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Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery

Author

Listed:
  • Holly A. Rees

    (Harvard University
    Howard Hughes Medical Institute, Harvard University
    Broad Institute of MIT and Harvard)

  • Alexis C. Komor

    (Harvard University
    Howard Hughes Medical Institute, Harvard University
    Broad Institute of MIT and Harvard)

  • Wei-Hsi Yeh

    (Harvard University
    Howard Hughes Medical Institute, Harvard University
    Broad Institute of MIT and Harvard
    Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary)

  • Joana Caetano-Lopes

    (Orthopaedic Research Laboratories, Boston Children’s Hospital
    Harvard Medical School)

  • Matthew Warman

    (Orthopaedic Research Laboratories, Boston Children’s Hospital
    Harvard Medical School)

  • Albert S. B. Edge

    (Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary
    Program in Speech and Hearing Bioscience and Technology, Harvard Medical School
    Harvard Medical School)

  • David R. Liu

    (Harvard University
    Howard Hughes Medical Institute, Harvard University
    Broad Institute of MIT and Harvard)

Abstract

We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo.

Suggested Citation

  • Holly A. Rees & Alexis C. Komor & Wei-Hsi Yeh & Joana Caetano-Lopes & Matthew Warman & Albert S. B. Edge & David R. Liu, 2017. "Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery," Nature Communications, Nature, vol. 8(1), pages 1-10, August.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_ncomms15790
    DOI: 10.1038/ncomms15790
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    Cited by:

    1. Catarina Rebelo & Tiago Reis & Joana Guedes & Cláudia Saraiva & Artur Filipe Rodrigues & Susana Simões & Liliana Bernardino & João Peça & Sónia L. C. Pinho & Lino Ferreira, 2022. "Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications," Nature Communications, Nature, vol. 13(1), pages 1-16, December.

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