Hummel AW, Chauhan RD, Cermak T, Mutka AM, Vijayaraghavan A, Boyher A, Starker CG, Bart R, Voytas DF, Taylor NJ.

Plant Biotechnol J. 2017 Dec 9. doi: 10.1111/pbi.12868. [Epub ahead of print]

Abstract

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots (Doll and Piedrahita Cañola, 1978). Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA repair pathways, we precisely introduced the best performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS-edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T-DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava. This article is protected by copyright. All rights reserved.

See: https://www.ncbi.nlm.nih.gov/pubmed/29223136

Figure: a Circos plot showing cassava-castor bean colinearity. A sample 2.7 Mb region of the cassava genome was aligned against castor bean scaffolds with the Promer tool from Mummer v3.2, and visualized with Circos. Colored segments of the circle represent cassava while greyscale represents corresponding segments of castor bean genome. Green blocks in outer ring are contigs within the 2.7 Mb scaffold. The second ring represents repeat content in grey blocks, and the inner ring represents genes in red blocks. Grey lines linking cassava segments to castor bean segments represent homology at the protein level, and the fact that these lines do not cross over each other indicates colinearity. The central portion of the cassava scaffold, highlighted in yellow, is zoomed in to show more detail. b The same 2.7 Mb cassava scaffold as in (a; top) but aligned against other regions of the cassava genome that are highly similar (bottom). This demonstrates the presence of a duplication in the cassava genome