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

Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks, showing promising potential for genome engineering in all kingdoms of life. Despite the successful improvement in using prime editors in eukaryotic cells, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic.Herein, we report the development of a versatile prime editing platform in bacteria (termed BacPE) by inhibiting 3-5 DNA exonucleases.Comparative prime editing in different bacterial species identified that the bacterial genetic background is likely a key factor in restricting efficient prime editing. Genetic screening of 129 Escherichia coli transposon mutants of potential DNA repair-related genes identified sbcB, a 3-5 DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, and deletion of sbcB increased the prime editing efficiency by up to 9-fold. Combinational deletions of sbcB with two additional 3-5 DNA exonucleases, xseA and exoX, drastically enhanced the prime editing efficiency by up to 100-fold. We propose a 3-directed hydrolysis model for inhibiting prime editing via degradation of the prime editing intermediates with 3-5 DNA exonucleases and demonstrate that the 3-directed hydrolysis mechanism is conserved in other bacterial species. Efficient prime editing in wild-type bacteria can be achieved by simultaneously inhibiting sbcB, xseA, and exoX via CRISPRi. Our results unveil the key intrinsic genetic factors that are adverse to efficient prime editing in bacteria and pave the way for versatile applications of prime editing for bacterial genome engineering.