Tree Genetics and Molecular Breeding 2024, Vol.14, No.1, 32-42 http://genbreedpublisher.com/index.php/tgmb 32 Review and Progress Open Access CRISPR/Cas9 in Poplar Lignin Biosynthesis: Advances and Future Prospects Yongquan Lu State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou, 311300, Zhejiang, China Corresponding email: luyongquan@zafu.edu.cn Tree Genetics and Molecular Breeding, 2024, Vol.14, No.1 doi: 10.5376/tgmb.2024.14.0005 Received: 14 Jan., 2024 Accepted: 16 Feb., 2024 Published: 28 Feb., 2024 Copyright © 2024 Lu, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Lu Y.Q., 2024, CRISPR/Cas9 in poplar lignin biosynthesis: advances and future prospects, Tree Genetics and Molecular Breeding, 14(1): 32-42 (doi: 10.5376/tgmb.2024.14.0005) Abstract The CRISPR/Cas9 system has emerged as a revolutionary tool for genome editing, offering unprecedented precision and efficiency in modifying genetic material. This systematic review focuses on the application of CRISPR/Cas9 technology in the biosynthesis of lignin in poplar species, highlighting recent advances and future prospects. Lignin, a complex polymer in the cell walls of plants, plays a crucial role in providing structural integrity and resistance to pathogens. However, its recalcitrance poses challenges for industrial processes such as pulping and biofuel production. Recent studies have demonstrated the potential of CRISPR/Cas9 to target and modify genes involved in lignin biosynthesis, thereby reducing lignin content and altering its composition to enhance industrial utility. Several research efforts have successfully employed CRISPR/Cas9 to edit lignin biosynthesis genes in poplar. For instance, the efficient knockout of the phytoene desaturase gene in Populus alba × Populus glandulosa using a single guide RNA (sgRNA) has shown promising results in generating targeted mutations with high efficiency. Similarly, the application of CRISPR/Cas9 in Populus tomentosa Carr. has demonstrated the system's capability to create precise genomic edits, resulting in significant phenotypic changes. Moreover, studies have evaluated the efficiency of various guide RNAs (gRNAs) in poplars, identifying key factors that influence gene editing success, such as GC content and the accessibility of the seed region. The review also discusses the broader implications of CRISPR/Cas9 technology in plant research, including its potential to enhance disease resistance, improve nutritional content, and develop drought-tolerant varieties Despite these advancements, challenges such as off-target effects and the need for efficient delivery methods remain. Future research directions include the development of high-fidelity Cas9 variants and the optimization of delivery systems to minimize off-target modifications and enhance editing efficiency. Keywords CRISPR/Cas9; Poplar; Lignin biosynthesis; Genome editing; Phytoene desaturase; Guide RNA; Gene knockout; Plant biotechnology 1 Introduction Lignin is a complex phenolic polymer that is integral to the structural integrity of plant cell walls, particularly in woody species like poplar (Populus spp.). The biosynthesis of lignin involves a series of enzymatic reactions that convert phenylalanine into monolignols, which are then polymerized into lignin. Key enzymes in this pathway include caffeoyl shikimate esterase (CSE), cinnamoyl-CoA reductase (CCR), and 4-coumarate:CoA ligase (4CL) (Tsai et al., 2019; Jang et al., 2021; Meester et al., 2021). These enzymes are crucial for the formation of guaiacyl (G) and syringyl (S) lignin units, which contribute to the rigidity and resistance of the plant cell wall. Lignin's recalcitrance to degradation poses a significant challenge for the efficient conversion of lignocellulosic biomass into biofuels and other bio-based materials. Reducing lignin content or altering its composition can enhance the saccharification efficiency, thereby improving the yield of fermentable sugars (Meester et al., 2020; Jang et al., 2021; Meester et al., 2021). For instance, CRISPR/Cas9-mediated knockout of the CSE gene in hybrid poplar has been shown to reduce lignin content by up to 29.1%, significantly improving saccharification efficiency without affecting plant growth (Jang et al., 2021). Similarly, vessel-specific lignin biosynthesis has been employed to mitigate growth defects while maintaining high saccharification yields (Meester et al., 2021).
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