Multiple gene substitution by Target-AID base-editing technology in tomato

Johan Hunziker,Hiroshi Ezura,Sanae Kishimoto,Tohru Ariizumi,Akihiko Kondo,Keiji Nishida

doi:10.1038/s41598-020-77379-2

Abstract

The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.

Highlights

The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine
In the presence of a homologous DNA template containing the desired DNA sequence, such cleavage can be fixed through homology-directed repair (HDR) during the resorption of double-strand breaks (DSBs), which involves the introduction of the desired sequence through homologous recombination
For SlDET1, the original Slhp[2] mutation, consisting of an AG-to-TG substitution, is located at the junction of intron 10 and exon 11 inducing alternative splicing of the mRNA leading to the deletion of 3 amino acids (Gly, Pro and Glu) of exon 11 within the second Nuclear localization signal (NLS)

Summary

Introduction

The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. The two edited lines showed significant differences in carotenoid accumulation These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions. Some major BE technologies are based on an engineered Cas[9] protein fused with a single strand DNA-specific cytidine deaminase (CDA)[2,3,4] Another BE technique has been developed based adenosine deaminase (ADA)[5] but has only been tested in bacteria. The CDA and ADA technologies were fused in a single plasmid to form a protein complex that is able to modify n ucleotides[6] Such technologies have been studied in organisms such as bacteria3,6, animals[7,8,9] and plants[5,10,11,12], with a variable range of efficiency. Editing technologies using programmable nucleases appears to be a promising and efficient approach for both breeding and basic research involving forward and reverse genetics in tomato

Methods

Results

Conclusion