Tree Genetics and Molecular Breeding 2024, Vol.14, No.2, 81-94 http://genbreedpublisher.com/index.php/tgmb 83 3.1.1 CRISPR/Cas9 CRISPR/Cas9 has emerged as the most popular and widely used genome editing tool due to its simplicity, efficiency, and versatility (Liu et al., 2022). The system uses a guide RNA (gRNA) to target specific DNA sequences, which are then cleaved by the Cas9 enzyme, leading to targeted mutations. This technology has been successfully applied in Eucalyptus to knock out genes related to lignin biosynthesis and other traits, allowing researchers to study gene function and improve disease resistance (Dai et al., 2020). Dai et al. (2020) utilized CRISPR/Cas9 technology to target and knock out two key genes- CCR1 (cinnamoyl-CoA reductase 1) and IAA9A (auxin-dependent transcription factor)- and observed the mutation effects in the hairy roots of Eucalyptus (Figure 1). The results indicated that while the CRISPR/Cas9 system had a high success rate in editing both genes, there were significant differences in editing efficiency and mutation types between the two genes. Editing of the CCR1 gene resulted in a reduction in lignin content, whereas editing of the IAA9A gene primarily affected the formation of secondary xylem. This study demonstrated the potential of CRISPR/Cas9 technology in functional gene studies in Eucalyptus, providing an effective tool for further gene function validation. Figure 1 Mutation types and frequencies of CCR1 and IAA9A genes in transgenic Eucalyptus hairy roots (Adapted from Dai et al., 2020) Image caption: The PCR amplification products of different transgenic lines are displayed by electrophoresis gel, indicating the successful editing of the target gene by the CRISPR/Cas9 system (Adapted from Dai et al., 2020) 3.1.2 TALENs TALENs are another powerful tool for genome editing, involving engineered proteins that bind to specific DNA sequences and introduce double-strand breaks. TALENs are known for their high specificity and lower off-target effects compared to CRISPR/Cas9. Although TALENs have been less commonly used in Eucalyptus, they have proven effective in editing genes in other plant species and hold potential for future applications in Eucalyptus biotechnology (Borrelli et al., 2018). 3.1.3ZFNs Zinc-finger nucleases (ZFNs) were one of the first genome editing technologies developed (Kim and Lee, 2022). ZFNs use a combination of zinc-finger DNA-binding domains and a DNA-cleaving domain to introduce specific cuts in the DNA (Becker and Boch, 2021). While ZFNs have been largely overshadowed by the emergence of CRISPR/Cas9, they remain a valuable tool for certain applications, particularly where CRISPR technology may be less effective. Their use in Eucalyptus is limited, but they offer a complementary approach to the more commonly used CRISPR system (Mushtaq et al., 2019).
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