Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
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DOI：
10.1038/nature17946
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发表时间：
2016-05-19
期刊：
影响因子：
64.8
通讯作者：
Liu DR
中科院分区：
文献类型：
--
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
目前的基因组编辑技术在目标基因座引入双链(DS)DNA断裂，作为基因校正的第一步。虽然大多数遗传病都是由点突变引起的，但目前的点突变纠正方法效率低下，通常会在细胞对dsDNA断裂的反应中在目标位置诱导大量随机插入和缺失(Indels)。在这里，我们报道了碱基编辑的发展，这是一种新的基因组编辑方法，能够以可编程的方式将一个目标DNA碱基直接、不可逆地转换为另一个碱基，而不需要dsDNA骨架切割或供体模板。我们设计了CRISPR/Cas9和胞苷脱氨酶的融合，它保留了被引导RNA编程的能力，不会诱导双链DNA断裂，并介导胞苷直接转化为尿苷，从而实现C→T(或G→A)取代。由此产生的“碱基编辑”在大约5个核苷酸(NT)的窗口内转换胞苷，并可以有效地纠正与人类疾病相关的各种点突变。在四个转化的人类和小鼠细胞系中，第二代和第三代碱基编辑融合尿嘧啶糖基酶抑制剂(UGI)，并使用针对未编辑链的Cas9镍酶，操纵细胞DNA修复反应，以支持预期的碱基编辑结果，导致永久纠正细胞总DNA的∼15%-75%，最少(通常为≤1%)INDel形成。碱基编辑扩大了点突变基因组编辑的范围和效率。
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. 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 from the cellular response to dsDNA breaks. 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 (nt), 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 (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of ∼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.