Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.
| Authors | |
| 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. |
| Year of Publication | 2017
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| Journal | Nat Commun
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| Volume | 8
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| Pages | 15790
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| Date Published | 2017 Jun 06
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| ISSN | 2041-1723
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| DOI | 10.1038/ncomms15790
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| PubMed ID | 28585549
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| PubMed Central ID | PMC5467206
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| Links | |
| Grant list | R01 DC007174 / DC / NIDCD NIH HHS / United States
R35 GM118062 / GM / NIGMS NIH HHS / United States
R01 GM065400 / GM / NIGMS NIH HHS / United States
F32 GM112366 / GM / NIGMS NIH HHS / United States
R01 DC014089 / DC / NIDCD NIH HHS / United States
R01 EB022376 / EB / NIBIB NIH HHS / United States
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