Talen Mediated Multi-Allelic Mutagenesis of COMT Improves Saccharification Efficiency from Lignocellulosic Biomass of Field Grown Sugarcane

Sugarcane is a prime feedstock for commercial production of bioethanol. Utilizing both sucrose and cell wall bound sugars for fermentation will enhance the biofuel yield per unit area. We recently demonstrated that RNAi mediated downregulation of lignin biosynthetic gene Caffeic acid O-methyltransferase (COMT) is a successful strategy to improve bioethanol production from lignocellulosic sugarcane biomass. Targeted mutagenesis with genome editing tools has been successful in a number of crops for creation of loss of function phenotypes but is very challenging in sugarcane due to its highly polyploid genome. In this study, a conserved region of COMT was targeted with a single TALEN pair for multi-allelic mutagenesis to modify lignin biosynthesis in sugarcane. Targeted mutations in regenerated sugarcane lines were identified by capillary electrophoresis of the COMT amplicon. Mutations were confirmed by high-throughput sequencing of the COMT amplicon which revealed the presence of insertions and deletions at the target region. Long COMT amplicons of vegetative progenies, which combined excellent agronomic performance with altered cell wall composition and drastically improved saccharification efficiency were analyzed with Sanger sequencing. Sequencing revealed the extent of co-editing of the large number of COMT copies/alleles and confirms the success of this strategy in a highly polyploid crop.