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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Cell Line
  • DNA / genetics*
  • DNA / metabolism
  • Gene Editing
  • Mice
  • Protein Engineering
  • Ribonucleoproteins / genetics*
  • Ribonucleoproteins / metabolism
  • Zebrafish


  • Ribonucleoproteins
  • DNA