Molecular Plant Breeding 2024, Vol.15, No.2, 81-89 http://genbreedpublisher.com/index.php/mpb 85 degrees of chlorosis. This indicates that CRISPR/Cas9-mediated gene editing is effective in poplar, and different sgRNA targets result in different mutation effects. This study provides important technical support for future improvements in the stress resistance traits of poplar. Another example demonstrates that applying CRISPR/Cas9 to edit drought-resistant genes in poplar has yielded significant results. Compared to other programmable nucleases such as ZFNs and TALENs, the CRISPR/Cas9 system offers greater simplicity and efficiency. The research covers various plant-specific CRISPR/Cas9 vector systems, multiplex editing strategies, mutation analysis methods, and factors affecting editing efficiency (Figure 3). These case studies showcase the potential of CRISPR/Cas9 for precise and efficient gene editing in poplar, paving the way for the development of drought-resistant varieties. Figure 3 Strategies for generation of multiple sgRNA expression cassettes in a binary vector (Adopted from Ma et al., 2016) Image caption: (A) Cloning of multiple sgRNA expression cassettes using multiple restriction enzymes (RE1–RE4). (B) Assembly of multiple (three as an example) sgRNA expression cassettes using Golden Gate ligation. (C) Cloning multiple (three as an example) sgRNA expression cassettes using Gibson assembly. (D) Generation of multiple sgRNAs by the polycistronic tRNA-gRNA gene (Adopted from Ma et al., 2016) Figure 3 illustrates various strategies for generating multiple sgRNA expression cassettes in a binary vector. The figure details the implementation steps of four methods: multiple restriction enzyme cloning, Golden Gate assembly, Gibson assembly, and a polycistronic tRNA-gRNA system. These methods employ different technical approaches to connect multiple sgRNA expression cassettes, enabling multiplex genome targeting. These strategies allow for the simultaneous mutation of multiple genes or gene family members, which is crucial for studying complex traits and improving crop varieties. The application of these methods not only enhances the efficiency of gene editing but also broadens the scope of the CRISPR/Cas9 system in plant research. 5 Challenges and Considerations in CRISPR/Cas9-Mediated Gene Editing 5.1 Off-target effects and their mitigation One of the primary challenges in CRISPR/Cas9-mediated gene editing is the occurrence of off-target effects, where the Cas9 enzyme cuts at unintended genomic locations. This can lead to unintended mutations, which may have deleterious effects on the plant's phenotype and overall health. Various strategies have been developed to
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