Molecular Plant Breeding 2024, Vol.15, No.6, 391-402 http://genbreedpublisher.com/index.php/mpb 394 In addition to CRISPR-Cas9, other gene-editing tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have also been used for genetic improvement. Although these tools are effective, they are generally more complex and less efficient compared to CRISPR-Cas9. The development of these technologies has significantly advanced our ability to manipulate plant genomes, including the Eucommia ulmoides genome, thereby promoting the improvement of desirable traits such as growth rate, stress resistance, and production of metabolites (Li et al., 2020; Sharma et al., 2020). The establishment of the CRISPR/Cas9 gene-editing system in Eucommia ulmoides successfully achieved gene editing in Eucommia cells for the first time. This research provides a new approach for gene function identification and genetic improvement in Eucommia ulmoides. 3.2 Potential target genes for modification Identifying and targeting specific genes associated with key traits is crucial for the successful genetic improvement of Eucommia ulmoides (Figure 1). Genes involved in growth regulation, stress resistance, and metabolite biosynthesis are priority candidates for modification. For instance, genes regulating hormone pathways, such as those involved in gibberellin and auxin signaling, can be targeted to enhance growth rate. Similarly, stress resistance genes, including those responsible for synthesizing heat shock proteins or antioxidant enzymes, can be modified to improve the plant’s adaptability to environmental stresses (Han et al., 2020; Li et al., 2020a; Sharma et al., 2020). Figure 1 Stagewise schematic representations of target site recognition in CRISPR/Cas9-mediated genome editing with modifications to the sgRNA and Cas9 endonuclease to reduce OTEs (Adopted from Han et al., 2020) Image caption: a The Cas9 endonuclease first scans the genomic DNA and binds to canonical PAM sequences (I). This induces a structural change in the sgRNA that allows the guide sequence to search and hybridise to complementary target sites upstream of the PAM (II). sgRNA-DNA hybridisation activates the Cas9 nuclease domains which then cleaves both strands of DNA (III). b sgRNA can be truncated at the 5′-end by 2–3 nucleotides or c modified at the 5′-end to contain 2 guanine nucleotides to improve the specificity of the guide sequence. d Cas9 nickase with only 1 active catalytic domain can be paired and e catalytically deactivated Cas9 fused to FokI nuclease that requires dimerisation for nuclease activity can be used to minimise off-target indels (insertion/deletion). f Base editors that convert a single cytosine base to thymine without requiring DSBs are less promiscuous at off-target sites. g Cas9 orthologs from other bacteria such as SaCas9 and h other Cas nucleases such as Cas12a that recognises alternative PAMs can be used to target novel DNA sequences and improve specificity (Adopted from Han et al., 2020)
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