Legume Genomics and Genetics 2025, Vol.16, No.3, 135-142 http://cropscipublisher.com/index.php/lgg 137 Gao et al., 2022). Research shows that epigenetic mechanisms which control gene presence/absence variation and expression levels contribute to the evolution of early flowering traits (Liu et al., 2022; Chiteri et al., 2024). The hormonal and epigenetic factors operate as additional control mechanisms which work together with genetic and environmental factors to regulate mung bean flowering. 3 CRISPR Activation (CRISPRa) Systems: Concepts and Mechanisms 3.1 CRISPRa operates through dCas9 fusion with transcriptional activators to achieve its mechanism of action The CRISPR activation system depends on dCas9 as its protein component because this enzyme variant maintains DNA targeting ability through single guide RNAs (sgRNAs) but lacks DNA cutting capability. The CRISPRa system enables gene expression enhancement through dCas9 connection to transcriptional activator domains which modify gene expression without modifying DNA sequences. The dCas9/sgRNA complex guides itself to promoter or enhancer regions to activate transcription by bringing transcriptional machinery through attached activator domains which results in elevated target gene expression (Karlson et al., 2021; Ding et al., 2022). Scientists use this method to precisely activate particular genes in plants and other biological systems. 3.2 CRISPR knockout and CRISPR interference (CRISPRi) systems are compared to the CRISPR-Cas13 system The CRISPR knockout system employs active Cas9 to generate double-strand breaks which result in gene disruption through the error-prone DNA repair mechanism. The CRISPR interference system (CRISPRi) employs dCas9 linked to KRAB repressor domains to prevent gene transcription which results in gene silencing. CRISPRa functions differently from CRISPR because it works to increase gene expression rather than interrupting or blocking it. The regulatory outcome of CRISPRa and CRISPRi systems depends on the effector domain selection between activator and repressor because both systems use dCas9 for DNA binding (Lo and Qi, 2017; Karlson et al., 2021). The reversible nature of CRISPRa and CRISPRi systems allows them to function without creating lasting genetic alterations which makes them suitable for functional genomics and crop development applications. 3.3 Common activation domains (VP64, VPR, SAM, SunTag) and their efficiency Researchers have established multiple activation domains which function to boost CRISPRa performance. The VP64 protein consists of four VP16 activation domains which function as a tetramer to activate genes at a moderate level (Park et al., 2017; Karlson et al., 2021). The tripartite activator VPR unites VP64 with p65 and Rta to achieve higher activation levels than VP64 operates independently (Liu et al., 2023). The SAM system (Synergistic Activation Mediator) enables gene activation through sgRNAs that include MS2 aptamers which bind p65 and HSF1 activators through MS2 coat proteins to achieve strong gene activation (Ding et al., 2022). SunTag: Employs a repeating peptide array fused to dCas9, which recruits multiple copies of an antibody-activator fusion, amplifying transcriptional activation (Ding et al., 2022). The study shows that VPR, SAM and SunTag systems generate superior results than dCas9-VP64 because they achieve 10 to over 1000-fold gene expression increases due to their precise targeting capabilities and optimized system architecture (Lo and Qi, 2017; Ding et al., 2022). The research of Liu et al. (2023) presents dCas9-VPRF as a new phase-separation protein fusion which enhances both activation efficiency and system design simplicity according to recent studies. 4 Applications of CRISPRa in Plant Flowering Regulation 4.1 Evidence from model plants (Arabidopsis, rice, tomato) The CRISPR activation (CRISPRa) systems show promise for model plant applications to control flowering time by enabling the activation of vital floral regulators. The CRISPRa system in Arabidopsis used dCas9-VP64 and
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