Precision agriculture is advancing through innovative genome modification techniques, with CRISPR/Cas9 emerging as a groundbreaking approach. This transformative technology enables targeted genetic alterations in plants, potentially enhancing agricultural productivity by improving critical characteristics such as crop resilience, nutritional composition, and environmental adaptability. The method addresses crucial challenges in global food sustainability by precisely manipulating plant DNA to develop more robust and efficient crop varieties. This article will introduce in detail the principles and applications of CRISPR/Cas9 technology in plant genome editing.
What is Genome Editing in Plants?
Genome editing in plants employs specialized nucleases like CRISPR/Cas9 to precisely manipulate genetic material. Researchers can strategically introduce genetic modifications at targeted genome locations, enabling trait enhancement. The CRISPR/Cas9 methodology utilizes guide RNA to direct the Cas9 protein, which identifies and cleaves specific DNA sequences, subsequently leveraging the plant's inherent DNA repair mechanisms to complete genetic alterations.
Figure 1.CRISPR/Cas9 makes site-specific modifications to the genome.(Yasmeen,et.al ,2023)
Compared with traditional breeding methods, genome editing is efficient, rapid and accurate. Traditional breeding takes decades to introduce new traits into crops, while genome editing can accomplish the same task in a few years. In addition, CRISPR/Cas9 technology can also avoid foreign DNA residue problems that may occur in traditional breeding, thereby reducing potential genomic instability.
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Why Precision Agriculture Matters
Addressing global food security requires innovative agricultural strategies amid escalating population growth and climate challenges. Genome editing technology empowers researchers to develop resilient crop varieties that optimize resource utilization and enhance agricultural sustainability. By creating adaptable plant strains, scientists can mitigate mounting pressures on global food production systems.
CRISPR/Cas9 Technology
Mechanism of CRISPR/Cas9
The core mechanism of the CRISPR/Cas9 system is based on the immune defense systems of bacteria and archaea, which enables precise editing of specific DNA sequences through Cas9 protein and guide RNA (gRNA). The specific process is as follows:
Guide RNA (gRNA): A gRNA is a synthetic single-stranded RNA molecule designed to recognize a target DNA sequence. The gRNA guides the Cas9 protein to a specific location by complementing the target DNA.
Cas9 protein: Cas9 is a nuclease that recognizes and cleaves double strands of DNA. When Cas9 binds to gRNA, it creates a double strand break (DSB) at the target DNA sequence, triggering the DNA repair mechanism within the cell.
Figure 2.CRISPR/Cas9 mechanism.(Khan,et.al ,2018)
DNA repair mechanism: Cells repair DSB through non-homologous end joining (NHEJ) or homologous directed repair (HDR). NHEJ usually causes insertions or deletions (Indels) to achieve gene knockouts, while HDR allows scientists to introduce new DNA fragments to achieve gene insertions or substitutions.
Advantages and Limitations
CRISPR/Cas9 offers significant advantages, including high precision due to the specificity of gRNA, cost-effectiveness compared to traditional gene-editing methods, and wide applicability across various organisms such as plants, animals, and microorganisms. These features have made it a powerful and versatile tool in genome editing.
However, the technology also has limitations. Off-target effects pose a risk of unintended DNA modifications, while efficient and safe delivery of the CRISPR/Cas9 system to target cells remains a challenge.Towards this end, we provides sophisticated CRISPR off-target validation services. Additionally, ethical controversies and regulatory hurdles, particularly regarding its use in human embryo editing, complicate its application in certain fields.
Applications of CRISPR/Cas9 in Plant Genome Editing
The role of CRISPR/Cas9 technology in modern agriculture is mainly reflected in the efficiency and accuracy of genome editing. It provides a revolutionary tool for agricultural research and plant breeding.
Crop improvement
CRISPR/Cas9 technology shows great potential in crop improvement, including increasing yield, improving quality and enhancing stress resistance. For example, researchers at Purdue University utilized CRISPR/Cas9 to silence specific genes in rice, resulting in a variety that produced 25-31% more grain than traditional strains. This was achieved by targeting genes associated with the phytohormone abscisic acid, which plays a crucial role in plant stress tolerance and growth regulation.
Figure 3.Group I pyl mutations promote rice growth.(Miao,et.al ,2018)
Disease resistance research
CRISPR/Cas9 technology has made significant progress in the research of plant disease resistance. For instance, a team from UC Davis successfully edited rice plants to enhance their resistance to rice blast disease, a major threat to global rice production. By identifying and modifying a gene associated with disease resistance, they developed a rice variety that yielded five times more than control plants affected by the fungus(Sha, G., et.al,2023).
Increase in output
CRISPR/Cas9 technology can optimize the growth and development characteristics of crops by accurately editing genes, thereby increasing yields. In one notable example, scientists generated mutations in tomato plants by targeting the SELF-PRUNING5G gene, which resulted in rapid flowering and compact growth habits. This led to earlier yields and increased productivity for field tomatoes(Soyk, S., et.al,2017).
Figure 4.CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering.(Soyk, S,et.al ,2017)
Environmental adaptability
CRISPR/Cas9 technology has also been used to enhance the adaptability of crops to environmental stresses. For instance, researchers have successfully edited the OsProDH gene in rice to increase proline accumulation, which helps protect plants from abiotic stresses such as drought and high temperatures. This genetic modification resulted in higher thermotolerance and improved overall plant resilience under challenging conditions.
Multiplex Genome Editing
Multiplex Genome Editing is a technology that uses the CRISPR/Cas9 system to edit multiple genes or DNA sequences simultaneously. This technology has attracted widespread attention due to its efficiency, flexibility and wide application potential.
Introduction to Multiplex Editing
Multiple genome editing refers to the simultaneous targeting of multiple genes or DNA sequences through the CRISPR/Cas9 system, thereby achieving the knockout, insertion or modification of multiple genes. This method overcomes the limitations of traditional single gene editing technology and can more efficiently study gene functions and perform complex genetic operations. For example, by using multiple guide RNAs (gRNAs), multiple sites can be edited simultaneously, resulting in multiple gene mutations.
Case Study of Multiplex Genome Editing in Plant
Background
Although thousands of genes have been identified or cloned in rice over the past two decades, most genes have only been individually characterized in the context of specific varieties or single gene modifications, limiting their practical application.
Methods
MGE toolbox development: Create a toolbox that can edit multiple genes simultaneously to improve the efficiency of gene editing.
Establishment of MRDI strategy: Combining MGE and phenotypic based field breeding, a rapid and practical improvement process is achieved through sgRNA assembly, transgenic-free screening and creation of excellent germplasm.
Apply MRDI strategy: Select rice variety FXZ, improve its growth cycle and plant structure through MRDI strategy, and produce plants with ideal characteristics while maintaining yield, rice blast resistance and rice quality.
Results
Through the MRDI strategy, ideal plants with desired traits such as early heading, reduced plant height and more efficient ears were successfully generated without affecting yield, blast resistance, and rice quality. Whole genome sequencing (WGS) analysis, including analysis of structural variants (SVs) and single nucleotide variants (SNVs), confirmed that this strategy has high specificity and low frequency of unwanted mutations. The MRDI breeding strategy is a powerful way to explore and apply key agronomic genes and create new and superior germplasm in the future.
Figure 5.MGE-based Rapid Directional Improvement (MRDI) in the study.(Wei, Y., et.al ,2024)
This study provides a new rice breeding method that can quickly improve multiple agronomic traits of rice while leaving other important traits unaffected. This is important for improving crop yield, adaptability and stability, and provides a powerful tool for future crop improvement.
Roadmap for Plant Genome Editing
Plant genome editing technology provides a powerful tool for crop improvement, but its application requires comprehensive strategic planning, scientifically rigorous target gene selection, and reasonable regulatory considerations. Future research should continue to optimize editing tools and strategies, while strengthening international cooperation to promote the sustainable development of this technology and address global food security challenges.
Strategic Planning for Genome Editing Projects
Plant genome editing technology provides a revolutionary tool for crop improvement, achieving the regulation of target traits by accurately introducing single point mutations or new DNA sequences. The application of this technology requires comprehensive strategic planning, including clarifying research goals, selecting appropriate editing tools, and assessing potential risks.
Selection of Target Genes and Traits
In plant genome editing, selecting the right target genes and traits is the key to success. Target genes are usually selected based on their important role in plant growth, development and stress resistance, such as disease resistance, environmental adaptability or nutritional improvement. In addition, researchers need to evaluate natural variations in the target gene or other related genes within the systemic genome to ensure editing efficiency and stability of the target trait.
Conclusion
CRISPR/Cas9 technology represents a transformative approach to agricultural innovation. By enabling precise genetic modifications, it offers unprecedented potential to address global challenges in food security, crop resilience, and sustainable agriculture.
References:
- Yasmeen, E., Wang, J., Riaz, M., Zhang, L., & Zuo, K. (2023). Designing artificial synthetic promoters for accurate, smart, and versatile gene expression in plants. Plant communications, 4(4), 100558. https://doi.org/10.1016/j.xplc.2023.100558
- Khan, S., Mahmood, M. S., Rahman, S. U., et.al. (2018). CRISPR/Cas9: the Jedi against the dark empire of diseases. Journal of biomedical science, 25(1), 29. https://doi.org/10.1186/s12929-018-0425-5
- Miao, C., Xiao, L., Hua, K.,et.al. (2018). Mutations in a subfamily of abscisic acid receptor genes promote rice growth and productivity. Proceedings of the National Academy of Sciences of the United States of America, 115(23), 6058–6063. https://doi.org/10.1073/pnas.1804774115
- Sha, G., Sun, P., Kong, X.,et.al. (2023). Genome editing of a rice CDP-DAG synthase confers multipathogen resistance. Nature, 618(7967), 1017–1023. https://doi.org/10.1038/s41586-023-06205-2
- Soyk, S., Müller, N. A., Park, S. J., et.al. (2017). Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato. Nature genetics, 49(1), 162–168. https://doi.org/10.1038/ng.3733
- Wei, Y., Zhang, H., Fan, J., Cai, Q.,et.al. (2024). Multiplex-genome-editing based rapid directional improvement of complex traits in rice. Plant biotechnology journal, 22(9), 2624–2628. https://doi.org/10.1111/pbi.14375
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