Engineering of high-precision base editors for site-specific single nucleotide replacement
Engineering of high-precision base editors for site-specific single nucleotide replacement
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DOI：
10.1038/s41467-018-08034-8
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发表时间：
2019-01-25
影响因子：
16.6
通讯作者：
Bock, Ralph
中科院分区：
文献类型：
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作者：
Tan, Junjie;Zhang, Fei;Bock, Ralph
CRISPR/Cas类型的RNA引导的核酸酶可以被重新利用为可编程的核苷酸脱氨酶以介导靶向的核苷酸取代。这种碱基编辑器在基因组编辑、基因治疗和精准育种方面具有巨大的潜力。然而，目前的编辑器具有有限的特异性，因为它们在较大的序列窗口内编辑不同的和/或多个碱基。使用引起C到T突变的胞苷脱氨酶碱基编辑器，我们在这里表明，可以通过工程化编辑器的脱氨酶结构域和Cas结构域之间的连接来实现高编辑精度。通过系统地测试不同的接头序列并从脱氨酶中去除非必需序列，我们获得了具有窄活性窗口的高精度碱基编辑器，其可以高精度和高效率地选择性编辑特定位置处的单个胞苷。这些碱基编辑器将使基因组编辑能够在需要单核苷酸变化并且不能容忍相邻核苷酸的脱靶编辑的应用中使用。
RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.