Genome editing and metabolic engineering of sugarcane to fuel the emerging bioeconomy

Saroj Parajuli1, Tufan Mehmet Oz1,6,  Ayman Eid1,6, Chakravarthi Mohan1, Baskaran Kannan1,6, Duoduo Wang1,6, Sara Sanchez1,6, Ratna Karan1, Aldo Merotto1, Hui Liu2,6, Eva Garcia-Ruiz3, Deepak Kumar4, Vijay Singh4,6, Huimin Zhao3,6, Steve Long5,6, John Shanklin2,6 and  Fredy Altpeter1,6

1** Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, IFAS, Gainesville, FL, USA.

2** Biosciences Dept., Brookhaven National Lab, Upton, NY, USA.

3** Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

4** Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

5** Departments of Plant Biology and Crop Sciences, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

6** DOE Center for Advanced Bioenergy and Bioproducts Innovation, USA.

altpeter@ufl.edu

Abstract

Sugarcane is a prime feedstock for commercial production of biofuel and table sugar. We recently demonstrated that metabolic engineering successfully resulted in accumulation of oil in vegetative sugarcane biomass for increased energy density and development of an advanced biofuel. We are exploring strategies to divert more of the carbon flux from sucrose to triacylglycerol (TAG). This strategy involves the analysis of combinations of gene expression cassettes for metabolic engineering, supporting biosynthesis and storage of TAG. Correlations between TAG accumulation and gene combinations and their expression levels will be presented. These data indicate the feasibility of genetically engineering the high biomass crop sugarcane to produce TAG, which can be readily converted to biodiesel transportation fuel.Genome editing tools such as CRISPR/Cas9 have been employed in several crop genomes. They enable precise targeting and introduction of double stranded DNA breaks in vivo. Subsequent cellular repair mechanisms, predominantly non-homologous end joining (NHEJ), act as critical steps to endogenous gene editing or correction. However, there is very limited control over these mechanisms, which generate an abundance of random insertions and deletions (indels). Frameshift mutations associated with these indels of unspecified size and sequence might result in loss of function phenotypes of agronomic importance. Gain of function mutations, on the other hand, generally require precise nucleotide substitutions in the target locus. This can be accomplished with the aid of a homologous repair template and involves the cellular homology directed repair (HDR) mechanism. We will present two rapid readout platforms that support the development of efficient multi-allelic NHEJ and HDR mediated precision editing in the highly polyploid sugarcane.

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Keywords: Genome editing, metabolic engineering, sugarcane, biofuel