Zhang Hongyuan, Ma Jiacheng, Wu Zhaowei, Chen Xiaoyang, Qian Yangyang, Chen Weizhong, Wang Zhipeng, Zhang Ya, Zhu Huanhu, Huang Xingxu, Ji Quanjiang
School of Physical Science and Technology & State Key Laboratory of Advanced Medical Materials and Devices, ShanghaiTech University, Shanghai, 201210, China.
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
Nat Commun. 2024 Jan 27;15(1):825. doi: 10.1038/s41467-024-45114-4.
Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks in eukaryotic cells. However, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic. Here, we report the determination of vital cellular factors that affect prime editing in bacteria. Genetic screening of 129 Escherichia coli transposon mutants identified sbcB, a 3'→5' DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, combinational deletions of which with two additional 3'→5' DNA exonucleases, xseA and exoX, drastically enhanced the prime editing efficiency by up to 100-fold. Efficient prime editing in wild-type E. coli can be achieved by simultaneously inhibiting the DNA exonucleases via CRISPRi. Our results pave the way for versatile applications of prime editing for bacterial genome engineering.
碱基编辑允许在真核细胞中精确安装任何单个碱基替换以及小的插入和缺失，而无需同源重组或双链DNA断裂。然而，其在细菌中的应用受到阻碍，阻碍高效碱基编辑的潜在机制仍然不明。在此，我们报告了影响细菌碱基编辑的重要细胞因子的确定。对129个大肠杆菌转座子突变体进行遗传筛选，确定了一种3'→5' DNA核酸外切酶sbcB是阻碍大肠杆菌碱基编辑的关键遗传决定因素，将其与另外两种3'→5' DNA核酸外切酶xseA和exoX进行组合缺失，可将碱基编辑效率大幅提高至100倍。通过CRISPRi同时抑制DNA核酸外切酶，可在野生型大肠杆菌中实现高效碱基编辑。我们的结果为碱基编辑在细菌基因组工程中的广泛应用铺平了道路。
