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

Abstract

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. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (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 CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

CRISPR/Cas9‐mediated P‐CR domain‐specific engineering of CESA4 heterodimerization capacity alters cell wall architecture and improves saccharification efficiency in poplar

“…The lignocellulose composition, high cellulose DP and CrI could increase the biomass recalcitrance and hinder saccharification efficiency (De Meester et al ., 2020 ; Himmel et al ., 2007 ; Hori et al ., 2020 ; Hu et al ., 2018 a; Hu et al ., 2018 b; Jang et al ., 2021 ; Martarello et al ., 2021 ; Speicher et al ., 2018 ). Here, the saccharification potential of WT and three T0 mutant plants were further investigated based on cellulose‐to‐glucose conversion efficiency under the limited saccharification conditions, including acidic (1 M of HCl, 80 °C, 2 h), alkaline (62.5 m m of NaOH, 90 °C, 3 h), and no pretreatment.…”

Lignin engineering in forest trees: From gene discovery to field trials

“…(2021a) P. tremula × P. alba CSE1 and CSE2 CRISPR-Cas9 ↓35% ↓S/G ↑ n.d. ↓ de Vries et al. (2021a) P. alba × P. glandulosa CSE1 CRISPR-Cas9 ↓16% n.d. n.d. n.d. WT Jang et al. (2021) P. alba × P. glandulosa CSE2 CRISPR-Cas9 ↓16% n.d. n.d. n.d. WT Jang et al.…”

Spatio-Temporal Modification of Lignin Biosynthesis in Plants: A Promising Strategy for Lignocellulose Improvement and Lignin Valorization

“…In the past several years, genome editing with CRISPR/Cas9 has been demonstrated in various plants, including biofuel species such as poplar and switchgrass (Table 3). For example, Jang et al (2021) established a CRISPR/Cas9 system in poplar to target both the CSE1 and CSE2 genes related to lignin biosynthesis, and achieved reduced lignin content. Park et al (2017) also employed the CRISPR/Cas9 system to knockout the Pv4CL1 gene for the generation of switchgrass with low lignin content, and the resulted mutant switchgrass also exhibited an increased glucose and xylose release upon saccharification.…”