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

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