Molecular Plant Breeding 2024, Vol.15, No.2, 70-80 http://genbreedpublisher.com/index.php/mpb 75 One promising approach involves the use of base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), which enable precise genome modifications without introducing double-strand breaks (DSBs). This method reduces the risk of genome instability and unpredictable outcomes associated with DNA repair mechanisms. For instance, researchers have successfully adapted ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). The TATA-edited plants exhibited resistance to the canker pathogen Xanthomonas citri subsp. Citri (Xcc) (Huang et al., 2021). Additionally, CBE has been employed to edit the acetolactate synthase (ALS) gene in citrus, resulting in herbicide-resistant plants. Notably, the ALS-edited plants were transgene-free, as the Cas9 gene was undetectable in the herbicide-resistant citrus plants. This represents a significant milestone, as it demonstrates the potential for developing transgene-free, gene-edited citrus varieties using CRISPR technology (Huang et al., 2021). 4.2 Forest trees 4.2.1 Poplar genome editing for bioenergy production Poplar trees have emerged as a significant focus for genome editing due to their potential in bioenergy production. The application of CRISPR technology in poplar has demonstrated promising results in enhancing wood properties, which are crucial for efficient bioenergy production (Figure 3). Figure 3 Transgenic CSE-CRISPR hybrid poplars have irregularly shaped xylem vessel cells (Adopted from Jang et al., 2021) Image caption: Stem anatomy of hybrid poplars (8-month-old LMO field grown) was assessed by (a) toluidine blue, (b) phloroglucinol-HCl, and (c) Mäule staining. Collapsed irregular vessels are marked with asterisks. Scale bars represent 50 µm (Adopted from Jang et al., 2021) Jang et al. (2021) illustrates the structural and compositional changes in xylem vessels of CRISPR/Cas9-edited hybrid poplars with CSE gene knockouts. Both CSE1-sg2 and CSE2-sg3 transgenic lines show significant abnormalities in xylem vessel formation, characterized by collapsed and irregularly shaped cells. This is a clear indication of compromised secondary cell wall development. Additionally, the reduced red staining intensity in both phloroglucinol and Mäule staining assays suggests a substantial decrease in overall lignin content and S-lignin content, respectively. This study highlights the effectiveness of CRISPR/Cas9 in elucidating gene functions and modifying plant traits, offering potential applications in improving wood quality and biofuel production through targeted genetic modifications. One notable study utilized multiplex CRISPR editing to target lignin biosynthesis genes in poplar. Lignin, a complex biopolymer in wood, poses a challenge for bioenergy production due to its resistance to chemical and enzymatic degradation. By editing multiple genes involved in lignin biosynthesis, researchers were able to significantly alter the lignin composition and improve the wood's carbohydrate-to-lignin ratio. This modification led to a 228% increase in the wood carbohydrate-to-lignin ratio compared to wild-type poplar, resulting in more efficient fiber pulping and reduced carbon emissions (Sulis et al., 2023).
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