Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. 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 nonhomologous 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.
Bibliographical noteFunding Information:
We thank R. Douglas Sammons for consultation on glyphosate tolerance in plants. We thank Monsanto Company for sharing the shikimate assay protocol and reagents and Jeff Berry for quantifying the shikimate assay data. We thank Jacob Jerrard, Rebecca Greenstein, Jacquelyn Leise, Jenny Tran, Stephanie Lamb and Claire Albin for excellent technical assistance and K. Leffler for help in generating the figures. This work was supported by a grant from the Bill & Melinda Gates Foundation.
- gene editing
- gene replacement
- herbicide tolerance