Expanded MutaT7 toolkit efficiently and simultaneously accesses all possible transition mutations in bacteria.
Expanded MutaT7 toolkit efficiently and simultaneously accesses all possible transition mutations in bacteria.
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DOI：
10.1093/nar/gkad003
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发表时间：
2023-04-11
影响因子：
14.9
通讯作者：
Shoulders, Matthew D.
中科院分区：
文献类型：
--
作者：
Mengiste, Amanuella A.;Wilson, Robert H.;Weissman, Rachel F.;Papa III, Louis J.;Hendel, Samuel J.;Moore, Christopher L.;Butty, Vincent L.;Shoulders, Matthew D.
由核苷酸脱氨酶-T7 RNA聚合酶融合介导的靶向诱变最近已成为一种新的和广泛有用的策略，以在体内定向进化运动的背景下提供遗传多样性。在这里，我们通过引入高活性的腺苷脱氨酶-T7 RNA聚合酶融合蛋白（eMutaT 7A →G）来扩展这种方法的实用性，从而导致更高的突变频率，以实现更快速的定向进化。我们还评估了使用这种更活跃的增变因子的好处和潜在的缺点。我们继续在大肠杆菌中证明，携带腺苷脱氨酶的突变体（MutaT 7A →G或eMutaT 7A →G）可以与携带胞苷脱氨酶的突变体（MutaT 7 C →T）串联使用，以同时引入所有可能的转换突变。我们通过探索抗菌药物耐药性的突变途径来说明这种体内诱变方法的有效性。这项工作为优化的MutaT 7工具的普遍应用奠定了基础，这些工具能够在不同生物体的体内定向进化活动中诱导所有类型的过渡突变。
Targeted mutagenesis mediated by nucleotide base deaminase–T7 RNA polymerase fusions has recently emerged as a novel and broadly useful strategy to power genetic diversification in the context of in vivo directed evolution campaigns. Here, we expand the utility of this approach by introducing a highly active adenosine deaminase–T7 RNA polymerase fusion protein (eMutaT7A→G), resulting in higher mutation frequencies to enable more rapid directed evolution. We also assess the benefits and potential downsides of using this more active mutator. We go on to show in Escherichia coli that adenosine deaminase-bearing mutators (MutaT7A→G or eMutaT7A→G) can be employed in tandem with a cytidine deaminase-bearing mutator (MutaT7C→T) to introduce all possible transition mutations simultaneously. We illustrate the efficacy of this in vivo mutagenesis approach by exploring mutational routes to antibacterial drug resistance. This work sets the stage for general application of optimized MutaT7 tools able to induce all types of transition mutations during in vivo directed evolution campaigns across diverse organisms.
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