Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems
Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems
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DOI：
10.1126/science.aaf8729
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发表时间：
2016-09-16
期刊：
影响因子：
56.9
通讯作者：
Kondo, Akihiko
中科院分区：
文献类型：
--
作者：
Nishida, Keiji;Arazoe, Takayuki;Kondo, Akihiko
遗传变异（体细胞超突变）的产生是脊椎动物适应性免疫系统的一个重要过程。我们证明了有针对性的单核苷酸取代的DNA使用杂交脊椎动物和细菌免疫系统的组成部分。核酸酶缺陷型II型CRISPR/Cas9（成簇的规则间隔的短回文重复序列/CRISPR相关的）和活化诱导的胞苷脱氨酶（AID）直系同源物PmCDA 1被工程化以形成进行高效靶特异性诱变的合成复合物（Target-AID）。特异性点突变主要在5个碱基的目标范围内的胞苷上诱导。与基于核酸酶的CRISPR/Cas9系统相关的毒性大大降低。尽管切口酶Cas9（D10 A）和脱氨酶的组合在酵母中是高度有效的，但它也在哺乳动物细胞中诱导插入和缺失（indel）。使用尿嘧啶DNA糖基化酶抑制剂抑制了indel的形成，提高了效率。
The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.