Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

被引:3385
|
作者
Komor, Alexis C. [1 ,2 ]
Kim, Yongjoo B. [1 ,2 ]
Packer, Michael S. [1 ,2 ]
Zuris, John A. [1 ,2 ]
Liu, David R. [1 ,2 ]
机构
[1] Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
[2] Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA
基金
加拿大自然科学与工程研究理事会; 美国国家卫生研究院;
关键词
CRISPR-CAS9; NUCLEASES; ENZYME APOBEC1; ENDONUCLEASE; PROTEINS;
D O I
10.1038/nature17946
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction1,2. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks(1,2). Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C -> T (or G -> A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second-and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of similar to 15-75% of total cellular DNA with minimal (typically <= 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
引用
收藏
页码:420 / +
页数:17
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