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Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope

Author

Listed:
  • Nathan Bamidele

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School)

  • Han Zhang

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School)

  • Xiaolong Dong

    (Tessera Therapeutics)

  • Haoyang Cheng

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School)

  • Nicholas Gaston

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School)

  • Hailey Feinzig

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School)

  • Hanbing Cao

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School)

  • Karen Kelly

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School)

  • Jonathan K. Watts

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School
    University of Massachusetts Chan Medical School
    NeuroNexus Institute, University of Massachusetts Chan Medical School)

  • Jun Xie

    (Horae Gene Therapy Center, University of Massachusetts Chan Medical School
    Viral Vector Core, University of Massachusetts Chan Medical, School
    University of Massachusetts Chan Medical School
    Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School)

  • Guangping Gao

    (Horae Gene Therapy Center, University of Massachusetts Chan Medical School
    Viral Vector Core, University of Massachusetts Chan Medical, School
    University of Massachusetts Chan Medical School
    Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School)

  • Erik J. Sontheimer

    (RNA Therapeutics Institute, University of Massachusetts Chan Medical School
    Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School
    Program in Molecular Medicine, University of Massachusetts Chan Medical School)

Abstract

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first use domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibit shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expand the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We then use these enhancements to introduce therapeutically relevant edits in a variety of cell types. Finally, we validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

Suggested Citation

  • Nathan Bamidele & Han Zhang & Xiaolong Dong & Haoyang Cheng & Nicholas Gaston & Hailey Feinzig & Hanbing Cao & Karen Kelly & Jonathan K. Watts & Jun Xie & Guangping Gao & Erik J. Sontheimer, 2024. "Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45763-5
    DOI: 10.1038/s41467-024-45763-5
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