Tree Genetics and Molecular Breeding 2024, Vol.14, No.1, 32-42 http://genbreedpublisher.com/index.php/tgmb 37 content and a 7%~11% increase in glucose release, along with a 23%~32% increase in xylose release, demonstrating improved saccharification efficiency without a significant growth penalty (Figure 4) (Park et al., 2017). Figure 4 Defined tetra-allelic gene disruption of the 4-coumarate:coenzyme A ligase 1 (Pv4CL1) gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release (Adopted from Park et al., 2017) Image caption: Histological staining for lignin deposition in control a, b, pv4cl1-25 c, d and pv4cl1-26 e, f. Second internode was sampled at R1 stage. Scale bars: 50 µm. Thickness of the cross section is 30 µm (Adopted from Park et al., 2017) 4.2 Case study on CSE gene knockouts Caffeoyl shikimate esterase (CSE) is another key enzyme in lignin biosynthesis. In hybrid poplar (Populus alba × P. glandulosa), CRISPR/Cas9 was used to knockout the CSE1 and CSE2 genes. Three single guide RNAs were designed, resulting in transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. The CSE1-sg2 and CSE2-sg3 poplars showed up to a 29.1% reduction in lignin deposition and up to a 25% higher saccharification efficiency compared to wild-type controls. These knockouts did not exhibit significant morphological differences or growth penalties in long-term field tests, indicating that precise editing of CSE genes can enhance lignocellulosic biomass without adverse effects on growth (Figure 5) (Jang et al., 2021). Figure 5 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)
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