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Nat Biotechnol. 2017 Apr;35(4):371-376. doi: 10.1038/nbt.3803. Epub 2017 Feb 13.

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

Kim YB1,2, Komor AC1,2, Levy JM1,2, Packer MS1,2, Zhao KT1,2, Liu DR1,2,3.

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Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.


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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