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Make-or-break prime editing for genome engineering in Streptococcus pneumoniae

Author

Listed:
  • Monica Rengifo-Gonzalez

    (CH-)

  • Maria-Vittoria Mazzuoli

    (CH-)

  • Axel B. Janssen

    (CH-)

  • Anne-Stéphanie Rueff

    (CH-)

  • Jessica Burnier

    (CH-)

  • Xue Liu

    (Shenzhen)

  • Jan-Willem Veening

    (CH-)

Abstract

CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.

Suggested Citation

  • Monica Rengifo-Gonzalez & Maria-Vittoria Mazzuoli & Axel B. Janssen & Anne-Stéphanie Rueff & Jessica Burnier & Xue Liu & Jan-Willem Veening, 2025. "Make-or-break prime editing for genome engineering in Streptococcus pneumoniae," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-59068-8
    DOI: 10.1038/s41467-025-59068-8
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    1. Xiangyang Li & Guiquan Zhang & Shisheng Huang & Yao Liu & Jin Tang & Mingtian Zhong & Xin Wang & Wenjun Sun & Yuan Yao & Quanjiang Ji & Xiaolong Wang & Jianghuai Liu & Shiqiang Zhu & Xingxu Huang, 2023. "Development of a versatile nuclease prime editor with upgraded precision," Nature Communications, Nature, vol. 14(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. Yutaro Shuto & Ryoya Nakagawa & Shiyou Zhu & Mizuki Hoki & Satoshi N. Omura & Hisato Hirano & Yuzuru Itoh & Feng Zhang & Osamu Nureki, 2024. "Structural basis for pegRNA-guided reverse transcription by a prime editor," Nature, Nature, vol. 631(8019), pages 224-231, July.
    4. 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.
    5. Daphne Collias & Elena Vialetto & Jiaqi Yu & Khoa Co & Éva d. H. Almási & Ann-Sophie Rüttiger & Tatjana Achmedov & Till Strowig & Chase L. Beisel, 2023. "Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    6. Jonathan Y. Hsu & Julian Grünewald & Regan Szalay & Justine Shih & Andrew V. Anzalone & Kin Chung Lam & Max W. Shen & Karl Petri & David R. Liu & J. Keith Joung & Luca Pinello, 2021. "PrimeDesign software for rapid and simplified design of prime editing guide RNAs," Nature Communications, Nature, vol. 12(1), pages 1-6, December.
    7. 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.
    8. Daphne Collias & Chase L. Beisel, 2021. "CRISPR technologies and the search for the PAM-free nuclease," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    9. Martin Peterka & Nina Akrap & Songyuan Li & Sandra Wimberger & Pei-Pei Hsieh & Dmitrii Degtev & Burcu Bestas & Jack Barr & Stijn Plassche & Patricia Mendoza-Garcia & Saša Šviković & Grzegorz Sienski &, 2022. "Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing," Nature Communications, Nature, vol. 13(1), pages 1-9, December.
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