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

• Published in: Nature communications Vol. 15; no. 1; pp. 825 - 9
• Main Authors: Zhang, Hongyuan, Ma, Jiacheng, Wu, Zhaowei, Chen, Xiaoyang, Qian, Yangyang, Chen, Weizhong, Wang, Zhipeng, Zhang, Ya, Zhu, Huanhu, Huang, Xingxu, Ji, Quanjiang
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 27-01-2024; Nature Publishing Group; Nature Portfolio
• Subjects: 42/41; 45/70; 631/1647/1511; 631/208/325; 631/208/4041/3196; Bacteria; Coliforms; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA - genetics; DNA Breaks, Double-Stranded; DNA damage; E coli; Editing; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Exodeoxyribonucleases - genetics; Exonuclease; Genetic factors; Genetic screening; Genome editing; Genomes; Homologous recombination; Humanities and Social Sciences; multidisciplinary; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• 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 3ʹ→5ʹ DNA exonuclease, as a key genetic determinant in impeding prime editing in
E. coli
, combinational deletions of which with two additional 3ʹ→5ʹ 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.
Prime editing in bacteria is currently inefficient. Here the authors report BacPE, a versatile prime editing platform in
Escherichia coli
that works by inhibiting 3′→5′ DNA exonucleases, highlighting the intrinsic genetic factors that are adverse to efficient prime editing.