Bioscience Methods 2024, Vol.15, No.6, 315-326 http://bioscipublisher.com/index.php/bm 322 (Liang et al., 2017). Furthermore, advancements in base-editing tools that enable targeted nucleotide substitutions have also contributed to the increased specificity and accuracy of CRISPR/Cas9-mediated genome editing in plants (Chen et al., 2019). 7.3 Strategies and Tool Development for Multi-Gene Editing The development of robust CRISPR/Cas9 systems for multiplex genome editing has opened new avenues for studying gene functions and improving crop traits. A notable example is the creation of a CRISPR/Cas9 vector system that allows for convenient and high-efficiency multiplex genome editing in both monocot and dicot plants. This system utilizes a plant codon-optimized Cas9 gene and PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into binary CRISPR/Cas9 vectors in a single cloning step. This approach has been successfully used to edit multiple target sites in rice and Arabidopsis, demonstrating its potential for multi-gene editing in wheat as well (Ma et al., 2015). Another strategy for multi-gene editing involves the use of co-expressed pairs of sgRNAs targeting different sites within the same gene. This method has been shown to produce large deletions and facilitate the identification of homoeolog-specific editing events in wheat (Cui et al., 2019). Additionally, the application of CRISPR/Cas9 ribonucleoprotein (RNP) complexes has been explored for multi-gene editing, offering a DNA-free approach that reduces the risk of off-target effects and transgene integration (Liang et al., 2017). These advancements in multi-gene editing strategies and tool development are crucial for the efficient manipulation of complex genomes like that of wheat, ultimately contributing to the improvement of crop traits and agricultural productivity. 8 Challenges in Wheat Breeding Applications 8.1 Low editing efficiency and technical barriers (e.g., off-target effects) One of the primary challenges in applying CRISPR/Cas9 technology to wheat breeding is the relatively low editing efficiency. Wheat, being a polyploid with a complex genome, presents significant difficulties in achieving precise and efficient genome edits. The efficiency of CRISPR/Cas9 in wheat is often lower compared to diploid plants, which complicates the breeding process and reduces the likelihood of obtaining the desired traits in a timely manner (Kim et al., 2017). Additionally, the identification and characterization of edited events in wheat are challenging due to its complex genome structure, which requires robust and efficient genotyping protocols to confirm successful edits (Cui et al., 2019). Another significant technical barrier is the occurrence of off-target effects, where the CRISPR/Cas9 system inadvertently edits regions of the genome other than the intended target. This can lead to unintended mutations that may affect plant growth and development negatively. Although methods such as the use of CRISPR/Cas9 ribonucleoproteins (RNPs) have been developed to reduce off-target mutations, the risk still exists and needs to be carefully managed (Liang et al., 2017). Ensuring high specificity and minimizing off-target effects are crucial for the successful application of CRISPR/Cas9 in wheat breeding (Li et al., 2021). 8.2 Ethical and regulatory challenges in gene editing The application of CRISPR/Cas9 in wheat breeding also faces significant ethical and regulatory challenges. Public perception of genetically modified organisms (GMOs) remains a contentious issue, with concerns about the safety and long-term impacts of consuming genetically edited crops. These concerns are often amplified by a lack of understanding of the technology and its benefits, leading to resistance against the adoption of CRISPR/Cas9-edited crops (Eş et al., 2019). Ethical considerations also include the potential for unintended ecological impacts, such as the spread of edited genes to wild relatives, which could disrupt local ecosystems. Regulatory frameworks for gene editing vary widely across different countries, creating a complex landscape for the commercialization of CRISPR/Cas9-edited wheat. In some regions, CRISPR/Cas9-edited crops are subject to the same stringent regulations as traditional GMOs, which can delay the approval process and increase the cost of bringing new varieties to market (Haque et al., 2018). Harmonizing these regulations and ensuring they are based on scientific evidence rather than public fear is ssential for the widespread adoption of CRISPR/Cas9 technology in wheat breeding (Liu et al., 2021).
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