Department of Chemistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.
Nucleic Acids Res. 2023 Jun 9;51(10):e59. doi: 10.1093/nar/gkad266.
Targeted in vivo hypermutation accelerates directed evolution of proteins through concurrent DNA diversification and selection. Although systems employing a fusion protein of a nucleobase deaminase and T7 RNA polymerase present gene-specific targeting, their mutational spectra have been limited to exclusive or dominant C:G→T:A mutations. Here we describe eMutaT7transition, a new gene-specific hypermutation system, that installs all transition mutations (C:G→T:A and A:T→G:C) at comparable frequencies. By using two mutator proteins in which two efficient deaminases, PmCDA1 and TadA-8e, are separately fused to T7 RNA polymerase, we obtained similar numbers of C:G→T:A and A:T→G:C substitutions at a sufficiently high frequency (∼6.7 substitutions in 1.3 kb gene during 80-h in vivo mutagenesis). Through eMutaT7transition-mediated TEM-1 evolution for antibiotic resistance, we generated many mutations found in clinical isolates. Overall, with a high mutation frequency and wider mutational spectrum, eMutaT7transition is a potential first-line method for gene-specific in vivo hypermutation.
靶向体内超突变通过同时进行 DNA 多样化和选择加速蛋白质的定向进化。尽管采用核碱基脱氨酶和 T7 RNA 聚合酶融合蛋白的系统具有基因特异性靶向，但它们的突变谱仅限于专一地或主导地 C:G→T:A 突变。在这里，我们描述了 eMutaT7transition，这是一种新的基因特异性超突变系统，可在可比频率下安装所有转换突变（C:G→T:A 和 A:T→G:C）。通过使用两种突变体蛋白，其中两种有效的脱氨酶 PmCDA1 和 TadA-8e 分别与 T7 RNA 聚合酶融合，我们在足够高的频率下获得了相似数量的 C:G→T:A 和 A:T→G:C 取代（在 80 小时的体内诱变过程中，在 1.3 kb 的基因中约有 6.7 个取代）。通过 eMutaT7transition 介导的 TEM-1 进化获得抗生素抗性，我们产生了许多在临床分离物中发现的突变。总体而言，eMutaT7transition 具有较高的突变频率和更广泛的突变谱，是基因特异性体内超突变的一种潜在首选方法。
