Targeted Base Editing with CRISPR-Deaminase in Tomato
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 wordsTargeted nucleotide substitution Activation-induced cytidine deaminase (AID) Target-AID CRISPR/Cas9 Tomato
Activation-induced cytidine deaminase
- AP endonuclease
Clustered regularly interspaced short palindromic repeats
Non-homologous end joining
Protospacer adjacent motif
Single guide RNA
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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