CRISPR/Cas 9-mediated gene editing generates glyphosate tolerant cassava (Manihot esculenta)

Weeds are a significant constraint to cassava production. Hand weeding is common in Africa but this is labor intensive and time consuming. Development of herbicide tolerant cassava would increase storage root yields and reduce required labor inputs. Inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by the herbicide glyphosate prevents synthesis of chorismate-derived aromatic amino acids and their secondary metabolites. To generate proof of concept for glyphosate tolerance in cassava the EPSPS coding sequence was mutated, placed under control of the 2x35S promoter and integrated into cassava by Agrobacterium-mediated transformation of cultivar TME 204. Transformants were screened by shikimate assays and subjected to glyphosate application in the greenhouse. Plants expressing the TIPA mutation were highly tolerant to glyphosate application at 50 mg active ingredient/plant. A parallel CRISPR/Cas9 approach was undertaken in which gRNAs were used to target the native cassava EPSPS locus and insert a 4.2 kb sequence consisting of the 2x35S promoter plus coding sequence carrying the TIPA mutation. NHEJ and HR editing tools were delivered by Agrobacterium into embryogenic cassava cells, with subsequent selection on glyphosate containing medium allowing recovery of somatic embryos and plants. Molecular analysis confirmed presence of repair DNA at the intended EPSPS site in regenerated plants. Shikimate and greenhouse glyphosate assays confirmed functionality of the promoter-TIPA “knockin” within gene edited plants at levels that equal the most glyphosate tolerant cassava plants generated by conventional transgenesis.