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A Cas3-base editing tool for targetable in vivo mutagenesis

Author

Listed:
  • Anna Zimmermann

    (VIB-KU Leuven Center for Microbiology
    KU Leuven)

  • Julian E. Prieto-Vivas

    (VIB-KU Leuven Center for Microbiology
    KU Leuven)

  • Charlotte Cautereels

    (VIB-KU Leuven Center for Microbiology
    KU Leuven)

  • Anton Gorkovskiy

    (VIB-KU Leuven Center for Microbiology
    KU Leuven)

  • Jan Steensels

    (VIB-KU Leuven Center for Microbiology
    KU Leuven)

  • Yves Peer

    (Ghent University
    VIB Center for Plant Systems Biology
    University of Pretoria
    Nanjing Agricultural University)

  • Kevin J. Verstrepen

    (VIB-KU Leuven Center for Microbiology
    KU Leuven)

Abstract

The generation of genetic diversity via mutagenesis is routinely used for protein engineering and pathway optimization. Current technologies for random mutagenesis often target either the whole genome or relatively narrow windows. To bridge this gap, we developed CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system), a tool that allows inducible and targetable, in vivo mutagenesis of genomic loci of up to 55 kilobases. CoMuTER employs the targetable helicase Cas3, signature enzyme of the class 1 type I-E CRISPR-Cas system, fused to a cytidine deaminase to unwind and mutate large stretches of DNA at once, including complete metabolic pathways. The tool increases the number of mutations in the target region 350-fold compared to the rest of the genome, with an average of 0.3 mutations per kilobase. We demonstrate the suitability of CoMuTER for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.

Suggested Citation

  • Anna Zimmermann & Julian E. Prieto-Vivas & Charlotte Cautereels & Anton Gorkovskiy & Jan Steensels & Yves Peer & Kevin J. Verstrepen, 2023. "A Cas3-base editing tool for targetable in vivo mutagenesis," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-39087-z
    DOI: 10.1038/s41467-023-39087-z
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    References listed on IDEAS

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    Cited by:

    1. Meiling Lu & Chenlin Yu & Yuwen Zhang & Wenjun Ju & Zhi Ye & Chenyang Hua & Jinze Mao & Chunyi Hu & Zhenhuang Yang & Yibei Xiao, 2024. "Structure and genome editing of type I-B CRISPR-Cas," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    2. Charlotte Cautereels & Jolien Smets & Peter Bircham & Dries De Ruysscher & Anna Zimmermann & Peter De Rijk & Jan Steensels & Anton Gorkovskiy & Joleen Masschelein & Kevin J. Verstrepen, 2024. "Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    3. Jing Guo & Luyao Gong & Haiying Yu & Ming Li & Qiaohui An & Zhenquan Liu & Shuru Fan & Changjialian Yang & Dahe Zhao & Jing Han & Hua Xiang, 2024. "Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells," Nature Communications, Nature, vol. 15(1), pages 1-16, December.

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