title: Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.
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.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Y Bill
authors.initials: YB
authors.lastname: Kim
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Alexis C
authors.initials: AC
authors.lastname: Komor
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Jonathan M
authors.identifier: 0000-0002-8061-7953
authors.initials: JM
authors.lastname: Levy
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Packer
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Zhao
authors.affiliation: 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.
authors.forename: David R
authors.initials: DR
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001482:Base Composition / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003564:Cytidine Deaminase / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D016678:Genome / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D003564:Cytidine Deaminase