This study introduces BacPE, a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases. Prime editing allows precise single base substitutions and small insertions/deletions without homologous recombination or double-strand breaks in eukaryotic cells. However, its application in bacteria is limited due to inefficiency. The researchers identified sbcB, a 3'→5' DNA exonuclease, as a key factor impeding prime editing in E. coli. Deletion of sbcB, along with xseA and exoX, significantly enhanced prime editing efficiency by up to 100-fold. Inhibiting these exonucleases via CRISPRi enabled efficient prime editing in wild-type E. coli. The study also shows that 3'→5' DNA exonucleases degrade prime editing intermediates, inhibiting editing. By inhibiting these exonucleases, the 3'-directed hydrolysis model is disrupted, allowing efficient prime editing. The BacPE system, which inhibits sbcB, xseA, and exoX, achieves efficient prime editing in E. coli and other bacteria. The findings reveal a novel mechanism for prime editing inhibition in bacteria, paving the way for versatile applications of prime editing in bacterial genome engineering. The study highlights the importance of intrinsic pathways and redundant genes in restricting prime editing, and provides insights for improving genome editing tools. The developed BacPE platform offers a template for prime editing system development in diverse bacterial species, making it valuable for bacterial genome engineering.This study introduces BacPE, a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases. Prime editing allows precise single base substitutions and small insertions/deletions without homologous recombination or double-strand breaks in eukaryotic cells. However, its application in bacteria is limited due to inefficiency. The researchers identified sbcB, a 3'→5' DNA exonuclease, as a key factor impeding prime editing in E. coli. Deletion of sbcB, along with xseA and exoX, significantly enhanced prime editing efficiency by up to 100-fold. Inhibiting these exonucleases via CRISPRi enabled efficient prime editing in wild-type E. coli. The study also shows that 3'→5' DNA exonucleases degrade prime editing intermediates, inhibiting editing. By inhibiting these exonucleases, the 3'-directed hydrolysis model is disrupted, allowing efficient prime editing. The BacPE system, which inhibits sbcB, xseA, and exoX, achieves efficient prime editing in E. coli and other bacteria. The findings reveal a novel mechanism for prime editing inhibition in bacteria, paving the way for versatile applications of prime editing in bacterial genome engineering. The study highlights the importance of intrinsic pathways and redundant genes in restricting prime editing, and provides insights for improving genome editing tools. The developed BacPE platform offers a template for prime editing system development in diverse bacterial species, making it valuable for bacterial genome engineering.