期刊
SCIENCE
卷 353, 期 6305, 页码 -出版社
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.aaf8729
关键词
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资金
- Ministry of Education, Culture, Sports and Technology (MEXT) of Japan
- commission for Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan
- JSPS [26119710, 16K14654]
- Cross-ministerial Strategic Innovation Promotion Program (SIP)
- Grants-in-Aid for Scientific Research [26119710, 15K18466, 16K14654, 26430198] Funding Source: KAKEN
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.
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