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Science. 2016 Sep 16;353(6305). pii: aaf8729. doi: 10.1126/science.aaf8729. Epub 2016 Aug 4.

Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.

Author information

1
Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
2
Synthetic Biology Division, Research Center for Advanced Science and Technology, the University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. Institute for Advanced Bioscience, Keio University, Tsuruoka, Yamagata, Japan. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Tokyo, Japan.
3
Department of Environmental Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
4
Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan. akondo@kobe-u.ac.jp.

Abstract

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.

PMID:
27492474
DOI:
10.1126/science.aaf8729
[Indexed for MEDLINE]
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