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

被引:629
作者
Kim, Y. Bill [1 ,2 ]
Komor, Alexis C. [1 ,2 ]
Levy, Jonathan M. [1 ,2 ]
Packer, Michael S. [1 ,2 ]
Zhao, Kevin T. [1 ,2 ]
Liu, David R. [1 ,2 ,3 ]
机构
[1] Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
[2] Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA
[3] Broad Inst Harvard & MIT, Cambridge, MA 02142 USA
基金
美国国家卫生研究院; 加拿大自然科学与工程研究理事会;
关键词
STAPHYLOCOCCUS-AUREUS CAS9; DNA CLEAVAGE; CRYSTAL-STRUCTURE; MAMMALIAN-CELLS; CRISPR-CAS9; APOBEC3G; DOMAIN;
D O I
10.1038/nbt.3803
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 similar to 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.
引用
收藏
页码:371 / +
页数:7
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