Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations
Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations
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消除碱基编辑器诱导的全基因组和转录组范围的脱靶突变
DOI：
10.1038/s41556-021-00671-4
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发表时间：
2021-05-10
影响因子：
21.3
通讯作者：
Chen, Jia
中科院分区：
文献类型：
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作者：
Wang, Lijie;Xue, Wei;Chen, Jia
CRISPR-Cas9与胞苷脱氨酶的融合导致能够进行可编程C-to-T编辑的碱基编辑器（BE），其在临床应用中具有潜力，但遭受脱靶（OT）突变。在这里，我们使用可切割的脱氧胞苷脱氨酶抑制剂（dCDI）结构域构建了一个Transformer BE（tBE）系统，该系统仅在全基因组和全转录组OT突变的背景水平下诱导有效的编辑。产生后，tBE在OT位点保持无活性，与可裂解的dCDI融合，因此消除了非预期的突变。当在靶位点结合时，tBE被转化以切割dCDI结构域并催化靶向脱氨基作用以进行精确的碱基编辑。在通过双腺相关病毒（AAV）系统递送到小鼠中后，tBE系统在Pcsk 9中产生了一个提前终止密码子，并显著降低了血清PCSK 9，导致总胆固醇降低了30-40%。tBE的开发建立了高度特异性的碱基编辑系统，其体内功效具有治疗应用的潜力。
The fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a similar to 30-40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.