Bioscience Methods 2024, Vol.15, No.4, 184-195 http://bioscipublisher.com/index.php/bm 1 88 range of NGN PAM sites, thereby illustrating its potential for more flexible and efficient genome editing in plants (Ren et al., 2019). The case studies presented here demonstrate the potential of CRISPR/Cas9 technology to facilitate advancements in functional genomics and crop improvement in rice, thereby paving the way for future innovations in plant biotechnology. 5 Functional Analysis of R2R3-MYB Genes Using CRISPR/Cas9 5.1 Target selection and design of CRISPR constructs The selection of target genes for CRISPR/Cas9-mediated knockout or knockdown is of paramount importance for elucidating the functional roles of R2R3-MYB genes in the anthocyanin biosynthesis pathway. In black rice, the R2R3-MYB gene OsMYB3 has been identified as a key regulator of anthocyanin biosynthesis, exerting a particularly pronounced influence on the expression of late ABP genes (Zheng et al., 2021). Similarly, other studies have underscored the significance of R2R3-MYB genes in regulating anthocyanin production in a range of plant species, including OjMYB1 in Oenanthe javanica and ASR genes in Petunia inflata (Feng et al., 2018; Zhang et al., 2019). These findings provide a foundation for the selection of specific R2R3-MYB genes for CRISPR/Cas9 targeting, thereby facilitating the investigation of their roles in black rice. 5.2 Generation of knockout and knockdown lines To generate knockout and knockdown lines, CRISPR/Cas9 constructs were designed to target the coding regions of selected R2R3-MYB genes. The constructs were introduced into rice plants via Agrobacterium-mediated transformation. The successful integration and expression of the CRISPR/Cas9 system were confirmed through molecular techniques, including polymerase chain reaction (PCR) and sequencing. Prior research has substantiated the efficacy of CRISPR/Cas9 in introducing targeted mutations in MYB genes, resulting in altered anthocyanin production in a range of plant species (Jung et al., 2019; Duan et al., 2022). These methodologies were adapted for the generation of knockout and knockdown lines in black rice, with a particular focus on the OsC1 gene and other candidate R2R3-MYB genes identified through comparative genomics and expression analysis. 5.3 Phenotypic and molecular characterization The phenotypic and molecular characterization of the generated knockout and knockdown lines entailed the assessment of anthocyanin content, expression levels of ABP genes, and other related phenotypic traits. Phenotypic assessments were conducted in the knockout (KO) and knockdown (KD) lines to determine the impact of specific R2R3-MYB gene disruptions on anthocyanin biosynthesis and related physiological traits (Figure 2) (Zheng et al., 2021). Molecular characterization was conducted through quantitative real-time polymerase chain reaction (qRT-PCR) analysis of ABP gene expression and high-performance liquid chromatography (HPLC) quantification of anthocyanins, as previously described in studies on other plant species (Naing and Kim, 2018; Yang et al., 2022). 5.4 Insights into the role of specific R2R3-MYB genes The functional analysis of R2R3-MYB genes using CRISPR/Cas9 provided valuable insights into their roles in the anthocyanin biosynthesis pathway in black rice. The disruption of OsC1 and other targeted R2R3-MYB genes resulted in significant alterations in anthocyanin content and the expression patterns of ABP genes, thereby confirming their regulatory roles (Zheng et al., 2019; Upadhyaya et al., 2021). Furthermore, the study demonstrated the involvement of these genes in broader physiological processes, such as the oxidative stress response and photosynthetic efficiency, as observed in OsC1-overexpressed plants (Upadhyaya et al., 2021). These findings contribute to a more profound comprehension of the molecular mechanisms that underpin anthocyanin biosynthesis and the potential for metabolic engineering of anthocyanin production in rice and other crops (Zhang et al., 2019; Yang et al., 2023).
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