CRISPR/cas9 genome editing in wheat
Open accessAbstract
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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Manipulating genes involved in root growth, such as TaDREB2 and TaERF3 in wheat, can significantly improve drought resilience.
“For instance, studies have shown that manipulating genes involved in root growth, such as TaDREB2 and TaERF3 in wheat, can significantly improve drought resilience (Kim et al., 2017).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kim et al., 2017
The Discussion reports that overexpressing DREB2 increased drought tolerance in wheat and barley, and links ERF3 to root development and drought resistance. The paper’s own wheat results show that TaDREB2 and TaERF3 expression rose during dehydration; they do not show that manipulating both genes improved root growth or drought resilience in wheat. No page numbers are available in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThird-generation base editors, with rAPOBEC1 fused to the N-terminus and UGI at the C-terminus of nickase Cas9 D10A, offered higher editing efficiency and reduced off-target effects but required an NGG PAM sequence, limiting their scope.
“Third-generation base editors, with rAPOBEC1 fused to the N-terminus and UGI at the C-terminus of nickase Cas9 D10A, offered higher editing efficiency and reduced off-target effects but required an NGG PAM sequence, limiting their scope (Kim et al., 2017).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kim et al., 2017
The Introduction identifies Cas9 D10A as a nickase variant used for more precise genome editing, and the Discussion describes selecting NGG PAM sites for the paper’s CRISPR/Cas9 wheat experiments. Neither passage addresses third-generation base editors, rAPOBEC1 or UGI fusions, or improved efficiency and reduced off-target effects for those editors. The NGG observation is not established specifically for the claimed base editor. (LLM, conf=0.82)
Experimental — accuracy not yet measuredTransgene-free plants produced through CRISPR/Cas9 do not contain foreign DNA in the final product, potentially allowing them to bypass GMO regulations.
“Transgene-free plants produced through CRISPR/Cas9 do not contain foreign DNA in the final product, potentially allowing them to bypass GMO regulations (Zetsche et al., 2017; Kim et al., 2017).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kim et al., 2017
The Abstract and Discussion describe transient CRISPR/Cas9 expression and targeted editing in wheat protoplasts, not the production of transgene-free plants. Searches for foreign DNA, transgene-free plants, and GMO regulation found no passage addressing whether a final plant contains foreign DNA or could bypass GMO regulations. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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“Transgene-free plants produced through CRISPR/Cas9 do not contain foreign DNA in the fi nal product, potentially allowing them to bypass GMO regulations (Zetsche et al., 2017; Kim et al., 2017).”
Influential
- Authors
- Kim, D., Alptekin, B., & Budak, H.
- Affiliations
- Montana State University (US)
- Corresponding author
- Budak, Hikmet
- Venue
- Functional & Integrative Genomics
- Published
- 2017-09-16
- Volume
- 18
- Issue
- 1
- Pages
- 31-41
- ISSN
- 1438-793X (ISSN-L)
- 1438-7948
- OpenAlex
- W2755144089
- Cited by
- 365
- References
- 47
- Citations by year
- 2026: 23 · 2025: 56 · 2024: 46 · 2023: 51 · 2022: 56 · 2021: 56 · 2020: 31 · 2019: 28 · 2018: 17 · 2017: 1
- FWCI
- 8.60
- Type
- journal article
- Language
- en
- Open access
- Locations
- Functional & Integrative Genomics (publishedVersion)
- PubMed (publishedVersion)
- Montana State University ScholarWorks (Montana State University) (submittedVersion)
- Funders
- Montana Wheat and Barley Committee
- Winifred-Asbjornson Plant Science Chair
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Wheat and Barley Genetics and Pathology
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Genetic and Mutation Studies
- Keywords
- CRISPR/Cas9 genome editing, wheat, protoplast transformation, DREB, ethylene response factor, stress-responsive transcription factors, small guide RNA, off-target analysis, crop improvement
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Transcription Factors, Triticum, Genome, Plant, CRISPR-Cas Systems
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