Tree Genetics and Molecular Breeding 2024, Vol.14, No.1, 32-42 http://genbreedpublisher.com/index.php/tgmb 33 The CRISPR/Cas9 system has revolutionized genetic engineering by enabling precise, targeted modifications in the genome. This technology utilizes a guide RNA (gRNA) to direct the Cas9 endonuclease to specific DNA sequences, where it introduces double-stranded breaks that can be repaired to create mutations or insertions (Bortesi and Fischer, 2015; Fan et al., 2015; Arora and Narula, 2017). In poplar, CRISPR/Cas9 has been successfully used to edit genes involved in lignin biosynthesis, such as CSE, CCR, and 4CL, demonstrating its potential for improving lignocellulosic biomass for biofuel production (Tsai et al., 2019; Meester et al., 2020; Jang et al., 2021; Meester et al., 2021). This systematic review aims to consolidate recent advancements in the application of CRISPR/Cas9 technology for modifying lignin biosynthesis in poplar. By synthesizing current research, this review will offer valuable insights into the potential of CRISPR/Cas9 technology to transform lignin biosynthesis in poplar, paving the way for more efficient and sustainable biofuel production. 2 Review on Research Background 2.1 Lignin biosynthesis pathway in poplar Lignin is a complex aromatic polymer found in the cell walls of plants, providing structural integrity and resistance to microbial attack. In poplar, lignin biosynthesis involves the phenylpropanoid pathway, where monolignols such as p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol are synthesized and subsequently polymerized into lignin. Key enzymes in this pathway include phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and caffeoyl shikimate esterase (CSE), among others (Figure 1) (Meester et al., 2021; Jang et al., 2021; Vries et al., 2021). 2.2 Previous methods used for lignin modification Traditional methods for lignin modification in poplar have included genetic engineering techniques such as overexpression or suppression of lignin biosynthetic genes. For instance, downregulation of 4-coumarate:CoA ligase (4CL) and caffeoyl shikimate esterase (CSE) has been shown to reduce lignin content and improve saccharification efficiency (Tsai et al., 2019; Vries et al., 2021). However, these methods often result in growth defects and other undesirable phenotypic changes, such as collapsed vessels and reduced biomass yield (Meester et al., 2021; Jang et al., 2021). 2.3 Introduction to CRISPR/Cas9 technology CRISPR/Cas9 is a revolutionary genome-editing tool that allows for precise, targeted modifications of DNA. The system consists of two main components: the Cas9 nuclease, which introduces double-stranded breaks in DNA, and a single guide RNA (sgRNA) that directs Cas9 to the specific genomic location (Figure 2) (Bortesi and Fischer, 2015; Fan et al., 2015; Bruegmann et al., 2019). This technology has been successfully applied in various plant species, including poplar, to achieve targeted gene knockouts and modifications (Fan et al., 2015; Bruegmann et al., 2019). 2.3.1 Mechanism of CRISPR/Cas9 The CRISPR/Cas9 system operates by utilizing the sgRNA to bind to a complementary DNA sequence in the genome. The Cas9 nuclease then introduces a double-stranded break at this location. The cell's natural repair mechanisms, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), subsequently repair the break, often resulting in insertions or deletions (indels) that can disrupt gene function (Figure 3) (Bortesi and Fischer, 2015; Fan et al., 2015; Bruegmann et al., 2019). 2.3.2 Advantages of using CRISPR/Cas9 over traditional methods CRISPR/Cas9 offers several advantages over traditional genetic engineering methods for lignin modification in poplar: (1) Precision: CRISPR/Cas9 allows for highly specific targeting of genes, reducing off-target effects and unintended genetic changes (Fan et al., 2015; Bruegmann et al., 2019). (2) Efficiency: The system can generate homozygous mutants in the first generation, significantly speeding up the breeding process (Fan et al., 2015).
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