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Targeted Base Editing with CRISPR-Deaminase in Tomato

  • Zenpei Shimatani
  • Tohru Ariizumi
  • Ushio Fujikura
  • Akihiko Kondo
  • Hiroshi Ezura
  • Keiji Nishida
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Targeted nucleotide substitution Activation-induced cytidine deaminase (AID) Target-AID CRISPR/Cas9 Tomato 

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Zenpei Shimatani
    • 1
  • Tohru Ariizumi
    • 2
  • Ushio Fujikura
    • 1
  • Akihiko Kondo
    • 1
    • 3
  • Hiroshi Ezura
    • 2
  • Keiji Nishida
    • 1
  1. 1.Graduate School of Science, Technology and InnovationKobe UniversityKobeJapan
  2. 2.Faculty of Life and Environmental SciencesGene Research Center, University of TsukubaTsukubaJapan
  3. 3.Department of Chemical Science and Engineering, Graduate School of EngineeringKobe UniversityKobeJapan

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