Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery
Open Access
- 6 June 2017
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Communications
- Vol. 8 (1), 15790
- https://doi.org/10.1038/ncomms15790
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.Keywords
This publication has 43 references indexed in Scilit:
- Highly efficient RNA-guided base editing in mouse embryosNature Biotechnology, 2017
- Directed evolution using dCas9-targeted somatic hypermutation in mammalian cellsNature Methods, 2016
- Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cellsNature Methods, 2016
- Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systemsScience, 2016
- Diphtheria Toxin-Induced Cell Death Triggers Wnt-Dependent Hair Cell Regeneration in Neonatal MiceJournal of Neuroscience, 2016
- Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticlesProceedings of the National Academy of Sciences of the United States of America, 2016
- Chemical Biology Approaches to Genome Editing: Understanding, Controlling, and Delivering Programmable NucleasesCell Chemical Biology, 2016
- The new frontier of genome engineering with CRISPR-Cas9Science, 2014
- Electroporation for the Delivery of DNA-based Vaccines and Immunotherapeutics: Current Clinical DevelopmentsMolecular Therapy, 2009
- Innate immune response to viral infectionCytokine, 2008