Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems

被引：1037

作者：

Nishida, Keiji ^{[1
]}

Arazoe, Takayuki ^{[1
]}

Yachie, Nozomu ^{[2
,3
,4
]}

Banno, Satomi ^{[1
]}

Kakimoto, Mika ^{[1
]}

Tabata, Mayura ^{[1
]}

Mochizuki, Masao ^{[1
]}

Miyabe, Aya ^{[1
]}

Araki, Michihiro ^{[1
]}

Hara, Kiyotaka Y. ^{[5
]}

Shimatani, Zenpei ^{[1
]}

Kondo, Akihiko ^{[1
,6
]}

机构：

[1] Kobe Univ, Grad Sch Sci Technol & Innovat, Nada Ku, 1-1 Rokkodai Cho, Kobe, Hyogo 6578501, Japan

[2] Univ Tokyo, Synthet Biol Div, Res Ctr Adv Sci & Technol, Meguro Ku, 4-6-1 Komaba, Tokyo 1538904, Japan

[3] Keio Univ, Inst Adv Biosci, Tsuruoka, Yamagata, Japan

[4] Japan Sci & Technol Agcy JST, Precursory Res Embryon Sci & Technol PRESTO, Tokyo, Japan

[5] Univ Shizuoka, Grad Sch Nutr & Environm Sci, Dept Environm Sci, Suruga Ku, 52-1 Yada, Shizuoka 4228526, Japan

[6] Kobe Univ, Grad Sch Engn, Dept Chem Sci & Engn, Nada Ku, 1-1 Rokkodai Cho, Kobe, Hyogo 6578501, Japan

来源：

SCIENCE | 2016年 / 353卷 / 6305期

关键词：

CRISPR-CAS SYSTEMS; STRANDED-DNA; CHO-CELLS; NUCLEASES; DEAMINASE; AID; DIVERSIFICATION; CLEAVAGE; ENZYME;

D O I：

10.1126/science.aaf8729

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：

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.

引用

页数：8

共 27 条

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