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Targeted Base Editing with CRISPR-Deaminase in Tomato

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Plant Genome Editing with CRISPR Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1917))

Abstract

The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.

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Abbreviations

AID:

Activation-induced cytidine deaminase

AP endonuclease:

Apurinic/apyrimidinic endonuclease

bp:

Base pair

Cas9:

CRISPR-associated protein9

CRISPR:

Clustered regularly interspaced short palindromic repeats

dCas9:

Deactivated Cas9

nCas9:

Nickase-Cas9

NHEJ:

Non-homologous end joining

PAM:

Protospacer adjacent motif

sgRNA:

Single guide RNA

References

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Acknowledgments

This work was supported by Cross-ministerial Strategic Innovation Promotion Program (SIP); “Technologies for creating next-generation agriculture, forestry and fisheries.” This work was also partly supported by a Special Coordination Fund for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan, the commission for Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan, by JSPS KAKENHI grant numbers 26119710 and 16K14654. The authors would like to thank Dr. C. Vavricka for critical reading of the manuscript.

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Authors and Affiliations

  1. Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Hyogo, Japan

    Zenpei Shimatani, Ushio Fujikura, Akihiko Kondo & Keiji Nishida

  2. Faculty of Life and Environmental Sciences, Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan

    Tohru Ariizumi & Hiroshi Ezura

  3. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Hyogo, Japan

    Akihiko Kondo

Authors
  1. Zenpei Shimatani
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  2. Tohru Ariizumi
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  3. Ushio Fujikura
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  4. Akihiko Kondo
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  5. Hiroshi Ezura
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  6. Keiji Nishida
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Corresponding author

Correspondence to Keiji Nishida .

Editor information

Editors and Affiliations

  1. Department of Plant Science and Landscape Architecture, University of Maryland College Park, College Park, MD, USA

    Yiping Qi

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Shimatani, Z., Ariizumi, T., Fujikura, U., Kondo, A., Ezura, H., Nishida, K. (2019). Targeted Base Editing with CRISPR-Deaminase in Tomato. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_22

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  • DOI: https://doi.org/10.1007/978-1-4939-8991-1_22

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8990-4

  • Online ISBN: 978-1-4939-8991-1

  • eBook Packages: Springer Protocols

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