Journal of Taiwan Agricultural Research

Author:Shuen-Chi You*, Yi-Wen Wang, Ssu-Yu Lin, Ching-Yi Huang, and Da-Gin Lin*

Abstract:

    The clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system is the most popular strategy for genomic editing technology in recent years, and has been a powerful tool for gene function verification and precision plant breeding. To replace the current time-consuming Agrobacterium-mediated method, this study aims to establish a rapid transient expression platform for the preliminary evaluation of genomic editing system. First of all, leaf protoplasts of Cucumis melo L. were used to establish the transient gene expression system, and the best transformation efficiency reached approximately 20%. Simultaneously, the melon eukaryotic translation initiation factors 4E (eIF4E) was used as a target gene to evaluate the genome editing efficiency in the transient expression system. Our results have demonstrated that the dual guide RNA (gRNA) CRISPR/Cas9 system could be introduced to mediate deletion of the genomic DNA fragment (135 bp) on the first exon of CmeIF4E in the transient expression system. Besides, this deletion could be rapidly detected by using the nested polymerase chain reaction (PCR) and PCR. These methods can significantly reduce the required staffing, time and costly processes in contrast to TA cloning and sequencing analysis for detecting the point mutation induced by using single gRNA CRISPR/Cas9 system. Furthermore, the system established in this study provides a foundation for further progress of the precise new-generation crop breeding platform.

Key words:Protoplast, Genomic editing, Dual gRNA, Cm-eIF4E

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