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

Author

Listed:
  • Hongyuan Zhang

    (ShanghaiTech University)

  • Jiacheng Ma

    (ShanghaiTech University)

  • Zhaowei Wu

    (ShanghaiTech University)

  • Xiaoyang Chen

    (ShanghaiTech University)

  • Yangyang Qian

    (ShanghaiTech University)

  • Weizhong Chen

    (ShanghaiTech University
    Ningbo University)

  • Zhipeng Wang

    (ShanghaiTech University)

  • Ya Zhang

    (ShanghaiTech University)

  • Huanhu Zhu

    (ShanghaiTech University)

  • Xingxu Huang

    (Zhejiang Lab
    ShanghaiTech University)

  • Quanjiang Ji

    (ShanghaiTech University
    ShanghaiTech University
    Shanghai Clinical Research and Trial Center)

Abstract

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.

Suggested Citation

  • Hongyuan Zhang & Jiacheng Ma & Zhaowei Wu & Xiaoyang Chen & Yangyang Qian & Weizhong Chen & Zhipeng Wang & Ya Zhang & Huanhu Zhu & Xingxu Huang & Quanjiang Ji, 2024. "BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases," Nature Communications, Nature, vol. 15(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45114-4
    DOI: 10.1038/s41467-024-45114-4
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    1. Pengpeng Liu & Shun-Qing Liang & Chunwei Zheng & Esther Mintzer & Yan G. Zhao & Karthikeyan Ponnienselvan & Aamir Mir & Erik J. Sontheimer & Guangping Gao & Terence R. Flotte & Scot A. Wolfe & Wen Xue, 2021. "Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
    2. Yaojun Tong & Tue S. Jørgensen & Christopher M. Whitford & Tilmann Weber & Sang Yup Lee, 2021. "A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing," Nature Communications, Nature, vol. 12(1), pages 1-11, December.
    3. Andrew V. Anzalone & Peyton B. Randolph & Jessie R. Davis & Alexander A. Sousa & Luke W. Koblan & Jonathan M. Levy & Peter J. Chen & Christopher Wilson & Gregory A. Newby & Aditya Raguram & David R. L, 2019. "Search-and-replace genome editing without double-strand breaks or donor DNA," Nature, Nature, vol. 576(7785), pages 149-157, December.
    4. Guiquan Zhang & Yao Liu & Shisheng Huang & Shiyuan Qu & Daolin Cheng & Yuan Yao & Quanjiang Ji & Xiaolong Wang & Xingxu Huang & Jianghuai Liu, 2022. "Enhancement of prime editing via xrRNA motif-joined pegRNA," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
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