|ZFIN ID: ZDB-PUB-170607-3|
Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery
Rees, H.A., Komor, A.C., Yeh, W.H., Caetano-Lopes, J., Warman, M., Edge, A.S.B., Liu, D.R.
|Source:||Nature communications 8: 15790 (Journal)|
|Keywords:||CRISPR-Cas9 genome editing, Genetic engineering, Protein delivery|
|PubMed:||28585549 Full text @ Nat. Commun.|
Rees, H.A., Komor, A.C., Yeh, W.H., Caetano-Lopes, J., Warman, M., Edge, A.S.B., Liu, D.R. (2017) Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nature communications. 8:15790.
ABSTRACTWe 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.