Genome Editing in Sugarcane to Improve Bioenergy Traits

Programmable endonucleases like transcription activator-like effector nucleases (TALENs) or RNA-guided nucleases (e.g. CRISPR/Cas9) enable precise genome modifications, such as targeted mutagenesis, gene replacement, or insertion. Sugarcane is a prime feedstock for bioethanol production, and utilizing both sucrose and cell wall bound sugars for fermentation will enhance the biofuel yield. We recently demonstrated that RNAi mediated downregulation of lignin biosynthetic genes is a successful strategy to improve bioethanol production from lignocellulosic sugarcane biomass. In this study, one of the lignin biosynthetic genes, Caffeic acid O-methyltransferase (COMT) was targeted for the TALEN induced mutagenesis to modify lignin biosynthesis in sugarcane. Targeted mutations following TALEN delivery were identified by capillary electrophoresis of the COMT amplicon. Events were confirmed by sequencing of the COMT amplicon which revealed the presence of insertions and deletions at the target site. Data describing the number of COMT copies that were co-editied in the highly polyploid sugarcane genome, saccharification and agronomic performance of field grown COMT mutants will be presented. The above described targeted mutagenesis through TALEN was the result of the error prone NHEJ DNA-repair pathway which created “loss of function” mutations following a targeted DNA double strand break in the COMT gene. Alternatively a precision gene-editing approach for allele replacement to confer “gain of function” will also be discussed, involving co-delivery of DNA repair template facilitating homology-directed repair (HDR) and programmable endonuclease.