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

Rees, Holly A., Komor, Alexis C., Yeh, Wei-Hsi, Caetano-Lopes, Joana, Warman, Matthew, Edge, Albert S. B., Liu, David R.·2017·Nature Communications
DOI 10.1038/ncomms15790PMC PMC5467206
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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 .

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Metadata

Authors
Rees, H. A., Komor, A. C., Yeh, W.-H., Caetano-Lopes, J., Warman, M., Edge, A. S. B., & Liu, D. R.
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-06-06
Volume
8
Issue
1
ISSN
2041-1723
DOI
10.1038/ncomms15790
PMC
PMC5467206
Cited by
412
References
14
Language
en
Licence

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