BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases

作者：Hongyuan Zhang, Jiacheng Ma, Zhaowei Wu, Xiaoyang Chen, Yangyang Qian, Weizhong Chen, Zhipeng Wang, Ya Zhang, Huanhu Zhu, Xingxu Huang, Quanjiang Ji · 发表于：Nature Communications · 年份：2024 · DOI：10.1038/s41467-024-45114-4 · 被引用次数：31 · 研究领域：CRISPR and Genetic Engineering、Bacterial Genetics and Biotechnology、Advanced biosensing and bioanalysis techniques

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.