Bioscience Methods 2024, Vol.15, No.4, 184-195 http://bioscipublisher.com/index.php/bm 1 87 3.3 Regulatory networks involving R2R3-MYB genes R2R3-MYB TFs play a pivotal role in the regulatory networks that control anthocyanin biosynthesis in rice. OsMYB3, an R2R3-MYB protein, is a pivotal regulator that modulates the expression of late ABP genes, thereby influencing anthocyanin accumulation (Zheng et al., 2021). The MYB-bHLH-WD40 (MBW) complex, which includes OsC1, plays a pivotal role in this regulatory network. In this complex, the bHLH protein S1 activates the expression of OsC1, which in turn activates the expression of the WD40 protein WA1, thereby establishing a cascading regulatory hierarchy (Sun et al., 2022). This hierarchical regulatory system ensures the efficient coordination of anthocyanin biosynthesis genes, thereby enabling precise control over anthocyanin production in different rice tissues. The regulatory role of R2R3-MYB genes is not limited to pigmentation. For example, the overexpression of OsC1 in white rice has been demonstrated to enhance oxidative stress tolerance by increasing anthocyanin content, which facilitates the reduction of reactive oxygen species and improves photosynthetic efficiency (Upadhyaya et al., 2021). This underscores the multifaceted function of R2R3-MYB genes in both anthocyanin synthesis and stress response pathways in rice. 4 CRISPR/Cas9 Technology in Functional Genomics 4.1 Principles of CRISPR/Cas9 genome editing CRISPR/Cas9 represents a revolutionary advance in the field of genome editing, offering the potential for highly precise modifications to the DNA of a diverse range of organisms. The system is comprised of two principal components: the Cas9 nuclease, which performs the cutting of the DNA, and a guide RNA (gRNA), which directs the Cas9 to the specific genomic location that is to be edited. The gRNA is designed to be complementary to the target DNA sequence, thereby ensuring specificity. Following the induction of a double-strand break by Cas9, the cell's intrinsic repair mechanisms, including non-homologous end joining (NHEJ) and homology-directed repair (HDR), are activated to repair the break, resulting in targeted mutations or insertions (Klimek-Chodacka et al., 2018; Kim et al., 2019). 4.2 Applications in plant research CRISPR/Cas9 has become a widely utilized tool in plant research, particularly in the fields of functional genomics and crop improvement (Mackon et al., 2023). The technique enables researchers to inactivate genes to elucidate their functions, introduce advantageous traits, and enhance resistance to diseases. For example, CRISPR/Cas9 has been employed to modify genes associated with anthocyanin biosynthesis in black rice, resulting in alterations in seed color and anthocyanin content (Jung et al., 2019; Mackon et al., 2024). Furthermore, it has been utilized to impart resistance to bacterial blight in rice by knocking out the Os8N3 gene, which enhanced resistance to Xanthomonas oryzae pv. oryzae without affecting other agronomic traits (Kim et al., 2019). 4.3 Case studies: successful CRISPR/Cas9 edits in rice Several successful applications of CRISPR/Cas9 in rice have been documented, demonstrating its efficacy and versatility: Anthocyanin Biosynthesis Pathway: A study was conducted to investigate the potential of CRISPR/Cas9 to alter the OsF3'H, OsDFR, and OsLDOX genes in black rice. This involved introducing mutations that were shown to affect seed color and anthocyanin content. The mutations were stably inherited, and the edited plants exhibited no discernible differences from non-GMO plants under the strictest GMO regulations (Jung et al., 2019). Disease Resistance: The Os8N3 gene in rice was inactivated using the CRISPR/Cas9 system to enhance resistance to bacterial blight caused by Xanthomonas oryzae pv. Oryza. The edited plants demonstrated stable transmission of the mutations across generations and exhibited no significant differences in agronomic traits when compared to non-transgenic controls (Kim et al., 2019). Expanded Targeting Scope: The development of Cas9-NG, a variant that recognizes a broader range of PAM sequences, has broadened the scope for targeting in rice using CRISPR/Cas9. The variant was employed to edit a
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