Tree Genetics and Molecular Breeding 2024, Vol.14, No.1, 32-42 http://genbreedpublisher.com/index.php/tgmb 36 In summary, the CRISPR/Cas9 system represents a powerful and efficient tool for modifying lignin biosynthesis in poplar, offering significant advantages over traditional genetic engineering methods. This technology holds great promise for improving lignocellulosic biomass for biofuel production and other industrial applications. 3 Recent Advancements in CRISPR/Cas9 Applications in Poplar Lignin Biosynthesis 3.1 Gene knockouts 3.1.1 Knockout of 4CL1 gene and its effects on lignin composition The knockout of the 4-coumarate: CoA ligase 1 (4CL1) gene in poplar has revealed significant insights into lignin biosynthesis. In a study by Tsai et al. (2019), CRISPR/Cas9-mediated knockout of 4CL1 in Populus tremula × alba resulted in a preferential reduction of syringyl (S) lignin, while guaiacyl (G) lignin levels were maintained. This alteration in lignin composition was accompanied by the upregulation of 4CL5, a low-affinity paralog of 4CL1, which helped sustain lignification. Interestingly, the knockout did not significantly affect biomass recalcitrance, suggesting a compensatory mechanism in lignin biosynthesis. 3.1.2 Knockout of CSEgenes to reduce lignin content and improve saccharification The caffeoyl shikimate esterase (CSE) genes are crucial targets for reducing lignin content in poplar. Research by Jang et al. (2021) demonstrated that CRISPR/Cas9-mediated knockout of CSE1 and CSE2 in hybrid poplar led to a significant reduction in lignin deposition, up to 29.1%, without affecting plant morphology or growth. This reduction in lignin content resulted in a 25% increase in saccharification efficiency, highlighting the potential of CSE gene knockouts for improving lignocellulosic biomass for biofuel production. Another study by Vries et al. (2021) confirmed the importance of CSE1 and CSE2 in lignification, showing that double mutants had a 35% reduction in lignin content and a fourfold increase in cellulose-to-glucose conversion. 3.2 Gene activation and base editing While the provided data does not include specific examples of dCas9-mediated gene activation in poplar, the potential for such applications is significant. The CRISPR/Cas9 system, particularly the use of deactivated Cas9 (dCas9) fused with transcriptional activators, can be employed to upregulate target genes involved in lignin biosynthesis. This approach could be used to enhance the expression of genes that promote desirable lignin traits or improve overall biomass quality. 3.3 Multigene targeting Recent advancements in CRISPR/Cas9 technology have enabled efficient multigene targeting in poplar. A study by Triozzi et al. (2021) described a robust and standardized CRISPR/Cas9 strategy for simultaneous editing of multiple genes in Populus tremula × alba. This approach utilized the Golden Gate MoClo cloning system to introduce mutations in two genes, YUC4 and PLT1, simultaneously. The high efficiency of this system was demonstrated by the successful generation of double mutants, with biallelic mutations detected in a significant proportion of transgenic roots. This multiplex genome editing capability is crucial for dissecting complex genetic pathways and improving lignin biosynthesis in poplar. In summary, the application of CRISPR/Cas9 in poplar lignin biosynthesis has made significant strides, particularly in gene knockouts, which have shown promising results in reducing lignin content and improving saccharification efficiency. Future research should explore gene activation techniques and further refine multigene targeting approaches to fully harness the potential of CRISPR/Cas9 in optimizing lignin biosynthesis for bioenergy applications. 4 Case Studies and Experimental Results 4.1 Case study on 4CL1 knockout The 4-coumarate:coenzyme A ligase (4CL) gene plays a crucial role in the lignin biosynthesis pathway. In a study on switchgrass, a CRISPR/Cas9 system was developed to target the Pv4CL1 gene, which is preferentially expressed in highly lignified stem tissues. Specific guide RNAs were designed to target Pv4CL1, and the construct was introduced into switchgrass calli. Out of 39 transgenic plants regenerated, four were confirmed to have tetra-allelic mutations. These Pv4CL1 knockout plants exhibited an 8%~30% reduction in total lignin
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