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Engineering self-deliverable ribonucleoproteins for genome editing in the brain

Author

Listed:
  • Kai Chen

    (University of California, Berkeley
    University of California)

  • Elizabeth C. Stahl

    (University of California, Berkeley
    University of California
    University of California, Berkeley)

  • Min Hyung Kang

    (University of California, Berkeley
    University of California
    University of California, Berkeley)

  • Bryant Xu

    (University of California, Berkeley
    University of California)

  • Ryan Allen

    (University of California, Berkeley
    University of California)

  • Marena Trinidad

    (University of California, Berkeley
    University of California
    University of California, Berkeley)

  • Jennifer A. Doudna

    (University of California, Berkeley
    University of California
    University of California, Berkeley
    University of California, Berkeley)

Abstract

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.

Suggested Citation

  • Kai Chen & Elizabeth C. Stahl & Min Hyung Kang & Bryant Xu & Ryan Allen & Marena Trinidad & Jennifer A. Doudna, 2024. "Engineering self-deliverable ribonucleoproteins for genome editing in the brain," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45998-2
    DOI: 10.1038/s41467-024-45998-2
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    References listed on IDEAS

    as
    1. Jennifer A. Doudna, 2020. "The promise and challenge of therapeutic genome editing," Nature, Nature, vol. 578(7794), pages 229-236, February.
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