Gene replacement and base substitution: The next generation of non-GM genome edited crops. Glyphosate tolerance in tomato as proof of concept

In addition to gene knockout and deletions, the USDA has approved additional modification as non-GM when the modified sequence is from the same gene pool as the crop or when it could have happened by a natural process. This assertion opens new horizons for plant genome editing applications such as gene replacement or altering enzyme activity by base substitution. Our general goal is to develop glyphosate-resistant tomato using a genome editing approach.

First, we applied traditional transgenesis to evaluate the impact of paired amino acid substitutions (TIPS, TIPA and TAP-IVS) in the S/EPSPS gene and promoters regulating the expression of the transformed gene enhancing the tomato plants’ tolerance to glyphosate (Fig 1). The plants of the first generation were tested for sensitivity to glyphosate (Fig. 2). Two constructs with mutations in the S/EPSPS gene, both under the control of the tomato Ubiquitin10 promoters (TIPS_U and TAP-IVS_U), provided high tolerance to glyphosate. TAP-IVS_U was selected for the genome editing approach.

We are currently testing base substitution methods to introduce the mutations TAP-IVS in the endogenous S/EPSPS gene, and the endogenous S/EPSPS promoter will be swapped by the strong tomato Ubiquitin10 promoter using gene targeting. This should lead to the development of cisgenic glyphosate-resistant tomato.

Project researchers: Dr. Evgenia Dor , Prof. Avraham Levy.

Research partner: Dr. Bedabrata Saha.