Repositorium
Journal Article / 2018
Kim, Dongjin; Alptekin, Burcu; Budak, Hikmet
Genome editing has been a long-term challenge for molecular biology research, particularly for plants possess complex genome. The recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a versatile tool for genome editing which enables editing of multiple genes based on the guidance of small RNAs. Even though the efficiency ofCRISPR/Cas9 system has been shown with several studies from diploid plants, its application remains a challenge for plants with polyploid and complex genome. Here, we applied CRISPR/Cas9 genome editing system in wheat protoplast to conduct the targeted editing of stress-responsive transcription factor genes, wheat dehydration responsive element binding protein 2 (TaDREB2) and wheat ethylene responsive factor 3 (TaERF3). Targeted genome editing ofTaDREB2 and TaERF3 was achieved with transient expression of small guide RNA and Cas9 protein in wheat protoplast. The effectiveness of mutagenesis in wheat protoplast was confirmed with restriction enzyme digestion assay, T7 endonuclease assay, and sequencing. Furthermore, several off-target regions for designed sgRNAs were analyzed, and the specificity of genome editing was confirmed with amplicon sequencing. Overall results suggested that CRISPR/Cas9 genome editing system can easily be established on wheat protoplast and it has a huge potentiality for targeted manipulation ofwheat genome for crop improvement purposes.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
715 |
Budak, Hikmet USA |
Triticum aestivum |
CRISPR/Cas9 DREB2 |
Increased drought resistance SDN1 |
Market-oriented Abiotic stress tolerance |
716 |
Budak, Hikmet USA |
Triticum aestivum |
CRISPR/Cas9 ERF3 |
Increased drought resistance SDN1 |
Market-oriented Abiotic stress tolerance |