Enhancing prime editor activity by directed protein evolution in yeast.
Enhancing prime editor activity by directed protein evolution in yeast.
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通过酵母中的定向蛋白质进化增强主要编辑器活性。
DOI：
10.1038/s41467-024-46107-z
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发表时间：
2024
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
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作者：
Weber,Yanik;Böck,Desirée;Ivașcu,Anastasia;Mathis,Nicolas;Rothgangl,Tanja;Ioannidi,EleonoraI;Blaudt,AlexC;Tidecks,Lisa;Vadovics,Máté;Muramatsu,Hiromi;Reichmuth,Andreas;Marquart,KimF;Kissling,Lucas;Pardi,Norbert;Jinek,Martin;
引体编辑是一种高度通用的基因组编辑技术，可以引入碱基替换、插入和删除。然而，与传统的Cas9核酸酶相比，引物编辑器的活性较低。在本研究中，我们使用基于酵母的定向蛋白进化平台OrthoRep来提高PEs的编辑效率。经过几轮不断增加的选择压力的进化，我们在酵母细胞和生化分析中发现了多个对PE活性有积极影响的突变。结合两个最有效的突变——nCas9中的A259D氨基酸替换和M-MLV RT中的K445T替换——产生变体PE_Y18。与PEmax相比，将编码在DNA、mRNA或核糖核蛋白复合物上的PE_Y18递送到哺乳动物细胞系中，编辑率可提高3.5倍。此外，PE_Y18在体内传递到肝脏或大脑时支持更高的初始编辑率。我们的研究证明了在真核细胞中应用OrthoRep优化基因组编辑工具的概念验证。
Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations – the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT – results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.