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A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing

Author

Listed:
  • Beverly Y. Mok

    (Broad Institute of MIT and Harvard
    Harvard University
    Harvard University)

  • Marcos H. de Moraes

    (University of Washington School of Medicine)

  • Jun Zeng

    (University of Washington School of Medicine)

  • Dustin E. Bosch

    (University of Washington School of Medicine
    University of Washington School of Medicine)

  • Anna V. Kotrys

    (Massachusetts General Hospital, Harvard Medical School
    Broad Institute of MIT and Harvard
    Institute of Biochemistry and Biophysics Polish Academy of Sciences)

  • Aditya Raguram

    (Broad Institute of MIT and Harvard
    Harvard University
    Harvard University)

  • FoSheng Hsu

    (University of Washington School of Medicine)

  • Matthew C. Radey

    (University of Washington School of Medicine)

  • S. Brook Peterson

    (University of Washington School of Medicine)

  • Vamsi K. Mootha

    (Massachusetts General Hospital, Harvard Medical School
    Broad Institute of MIT and Harvard)

  • Joseph D. Mougous

    (University of Washington School of Medicine
    University of Washington School of Medicine
    University of Washington)

  • David R. Liu

    (Broad Institute of MIT and Harvard
    Harvard University
    Harvard University)

Abstract

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

Suggested Citation

  • Beverly Y. Mok & Marcos H. de Moraes & Jun Zeng & Dustin E. Bosch & Anna V. Kotrys & Aditya Raguram & FoSheng Hsu & Matthew C. Radey & S. Brook Peterson & Vamsi K. Mootha & Joseph D. Mougous & David R, 2020. "A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing," Nature, Nature, vol. 583(7817), pages 631-637, July.
  • Handle: RePEc:nat:nature:v:583:y:2020:i:7817:d:10.1038_s41586-020-2477-4
    DOI: 10.1038/s41586-020-2477-4
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    Cited by:

    1. Li Mi & Ming Shi & Yu-Xuan Li & Gang Xie & Xichen Rao & Damu Wu & Aimin Cheng & Mengxiao Niu & Fengli Xu & Ying Yu & Ning Gao & Wensheng Wei & Xianhua Wang & Yangming Wang, 2023. "DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing," Nature Communications, Nature, vol. 14(1), pages 1-9, December.
    2. Yanan Zhao & Jiaojiao Hu & Shan-Shan Yang & Jing Zhong & Jianping Liu & Shuo Wang & Yuzhuo Jiao & Fang Jiang & Ruiyang Zhai & Bingnan Ren & Hua Cong & Yuwei Zhu & Fengtong Han & Jixian Zhang & Yue Xu , 2022. "A redox switch regulates the assembly and anti-CRISPR activity of AcrIIC1," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    3. Huawei Tong & Haoqiang Wang & Xuchen Wang & Nana Liu & Guoling Li & Danni Wu & Yun Li & Ming Jin & Hengbin Li & Yinghui Wei & Tong Li & Yuan Yuan & Linyu Shi & Xuan Yao & Yingsi Zhou & Hui Yang, 2024. "Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    4. Zhenzhen Chen & Qiankun He & Tiankun Lu & Jiayi Wu & Gaoli Shi & Luyun He & Hong Zong & Benyu Liu & Pingping Zhu, 2023. "mcPGK1-dependent mitochondrial import of PGK1 promotes metabolic reprogramming and self-renewal of liver TICs," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    5. Yuting Chen & Eriona Hysolli & Anlu Chen & Stephen Casper & Songlei Liu & Kevin Yang & Chenli Liu & George Church, 2022. "Multiplex base editing to convert TAG into TAA codons in the human genome," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    6. Jinchun Wu & Yang Liu & Liqiong Ou & Tingting Gan & Zhengrong Zhangding & Shaopeng Yuan & Xinyi Liu & Mengzhu Liu & Jiasheng Li & Jianhang Yin & Changchang Xin & Ye Tian & Jiazhi Hu, 2024. "Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    7. Friedrich Fauser & Bhakti N. Kadam & Sebastian Arangundy-Franklin & Jessica E. Davis & Vishvesha Vaidya & Nicola J. Schmidt & Garrett Lew & Danny F. Xia & Rakshaa Mureli & Colman Ng & Yuanyue Zhou & N, 2024. "Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    8. Ason C. Y. Chiang & Jan Ježek & Peiqiang Mu & Ying Di & Anna Klucnika & Martin Jabůrek & Petr Ježek & Hansong Ma, 2024. "Two mitochondrial DNA polymorphisms modulate cardiolipin binding and lead to synthetic lethality," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    9. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    10. Shao-Ming Gao & Han-Lan Fei & Qi Li & Li-Ying Lan & Li-Nan Huang & Peng-Fei Fan, 2024. "Eco-evolutionary dynamics of gut phageome in wild gibbons (Hoolock tianxing) with seasonal diet variations," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    11. Emily Zhang & Monica E. Neugebauer & Nicholas A. Krasnow & David R. Liu, 2024. "Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    12. Haifeng Sun & Zhaojun Wang & Limini Shen & Yeling Feng & Lu Han & Xuezhen Qian & Runde Meng & Kangming Ji & Dong Liang & Fei Zhou & Xin Lou & Jun Zhang & Bin Shen, 2023. "Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    13. Xiaoguang Pan & Kunli Qu & Hao Yuan & Xi Xiang & Christian Anthon & Liubov Pashkova & Xue Liang & Peng Han & Giulia I. Corsi & Fengping Xu & Ping Liu & Jiayan Zhong & Yan Zhou & Tao Ma & Hui Jiang & J, 2022. "Massively targeted evaluation of therapeutic CRISPR off-targets in cells," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    14. Pedro Silva-Pinheiro & Pavel A. Nash & Lindsey Van Haute & Christian D. Mutti & Keira Turner & Michal Minczuk, 2022. "In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue," Nature Communications, Nature, vol. 13(1), pages 1-9, December.
    15. Kayeong Lim & Sung-Ik Cho & Jin-Soo Kim, 2022. "Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    16. Julian C. W. Willis & Pedro Silva-Pinheiro & Lily Widdup & Michal Minczuk & David R. Liu, 2022. "Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    17. Young Geun Mok & Ji Min Lee & Eugene Chung & Jaesuk Lee & Kayeong Lim & Sung-Ik Cho & Jin-Soo Kim, 2022. "Base editing in human cells with monomeric DddA-TALE fusion deaminases," Nature Communications, Nature, vol. 13(1), pages 1-10, December.

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