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Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.

  • Author(s): Kim, Y Bill
  • Komor, Alexis C
  • Levy, Jonathan M
  • Packer, Michael S
  • Zhao, Kevin T
  • Liu, David R
  • et al.

Published Web Location

https://doi.org/10.1038/nbt.3803
Abstract

Base editing induces single-nucleotide changes in the DNA of living cells using a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair. This genome editing approach has the advantage that it does not require formation of double-stranded DNA breaks or provision of a donor DNA template. Here we report the development of five C to T (or G to A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing 2.5-fold. Additionally, we engineered base editors containing mutated cytidine deaminase domains that narrow the width of the editing window from ∼5 nucleotides to as little as 1-2 nucleotides. We thereby enabled discrimination of neighboring C nucleotides, which would otherwise be edited with similar efficiency, and doubled the number of disease-associated target Cs able to be corrected preferentially over nearby non-target Cs.

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