Abstract
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 of CRISPR/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 of TaDREB2 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 of wheat genome for crop improvement purposes.
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Acknowledgements
The authors acknowledge Montana Wheat and Barley Committee Grant #MDA/MWBC CY5416-462 and Winifred-Asbjornson Plant Science Endowment. The authors also thank the International Wheat Genome Sequencing Consortium (IWGSC) for pre-publication access to the wheat genome RefSeq v1.0.
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Supplementary Figure 1
The TaDREB2 gene sequence and structure. A. TaDREB2 consists one exon and 1122 bases long in length. B. The sequence of TaDREB2 CDS is from GeneBank (ID# DQ353852). The target sequence is shown in blue bold letters together with PAM sequence in the red bold letter. Yellow highlighted sites show the primers for amplification of 500 bases region for detection of mutation. Red highlights show the start and stop codons inside gene. (JPEG 616 kb).
Supplementary Figure 2
The TaERF3 gene structure and sequences. A. TaERF3 consists three exons and 2139 bases long in length. B. The sequence of TaERF3 CDS is from GeneBank (ID# EF570122). The target sequence is shown in blue bold letters together with PAM sequence in the red bold letter. Yellow highlighted sites show the primers for amplification of 500 bases region for detection of mutation. Red highlights show the start and stop codons inside gene. (JPEG 773 kb).
Supplementary Figure 3
Isolation of protoplasts from the wheat tissue. A. Representative fresh 15-day-old wheat (v. Chinese spring) seedling used for protoplast isolation. B. After 2 weeks of growth on ½ MS solid medium containing 3% sucrose and 0.01% inositol, plants were collected. C. Red circle indicates the best part of seedlings for high yield protoplast preparation. D. Selected part of plant was cut into strips of shorter than 0.5 mm with a sharp razor blade. E. The strips digested with cell wall dissolving enzyme for 5–6 h under dark. F. Protoplast cells were harvested by low speed (80 g) centrifuge and washed with the W5 solution. G & H. The final protoplast cell was observed and counted. The observed final concentration of protoplast solution was around 1 × 106 cells. (JPEG 358 kb).
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Kim, D., Alptekin, B. & Budak, H. CRISPR/Cas9 genome editing in wheat. Funct Integr Genomics 18, 31–41 (2018). https://doi.org/10.1007/s10142-017-0572-x
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DOI: https://doi.org/10.1007/s10142-017-0572-x