Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.
Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.
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DOI：
10.1038/ncomms15790
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发表时间：
2017-06-06
影响因子：
16.6
通讯作者：
Liu DR
中科院分区：
文献类型：
--
作者：
Rees HA;Komor AC;Yeh WH;Caetano-Lopes J;Warman M;Edge ASB;Liu DR
我们最近开发了碱基编辑，这是一种基因组编辑方法，可以将一个碱基对可编程地转换为另一个碱基对，而不需要双链DNA切割，过多的随机插入和缺失，或依赖于同源定向修复。碱基编辑的应用受到脱靶活性和依赖于细胞内DNA传递的限制。在这里，我们描述了解决这些限制的两项进展。首先，我们通过在第三代碱基编辑器（BE3）中安装突变来生成高保真碱基编辑器（HF-BE3），从而大大减少了脱靶碱基编辑。接下来，我们纯化并将BE3和HF-BE3作为核糖核蛋白（RNP）复合物递送到哺乳动物细胞中，建立无dna碱基编辑。RNP递送BE3甚至比质粒转染HF-BE3具有更高的特异性，同时保持相当的靶向编辑水平。最后，我们将这些进展应用于将BE3 RNPs传递到斑马鱼胚胎和活小鼠内耳中，以实现体内特异性的无dna碱基编辑。第三代碱基编辑器包括催化失能的Cas9与胞苷脱氨酶和碱基切除修复抑制剂融合，能够高效、精确地编辑DNA中的单个碱基对。在这里，作者描述了碱基编辑器的工程和蛋白质传递，以提高它们的DNA特异性，并在活体动物中实现特异性碱基编辑。
We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo. Third-generation base editors consist of a catalytically disabled Cas9 fused to a cytidine deaminase and a base excision repair inhibitor, enabling efficient, precise editing of individual base pairs in DNA. Here the authors describe engineering and protein delivery of base editors to improve their DNA specificity and enable specific base editing in live animals.