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.
Date: 2017
Source: Nature communications   8: 15790 (Journal)
Registered Authors:
Keywords: CRISPR-Cas9 genome editing, Genetic engineering, Protein delivery
MeSH Terms:
  • Animals
  • CRISPR-Cas Systems
  • Cell Line
  • DNA/genetics*
  • DNA/metabolism
  • Gene Editing
  • Mice
  • Protein Engineering
  • Ribonucleoproteins/genetics*
  • Ribonucleoproteins/metabolism
  • Zebrafish
PubMed: 28585549 Full text @ Nat. Commun.
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.