Molecular Plant Breeding 2024, Vol.15, No.4, 178-186 http://genbreedpublisher.com/index.php/mpb 179 The aim of this study is to provide a comprehensive overview of the applications of CRISPR/Cas9 in rice breeding, with a particular focus on yield trait improvement. This study will examine the various strategies employed to enhance yield through CRISPR/Cas9-mediated genome editing, including gene knockout, point mutations, and single base editing. Additionally, this study will discuss the recent advancements in delivery mechanisms and the challenges associated with the broader application of this technology in rice breeding. By synthesizing the current state of research, this study aims to highlight the potential of CRISPR/Cas9 to transform rice breeding and contribute to global food security. 2 Basics of CRISPR/Cas9 Technology 2.1 History and development of CRISPR/Cas9 The CRISPR/Cas9 system, originally discovered as a part of the adaptive immune system in bacteria, has revolutionized genetic engineering since its adaptation for use in eukaryotic cells. The system was first identified in the late 1980s, but its potential for genome editing was not realized until the early 2010s. The breakthrough came when researchers demonstrated that CRISPR/Cas9 could be programmed to target specific DNA sequences, allowing for precise genetic modifications. CRISPR/Cas9 can efficiently edit the genome of diverse organisms, including humans, animals and plants (Figure 1) (Fiaz et al., 2019). This technology has since been rapidly adopted in various fields, including plant science, due to its simplicity, efficiency, and versatility (Ahmad et al., 2020; Zegeye et al., 2022). 2.2 Mechanism of CRISPR/Cas9 function The CRISPR/Cas9 system functions through a guide RNA (gRNA) that directs the Cas9 nuclease to a specific DNA sequence. The gRNA binds to the target DNA through complementary base pairing, and the Cas9 protein induces a double-strand break at the target site. The cell's natural repair mechanisms then take over, either through non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or through homology-directed repair (HDR) if a repair template is provided. This precise targeting and cutting mechanism allows for the efficient editing of specific genes, making CRISPR/Cas9 a powerful tool for genetic research and breeding (Yimam et al., 2021; Zegeye et al., 2022). 2.3 Applications of CRISPR/Cas9 in plant science CRISPR/Cas9 has been widely applied in plant science to improve various traits, including yield, stress resistance, and quality. In rice, CRISPR/Cas9 has been used to edit genes associated with drought tolerance, grain size, and cold resistance. For instance, editing the OsSAP gene has shown potential in enhancing drought tolerance by modulating stress-related transcription factors (Park et al., 2022). Similarly, the OsSPL16 gene was edited to increase grain yield by affecting pyruvate metabolism and cell cycle proteins (Usman et al., 2020). Additionally, simultaneous editing of multiple genes, such as OsPIN5b, Grain Size 3 (GS3), and OsMYB30, has resulted in rice varieties with improved yield and cold tolerance (Zeng et al., 2020). These examples highlight the versatility and effectiveness of CRISPR/Cas9 in addressing complex breeding challenges and accelerating the development of superior rice cultivars (Bui, 2020; Peng et al., 2020; Usman et al., 2021). 3 CRISPR/Cas9 in Rice Breeding 3.1 Early successes in CRISPR/Cas9 rice research The application of CRISPR/Cas9 technology in rice breeding has shown significant promise in recent years. Early successes include the precise editing of genes associated with important agronomic traits, such as drought resistance and grain size. For instance, the editing of the Oryza sativa Senescence-associated protein (OsSAP) gene demonstrated the potential of CRISPR/Cas9 to enhance drought tolerance in rice. The edited plants exhibited improved survival rates and growth metrics under drought stress, as illustrated in Figure 2, highlighting the efficiency of CRISPR/Cas9 in developing stress-resistant rice varieties (Park et al., 2022). Additionally, the CRISPR/Cas9-mediated mutagenesis of the Grain Size 3 (GS3) gene resulted in rice mutants with significantly increased grain length and weight, showcasing the technology’s capability to improve yield-related traits (Usman et al., 2021).
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