOBEC1 (rA1) fused to nCas9, can indiscriminately deaminate multiple C’s within the editing window and also create substantial off-target edits on the transcriptome. A powerful countermeasure for the DNA off-target editing is to replace rA1 with APOBEC proteins which selectively edit C’s in the context of specific motifs, as illustrated in eA3A-BE3 which targets TC. However, analogous editors selective for other motifs have not been described. In particular, it has been challenging to target a particular C in C-rich sequences. Here, we sought to confront this challenge and also to overcome the RNA off-target effects seen in BE3. By replacing rA1 with an optimized human A3G (oA3G), we developed oA3G-BE3, which selectively targets CC and CCC and is also free of global off-target effects on the transcriptome. Furthermore, we created oA3G-BE4max, an upgraded version of oA3G-BE3 with robust on-target editing. Finally, we showed that oA3G-BE4max has negligible Cas9-independent off-target effects at the genome. oA3G-BE4max can edit C(C)C with high efficiency and selectivity, which complements eA3A-editors to broaden the collective editing scope of motif selective editors, thus filling a void in the base editing tool box.
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