An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities

被引:330
作者
Gehrke, Jason M. [1 ,2 ,3 ]
Cervantes, Oliver [1 ,2 ]
Clement, M. Kendell [1 ,2 ,4 ]
Wu, Yuxuan [5 ]
Zeng, Jing [5 ]
Bauer, Daniel E. [5 ]
Pinello, Luca [1 ,2 ,4 ]
Joung, J. Keith [1 ,2 ,4 ]
机构
[1] Massachusetts Gen Hosp, Ctr Canc Res, Mol Pathol Unit, Charlestown, MA 02129 USA
[2] Massachusetts Gen Hosp, Ctr Computat & Integrat Biol, Charlestown, MA 02129 USA
[3] Harvard Univ, Dept Mol & Cellular Biol, Cambridge, MA 02138 USA
[4] Harvard Med Sch, Dept Pathol, Boston, MA 02115 USA
[5] Harvard Med Sch, Dana Farber Canc Inst, Boston Childrens Hosp,Dept Pediat Oncol, Harvard Stem Cell Inst,Dept Pediat,Div Hematol On, Boston, MA USA
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
BETA-THALASSEMIA; STRUCTURAL BASIS; DNA; CRISPR-CAS9; CAS9; SPECIFICITY; DEAMINATION; NUCLEASES; CLEAVAGE; EMBRYOS;
D O I
10.1038/nbt.4199
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 editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations(1-6). However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human beta-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.
引用
收藏
页码:977 / +
页数:8
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