title: Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.
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.
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Keiji
authors: K
authors: Nishida
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Takayuki
authors: T
authors: Arazoe
authors: 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.
authors: Nozomu
authors: N
authors: Yachie
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Satomi
authors: S
authors: Banno
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Mika
authors: M
authors: Kakimoto
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Mayura
authors: M
authors: Tabata
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Masao
authors: M
authors: Mochizuki
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Aya
authors: A
authors: Miyabe
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Michihiro
authors: M
authors: Araki
authors: Department of Environmental Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors: Kiyotaka Y
authors: KY
authors: Hara
authors: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors: Zenpei
authors: Z
authors: Shimatani
authors: 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.
authors: Akihiko
authors: A
authors: Kondo
mesh_terms: D000483:Alleles; D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry; D016466:CHO Cells; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003412:Cricetulus; D003562:Cytidine / Q000737:chemistry / Q000235:genetics*; D003564:Cytidine Deaminase / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004720:Endonucleases / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D054643:INDEL Mutation; D007107:Immune System; D017354:Point Mutation; D012313:RNA / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D051981:Uracil-DNA Glycosidase / Q000037:antagonists & inhibitors; D014714:Vertebrates / Q000276:immunology; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D003562:Cytidine; D012313:RNA; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D003850:Deoxyribonuclease I; D051981:Uracil-DNA Glycosidase; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase