Bioscience Methods 2024, Vol.15, No.6, 315-326 http://bioscipublisher.com/index.php/bm 323 8.3 Limitations and potential of CRISPR/Cas9 in commercial breeding Despite its potential, CRISPR/Cas9 technology has limitations that need to be addressed for its successful application in commercial wheat breeding. One major limitation is the difficulty in achieving large deletions or complex genetic modifications, which are often required for significant trait improvements. While protocols have been developed to create large deletions using pairs of co-expressed sgRNAs, these methods are still not as efficient or reliable as needed for commercial applications (Cui et al., 2019). Additionally, the delivery of CRISPR/Cas9 components into wheat cells remains a technical challenge, with traditional methods often resulting in low transformation efficiencies (Arora and Narula, 2017). However, the potential of CRISPR/Cas9 in commercial wheat breeding is immense. The technology allows for precise modifications at specific genomic locations, enabling the development of wheat varieties with improved traits such as disease resistance, drought tolerance, and enhanced nutritional content (Chen et al., 2019). The use of DNA-free methods, such as CRISPR/Cas9 RNPs, also holds promise for producing transgene-free edited plants, which could alleviate some regulatory and public acceptance issues (Liang et al., 2017). As the technology continues to advance, it is expected that these limitations will be overcome, paving the way for the widespread adoption of CRISPR/Cas9 in commercial wheat breeding (Bortesi and Fischer, 2015). 9 Concluding Remarks The application of CRISPR/Cas9 technology in wheat genetic improvement is rapidly evolving, with significant advancements in multi-gene editing and precision breeding. The ability to target multiple genes simultaneously using multiplex sgRNA-CRISPR/Cas9 systems has been demonstrated to be highly effective in creating complex trait modifications. For instance, the successful editing of five TaSal1 homologous genes in wheat using three gRNAs showcases the potential of this technology to address traits like drought tolerance by inducing heritable mutations across multiple loci. Additionally, the use of CRISPR/Cas9 ribonucleoproteins (RNPs) has been shown to reduce off-target effects and avoid transgene integration, making the process more precise and acceptable for commercial applications. These trends indicate a move towards more sophisticated and precise breeding techniques that can address multiple traits simultaneously, thereby accelerating the breeding process and enhancing crop resilience and productivity. Combining CRISPR/Cas9 technology with traditional and modern breeding methods holds great promise for the future of wheat genetic improvement. Traditional breeding methods have long been used to enhance crop traits, but they are often time-consuming and less precise. The integration of CRISPR/Cas9 with these methods can significantly speed up the breeding process and increase precision. For example, the use of CRISPR/Cas9 in conjunction with marker-assisted selection can help in the rapid identification and incorporation of desirable traits. Moreover, the combination of CRISPR/Cas9 with other genome editing tools like TALENs and ZFNs can provide a more comprehensive approach to genome manipulation, allowing for the fine-tuning of gene expression and the development of crops with enhanced traits. This hybrid approach can lead to the development of wheat varieties that are not only high-yielding but also resistant to diseases and environmental stresses. The potential of CRISPR/Cas9 technology to contribute to global food security is immense. As the global population continues to rise, the demand for food is expected to increase significantly. Traditional breeding methods alone may not be sufficient to meet this demand. CRISPR/Cas9 offers a powerful tool to enhance crop yields, improve nutritional quality, and develop resistance to pests and diseases, thereby playing a crucial role in ensuring food security. For instance, the application of CRISPR/Cas9 in editing genes related to stress responses in wheat has shown promising results in developing drought-tolerant varieties, which are essential for maintaining crop productivity under changing climatic conditions. Furthermore, the ability to produce transgene-free mutants using CRISPR/Cas9 RNPs makes the technology more acceptable to regulatory bodies and consumers, paving the way for its widespread adoption in agriculture. By enabling the rapid and precise improvement of crop traits, CRISPR/Cas9 technology can help secure a stable and nutritious food supply for the growing global population. Acknowledgments We would like to thank CropSci Publisher continuous support throughout the development of this study.
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