International Journal of Horticulture, 2024, Vol.14, No.3, 142-155 http://hortherbpublisher.com/index.php/ijh 145 2 CRISPR/Cas9 Gene Editing Technology 2.1 Principles and mechanisms of CRISPR/Cas9 CRISPR/Cas9 is a powerful gene-editing technology that allows for precise modifications in the DNA of living organisms. The CRISPR/Cas9 system, derived from the adaptive immune system of prokaryotes, has revolutionized genome editing due to its simplicity, efficiency, and precision. The system consists of two key components: the Cas9 nuclease and a single-guide RNA (sgRNA). The sgRNA is designed to be complementary to a specific DNA sequence, guiding the Cas9 to the target site. When the gRNA binds to the complementary DNA sequence, the Cas9 enzyme induces a double-strand break at that location. The cell's natural repair mechanisms then kick in to repair the break. This repair process can be harnessed in two ways: non-homologous end joining (NHEJ), which often results in insertions or deletions that disrupt the gene, and homology-directed repair (HDR), which can be used to introduce precise genetic changes by providing a DNA template (Figure 2) (El-Mounadi et al., 2020). This precision and versatility make CRISPR/Cas9 an important tool for genetic research and engineering (Bao et al., 2019; Mao et al., 2019; El-Mounadi et al., 2020). Figure 2 Targeted genome editing using CRISPR-Cas9 (Adopted from El-Mounadi et al., 2020) Image caption: The figure illustrates three key steps in targeted genome editing using CRISPR-Cas9: (A) Components: The CRISPR-Cas9 system consists of the Cas9 protein and one or more guide RNAs (sgRNAs). The guide RNA determines the specificity of the target DNA through sequence complementarity; (B) Binary Complex Formation: The guide RNA binds with the Cas9 protein to form a binary complex. This complex can specifically recognize and bind to the target DNA sequence, introducing a double-strand break (DSB) at the target location; (C) DNA Repair Mechanisms: The cell utilizes its own DNA repair mechanisms to repair the DSB, primarily through non-homologous end joining (NHEJ) or homology-directed repair (HDR). During this process, insertions, deletions, replacements, or gene insertions may occur, thereby achieving targeted genome modification (Adapted from El-Mounadi et al., 2020)
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