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

Holly A. Rees, Alexis C. Komor, Wei-Hsi Yeh, Joana Caetano-Lopes, Matthew Warman, Albert S. B. Edge and David R. Liu ()
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Holly A. Rees: Harvard University
Alexis C. Komor: Harvard University
Wei-Hsi Yeh: Harvard University
Joana Caetano-Lopes: Orthopaedic Research Laboratories, Boston Children’s Hospital
Matthew Warman: Orthopaedic Research Laboratories, Boston Children’s Hospital
Albert S. B. Edge: Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary
David R. Liu: Harvard University

Nature Communications, 2017, vol. 8, issue 1, 1-10

Abstract: 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.

Date: 2017
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DOI: 10.1038/ncomms15790

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