Plant Genome Editing with CRISPR Systems pp 297-307 | Cite as
Targeted Base Editing with CRISPR-Deaminase in Tomato
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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 TomatoAbbreviations
- 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.
References
- 1.Hsu P-D, Lander E-S, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278. https://doi.org/10.1016/j.cell.2014.05.010CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Mitsunobu H, Teramoto J, Nishida K, Kondo A (2017) Beyond native Cas9: manipulating genomic information and function. Trends Biotechnol 35:983–996. https://doi.org/10.1016/j.tibtech.2017.06.004CrossRefPubMedGoogle Scholar
- 3.Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara K-Y, Shimatani Z, Kondo A (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353(6305):aaf8729. https://doi.org/10.1126/science.aaf8729CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Arazoe T, Nishida K, Kondo A (2016) Targeted nucleotide substitution in mammalian cell by Target-AID. Bio-protocol 7:11. https://doi.org/10.21769/BioProtoc.2339CrossRefGoogle Scholar
- 5.Banno S, Nishida K, Arazoe T, Mitsunobu H, Kondo A (2018) Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol 3:423–429. https://doi.org/10.1038/s41564-017-0102-6CrossRefPubMedGoogle Scholar
- 6.Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K, Ariizumi T, Kondo A (2017) Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol 35:441–443. https://doi.org/10.1038/nbt.3833CrossRefPubMedGoogle Scholar
- 7.Fauser H, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359. https://doi.org/10.1111/tpj.12554CrossRefPubMedGoogle Scholar
- 8.Sun H-J, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431. https://doi.org/10.1093/pcp/pci251CrossRefGoogle Scholar