Rice Genomics and Genetics 2024, Vol.15, No.1, 12-18 http://cropscipublisher.com/index.php/rgg 14 Once the Cas9 protein and RNA guide recognition and binding to the target DNA, it will guide the Cas9 protein to cleave DNA, resulting in DNA double strand breaks. This rupture activates the DNA repair mechanisms of cells, including non homologous end junctions (NHEJ) and homologous recombination (HDR). NHEJ typically causes small fragment insertions or deletions, while HDR allows for more precise DNA repair (Zhang et al., 2019). Ultimately, DNA double strand breaks and repair processes can lead to gene editing. If the goal of scientists is to knock out genes, the NHEJ repair process may result in inserted or missing bases, thereby disrupting gene function. If the goal is to repair genes or add new DNA sequences, HDR can be used to achieve more precise editing. This approach enables scientists to modify specific genes to achieve the desired genetic improvement (Zhang et al., 2016). The mechanism guided by Cas9 protein and RNA is the core of CRISPR-Cas9 technology, allowing scientists to achieve precise gene editing. The understanding of this mechanism is of crucial importance for the study of genetic improvement and reproductive organ development in rice. 2.3 Basic process of targeted gene editing Targeted gene editing is one of the core applications of CRISPR-Cas9 technology, allowing scientists to accurately modify the DNA sequence of organisms. Firstly, scientists need to determine the target genes they want to edit. This can be genes related to the development of specific reproductive organs, or other genes of interest. The selection of target genes depends on the research purpose and the required genetic improvements. Once the target gene is identified, scientists need to design an RNA guidance sequence, which typically consists of 20 nucleotides and complements the DNA sequence of the target gene. A well-designed RNA guidance sequence is key to achieving precise editing, as it determines where the Cas9 protein will cleave DNA. The designed RNA guide sequence is merged into the CRISPR-Cas9 system and used in conjunction with the Cas9 protein. This Cas9 RNA complex forms a highly specific complex through complementary pairing with the RNA guide sequence. This complex guides the Cas9 protein to a specific location of the target gene (Romero and Gatica Arias, 2019). Once the Cas9 RNA complex binds to the target DNA, the Cas9 protein will cleave the DNA, causing DNA double strand breaks. This rupture will activate the cell's DNA repair mechanism. There are two main repair mechanisms, non homologous terminal junction (NHEJ) and homologous recombination (HDR). NHEJ often leads to small fragment insertion or deletion, as it is a relatively imprecise repair process. This method is commonly used for gene knockout as it can cause loss of gene function. HDR allows for more precise DNA repair as it uses exogenous DNA templates for repair. This method is commonly used to repair damaged genes or add new DNA sequences. Scientists need to analyze the effectiveness of editing. This can be achieved through PCR, sequencing, or other molecular biology techniques. The analysis of editing effects helps to determine whether the required genetic improvements have been successfully achieved. This provides a precise method for modifying the DNA of living organisms in the basic process of gene editing, which is very useful for genetic improvement and reproductive organ development research in rice. The success of this process depends on the careful design of RNA guided sequences and the analysis of editing effects. 3 The Demand for Genetic Improvement in Rice 3.1 Challenges faced by genetic improvement of rice Although the CRISPR-Cas9 multi editing system provides new possibilities for genetic improvement in rice, this field still faces numerous challenges. These challenges involve ethics, regulations, technology, and the practical application of genetic improvement. Ethical and regulatory issues have always been one of the key challenges in the field of rice genetic improvement. How to ensure that gene editing does not pose potential environmental and health risks, as well as how to address potential ethical and social issues, are issues that need to be carefully considered. The regulations for gene editing vary in different countries, so researchers need to comply with local regulations to ensure that their work is legal and safe.
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