Gene replacement and base substitution: the next generation of non-GM genome edited crops. Glyphosate tolerance in tomato as proof of concept
Research project 22
Project researchers: Dr. Evgenia Dor and Prof. Avraham Levy

Our task is to replace the endogenous tomato EPSPS gene with the same coding sequence containing mutations that provide partial tolerance to glyphosate.

Firstly, we will design a synthetic gene (the donor) with a similar sequence as Solyc01g091190, but with base substitutions that generate the double or triple amino acid substitutions (Table 1). Secondly, we will swap the endogenous EPSPS Solyc01g091190 promoter with the strong and constitutive UBQ10 tomato promoter (Fig. 1) using gene targeting.

We will develop base substitution methods as a tool for genome editing in plants.
The current base editing methods can generate targeted C->T or A->G substitutions. This is achieved through a Cytosine or Adenine deaminase fusion to inactive dCas9 or nickase guided by gRNA. We will use a Cas9-deaminase adapted to plants to perform the relevant mutations.

Table 1: Double or triple amino acids substitutions that will be conducted.
Research project 22 - Dr. Evgenia Dor and Prof. Avraham Levy
Fig.1. Scheme of a synthetic gene (the donor) design. All combinations of endogenous EPSPS gene and edited genes, containing base substitutions that will generate the double or triple a. a. substitutions with endogenous EPSPS promoter or UBQ10 tomato promoter will be tested for glyphosate tolerance.
פרופ' אברהם לוי

Prof. Avraham Levy
Project researcher

Faculty of biochemistry, Weizmann institude of science.

Email: avi.levy@weizmann.ac.il

Phone: +972-8-9342734

Website: Prof. Avraham Levy

Research areas:

  • The connection between epigenetic modifications and the maintenance of genome integrity.
  • Harnessing DNA repair mechanisms for developing new approaches for precise genome editing for plant breeding.
ד"ר יבגניה דור

Dr. Evgenia Dor
Project researcher

Phytopathology and Weed Research, Newe Ya’ar, Agricultural Research Organization.

Email: evgeniad@volcani.agri.gov.il

Phone: +972-4-9539534

Website: Dr. Evgenia Dor

Research areas:

Weed research, plant resistance to herbicides, parasitic plants, mutagenesis, biological pesticides, strigolactones.

Research partner:

ד"ר בדברטה סחה

Dr. Bedabrata Saha
Postdoctoral researcher

Institution:
Plant Sciences, Agricultural Research Organization, Newe Ya’ar.