Molecular Plant Breeding 2024, Vol.15, No.2, 70-80 http://genbreedpublisher.com/index.php/mpb 71 This study aims to explore the revolutionary applications of precision genome editing technologies in tree breeding. By reviewing the latest advancements and their implications for tree genetics, this study provides a comprehensive understanding of how these technologies can overcome the limitations of traditional breeding methods and address the pressing challenges faced by modern forestry, and examine the current state of genome editing in trees, including successful applications and ongoing research, and discuss the potential for these technologies to transform tree breeding practices. Ultimately, this study aims to highlight the potential of precision genome editing to contribute to sustainable forestry and the development of climate-resilient tree species. 2 Genome Editing Technologies 2.1 Overview of genome editing tools 2.1.1 Zinc finger nucleases (ZFNs) Zinc Finger Nucleases (ZFNs) are engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks (DSBs) at specific locations. These breaks are then repaired by the cell's natural repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). ZFNs are composed of a DNA-binding domain, which can be customized to target specific DNA sequences, and a DNA-cleavage domain derived from the FokI restriction enzyme. This technology has been successfully applied in various plant species, including hexaploid bread wheat, where ZFN-mediated editing introduced specific amino acid changes to confer herbicide resistance (Ran et al., 2018). 2.1.2 Transcription activator-like effector nucleases (TALENs) Transcription Activator-Like Effector Nucleases (TALENs) are another class of engineered nucleases used for precise genome editing. Similar to ZFNs, TALENs consist of a customizable DNA-binding domain and a FokI nuclease domain. The DNA-binding domain of TALENs is derived from transcription activator-like effectors (TALEs) of plant pathogenic bacteria, which can be engineered to recognize specific DNA sequences. TALENs have been widely used in plant genome editing due to their high specificity and efficiency. They offer a versatile tool for introducing targeted modifications in plant genomes, facilitating the development of crops with desirable traits. 2.1.3 Clustered regularly interspaced short palindromic repeats (CRISPR/Cas) The CRISPR/Cas system, particularly the CRISPR/Cas9 and CRISPR/Cas12a variants, has revolutionized genome editing due to its simplicity, efficiency, and versatility. This system uses a guide RNA (gRNA) to direct the Cas nuclease to a specific DNA sequence, where it creates a DSB. The DSBs can be repaired by NHEJ, leading to insertions or deletions (indels), or by HDR if a donor template is provided. The CRISPR/Cas system has become the most popular method for plant genome editing, enabling precise modifications and the development of crops with improved traits (Kim and Kim, 2019). 2.2 Comparison of different genome editing technologies Each genome editing technology has its own advantages and limitations. ZFNs and TALENs offer high specificity due to their customizable DNA-binding domains, but their design and construction can be complex and time-consuming. In contrast, the CRISPR/Cas system is easier to design and implement, making it more accessible to researchers. CRISPR/Cas also allows for multiplexing, where multiple genes can be edited simultaneously, which is more challenging with ZFNs and TALENs (Ran et al., 2018; Kim and Kim, 2019). While ZFNs and TALENs have been successfully used in various plant species, the CRISPR/Cas system has rapidly become the preferred tool due to its efficiency and ease of use. The expanding CRISPR/Cas toolbox, including various Cas enzymes and engineered components, continues to enhance its capabilities and applications in plant genome editing (Kim and Kim, 2019). In summary, while ZFNs and TALENs have paved the way for precise genome editing, the CRISPR/Cas system has emerged as the most versatile and widely adopted technology, driving significant advancements in plant breeding and the development of crops with desirable traits.
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