Rees, HollyKomor, Alexis C.Yeh, Wei-HsiCaetano-Lopes, JoanaWarman, MatthewEdge, AlbertLiu, David2017-07-242017Rees, Holly A., Alexis C. Komor, Wei-Hsi Yeh, Joana Caetano-Lopes, Matthew Warman, Albert S. B. Edge, and David R. Liu. 2017. “Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.” Nature Communications 8 (1): 15790. doi:10.1038/ncomms15790. http://dx.doi.org/10.1038/ncomms15790.http://nrs.harvard.edu/urn-3:HUL.InstRepos:33490889We 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.en-USJournal Article2017-07-2410.1038/ncomms15790