CRISPR-Knockout of <i>CSE</i> Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

• Main Authors: Hyun-A Jang, Eun-Kyung Bae, Min-Ha Kim, Su-Jin Park, Na-Young Choi, Seung-Won Pyo, Chanhui Lee, Ho-Young Jeong, Hyoshin Lee, Young-Im Choi, Jae-Heung Ko
• Format: Article
• Language: English
• Published: MDPI AG 2021-09-01
• Series: International Journal of Molecular Sciences
• Subjects: biofuels; caffeoyl shikimate esterase (CSE); CRISPR/Cas9; hybrid poplar; lignin; saccharification
• Online Access: https://www.mdpi.com/1422-0067/22/18/9750
• ISSN: 1661-6596; 1422-0067

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. <i>Populus</i> spp. has two <i>CSE</i> genes (<i>CSE1</i> and <i>CSE2</i>) and, thus, the hybrid poplar (<i>Populus alba</i> × <i>P. glandulosa</i>) investigated in this study has four <i>CSE</i> genes. Here, we present transgenic hybrid poplars with knockouts of each <i>CSE</i> gene achieved by CRISPR/Cas9. To knockout the <i>CSE</i> genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either <i>CSE1</i> (CSE1-sg2), <i>CSE2</i> (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of <i>CSE</i> by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.