Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions
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Open Access
- 13 February 2017
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Biotechnology
- Vol. 35 (4), 371-376
- https://doi.org/10.1038/nbt.3803
Abstract
Genome editing of single bases is made more versatile and accurate with new fusions of mutated Cas9 and cytidine deaminase domains. 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 repair1. 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.Keywords
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