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Nat Commun. 2017 Jun 6;8:15790. doi: 10.1038/ncomms15790.

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

Rees HA1,2,3, Komor AC1,2,3, Yeh WH1,2,3,4,5, Caetano-Lopes J6,7, Warman M6,7, Edge ASB4,5,8, Liu DR1,2,3.

Author information

1
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
2
Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
3
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02141, USA.
4
Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA.
5
Program in Speech and Hearing Bioscience and Technology, Harvard Medical School, Boston, Massachusetts 02115, USA.
6
Orthopaedic Research Laboratories, Boston Children's Hospital, Boston, Massachusetts 02215, USA.
7
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02215, USA.
8
Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115, USA.

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.

PMID:
28585549
PMCID:
PMC5467206
DOI:
10.1038/ncomms15790
[Indexed for MEDLINE]
Free PMC Article

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