CRISPR Solution for Crop Improvement and Yield Enhancement    

Global agriculture production faces enormous problems. By 2050, the world's population will be 9.6 billion, resulting in a 60% rise in demand for staple foods. As the yield gains from the Green Revolution have slowly declined, and climate change is predicted to severely limit plant production, there is an urgent need for novel cultivars that can survive unfavorable climatic conditions while maintaining high yields and quality. Traditional breeding tactics are labor-intensive, time-consuming, and complicated; therefore, more efficient and time-saving breeding approaches are necessary.

With rapid advancements in sequencing technology, genomic information on an increasing number of plant species is becoming available, allowing for precise gene editing and opening up new avenues for crop improvement. Since 2013, the CRISPR/Cas system, derived from archaea and bacteria's adaptive phage immunity system, has been used to edit genomes in a variety of crops, introducing genes with significant agricultural value.

Advantages of CRISPR/Cas in Plant Gene Editing

CRISPR technology possesses multiple advantages and strengths, revolutionizing modern breeding techniques and providing feasible solutions to various future agricultural challenges. By continually optimizing and promoting CRISPR technology, it is expected to achieve more sustainable and efficient agricultural production systems.

1. High-Precision Gene Editing

The CRISPR-Cas system can introduce DNA double-strand breaks (DSBs) at target loci using sequence-specific nucleases and then repair these breaks through homology-directed repair (HDR) or non-homologous end joining (NHEJ), introducing precise genetic variations.

2. Simple Sequence Specificity

CRISPR-Cas relies on DNA-RNA recognition for sequence-specific nucleic acid cleavage, allowing easy programming to introduce DSBs at target sites at minimal cost, providing high flexibility and accuracy in gene editing.

3. Wide Application Range

CRISPR technology has been successfully applied to various crops, including rice, wheat, maize, tomatoes, and fruit crops, showing significant trait improvements such as increased yield, improved quality, and enhanced disease resistance.

4. Multiplexed Gene Editing

CRISPR technology can edit multiple genes simultaneously, regulating gene expression, stacking traits, and controlling regulatory pathways, facilitating crop improvement, breeding, and domestication. Multiplexed sgRNA expression systems and orthogonal editing strategies (e.g., using different Cas proteins) can edit multiple targets in a single experiment.

Applications in Crop Improvement

Unlike conventional breeding approaches, CRISPR-Cas technology provides a rapid way to generate ideal germplasms by deleting negative genetic elements responsible for undesired traits or introducing gain-of-function mutations through precise genome editing. In the past two years, the use of CRISPR-Cas has improved several crop characteristics, including yield, quality, disease resistance, and herbicide resistance.

• Increasing Yield

Manipulating cytokinin homeostasis is a practical way to increase cereal yield. For example, editing the C terminus of Oryza sativa LOGL5 (encoding a cytokinin-activation enzyme) enhanced grain yield in various environments. Knocking out genes such as cytokinin oxidase/dehydrogenase (CKX), which catalyzes cytokinin degradation, has produced high-yield wheat phenotypes. Editing genes like O. sativa PIN5b (panicle size), O. sativa GS3 (grain size), and various GW genes (grain weight) have increased yields in rice, wheat, and other cereal crops.

• Improving Quality

Traits other than yield are also critical for agricultural production. For instance, targeting granule-bound starch synthase 1 (GBSS1) has produced waxy maize and rice varieties with low amylose content, enhancing eating and cooking quality. CRISPR-Cas has also created high-quality crops with enriched carotenoid content, reduced phytic acid, and high oleic acid.

• Disease Resistance

Disrupting host susceptibility factors using CRISPR-Cas is a promising approach. For example, mutating promoter regions of SWEET genes has generated rice lines with broad-spectrum resistance to bacterial blight. Similarly, editing genes like EDR1 in wheat and MLO in tomatoes has produced resistance to powdery mildew. CRISPR-Cas9 can also cleave plant DNA viruses, providing resistance to geminivirus, caulimovirus, and RNA viruses.

• Herbicide Resistance

Developing herbicide-resistant germplasms using CRISPR-Cas is a cost-effective way of maintaining crop productivity. For instance, base editing of ALS genes to introduce specific mutations has conferred resistance to herbicides like sulfonylurea and imidazolinone. Similar strategies have been used to generate herbicide-resistant rice, wheat, and other crops.

Implementing CRISPR Solutions（Take CRISPR/Cas9 for example）

Through continual technical optimization and applied research, the CRISPR/Cas9 system will drive ongoing progress in crop improvement, addressing global food security issues, increasing agricultural output, and achieving sustainable agriculture goals. Here are the steps to implement it:

Figure 1. Flowchart of plant genome editing based on Cas9 system. (Chovatiya A, et al., 2024)

1. Target Gene Selection and Design

Select target genes for editing based on desired traits. Design corresponding sgRNA sequences to match the target DNA regions.

2. Construction of Gene Editing Vectors

Construct the gene editing vectors that will deliver the CRISPR/Cas9 system into plant cells. This involves assembling the sgRNAs and Cas9 into a suitable vector backbone.

3. Activity Validation in Protoplasts (Optional)

Validate the activity of the constructed vectors in protoplasts—plant cells without cell walls released from enzymatically digested tissues. This step ensures that the editing constructs work efficiently before moving on to whole-plant transformations.

4. Transformation

Introduce the gene editing vectors into plant cells. This can be done using techniques such as Agrobacterium-mediated transformation or gene gun methods to deliver the plasmid DNA into the plant genome.

5. Regeneration of Edited Cells into Plants

Use tissue culture methods to regenerate the edited cells into whole plants. This may involve cultivating callus tissue, rooting, and acclimatizing the plants to growing conditions.

6. Screening and Characterization

Screen the regenerated plants for successful genome editing events. This involves molecular techniques such as PCR, sequencing, and phenotypic analysis to confirm the desired genetic alterations and ensure the absence of off-target effects.

Creative Biogene's Support for Crop Improvement Projects

Creative Biogene provides comprehensive products and services to support various crop improvement projects. With expertise in CRISPR technology and access to advanced genomic resources, Creative Biogene assists in the design, construction, and validation of CRISPR constructs for targeted gene editing, enabling precision improvements in crop yield, quality, disease resistance, and environmental resilience. By offering end-to-end solutions from sgRNA design to transgenic plant production, Creative Biogene accelerates the development of superior crop varieties to meet the rising global agricultural demands.

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