Bioscience Methods 2024, Vol.15, No.6, 315-326 http://bioscipublisher.com/index.php/bm 321 Another example is the modification of genes involved in the biosynthesis of vitamins. By targeting specific genes, researchers have been able to increase the levels of vitamins such as vitamin E and provitamin A in wheat. These modifications not only enhance the nutritional value of wheat but also contribute to better health outcomes for consumers. The use of CRISPR/Cas9 in these studies highlights its potential to revolutionize the nutritional quality of wheat (Upadhyay et al., 2013; Eş et al., 2019; Wang et al., 2021a). 6.3 Target genes for nutritional content improvement and their editing strategies Several target genes have been identified for improving the nutritional content of wheat using CRISPR/Cas9. One key target is the TaABCC6 gene, which is involved in the transport of phytic acid. By knocking out this gene, researchers have been able to reduce phytic acid levels, thereby increasing the bioavailability of essential minerals such as iron and zinc (Cui et al., 2019; Wang et al., 2021a). Another important target is the TaNFXL1 gene, which has been edited to enhance the accumulation of beneficial nutrients in wheat grains (Cui et al., 2019). The editing strategies for these genes typically involve the use of single guide RNAs (sgRNAs) to direct the Cas9 endonuclease to specific genomic loci. In some cases, multiple sgRNAs are used to create larger deletions or to target multiple genes simultaneously. This multiplexing approach allows for more comprehensive modifications and can lead to more significant improvements in nutritional content. Additionally, the use of optimized Cas9 variants, such as pcoCas9, has been shown to increase the efficiency and precision of gene editing in wheat (Cui et al., 2019; Wang et al., 2021a). 7 Optimization and Innovation in CRISPR/Cas9 Technology 7.1 Methods to improve editing efficiency One of the primary methods to enhance the efficiency of CRISPR/Cas9 editing in wheat involves the use of modified Cas9 variants. For instance, a study demonstrated that a plant codon-optimized Cas9 (pcoCas9) yielded more consistent results compared to a codon-optimized Cas9 for expression in algae (crCas9) when targeting specific genes in wheat (Cui et al., 2019). This highlights the importance of using Cas9 variants that are specifically optimized for the target organism to achieve higher editing efficiency. Additionally, the use of ribonucleoprotein (RNP) complexes has been shown to significantly reduce the chances of off-target mutations, thereby increasing the overall efficiency of the genome editing process (Liang et al., 2017). Another approach to improve editing efficiency is the co-expression of multiple sgRNAs targeting the same gene. This method has been successfully employed to create large deletions in wheat by using pairs of co-expressed sgRNAs, which target different sites within the same gene. This strategy not only increases the frequency of desired editing events but also facilitates the identification and characterization of these events in complex genomes like that of wheat. Furthermore, the development of efficient genotyping protocols to identify edited events in hexaploid genomes has also contributed to the optimization of CRISPR/Cas9 technology in wheat (Cui et al., 2019). 7.2 Techniques for enhancing specificity and accuracy Enhancing the specificity and accuracy of CRISPR/Cas9-mediated genome editing is crucial to minimize off-target effects. One effective technique involves the optimization of single-guide RNA (sgRNA) parameters. Research has shown that the GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs) in the sgRNA is positively correlated with mutagenesis efficiency, suggesting that careful design of sgRNAs can significantly improve the specificity and efficiency of the CRISPR/Cas9 system. Additionally, the use of well-designed sgRNA plasmids at optimal concentrations has been demonstrated to efficiently generate mutations in multiple genes in a single step (Ren et al., 2014; Zhang et al., 2024). Another promising approach to enhance specificity is the development of novel Cas proteins and engineered variants. For example, the use of CRISPR/Cas9 ribonucleoprotein (RNP) complexes has been shown to produce transgene-free mutants with a much lower chance of off-target mutations compared to traditional DNA-based CRISPR/Cas9 methods (Liang et al., 2017). This method not only improves the specificity of genome editing but also addresses concerns related to transgene integration, making it a valuable tool for precision crop breeding
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