Development of self-excising T-DNA cassette using CPF1 for plants breeding
Research project 19
Project researchers: Prof. Avraham Levy, Prof. Martin Goldway and Dr. Amir Raz

Agrobacterium infection is the most commonly used method for plants transformation. However, the stable insertion of the T-DNA into the host genome makes it a Genetically Modified Organism (GMO), which in many countries is forbidden for commercial growth.

Using the CRISPR-Cas9 system to extract the T-DNA out of the genome by designing guides to its borders was proposed before. However, due to the nature of the Cas9 cleavage along the target, this will leave a “scar” of 20-30 bp of foreign DNA at the repair site. We propose to use the CPF1 for the excision of the T-DNA from the genome. Unlike Cas9, this enzyme cleaves the target at the 3’ end (while the PAM is at the 5’ end), dramatically improving the probability of a “clean” repair with no foreign DNA. Thus, we can create a generic self-excision cassette by carefully designing guides to the T-DNA borders (in addition to the gene of interest guides). In the first stage, we will examine the excision efficiency of three CPF1 variants by hybridization of M82 tomato plants carrying one of the enzymes and gRNAs against the borders of T-DNA cassette and Micro-Tom tomato plant harboring the betalain biosynthetic pathway, which gives it a purple color. Lose of purple color in the progenies will indicate the excision of the betalain cassette.

Next, we will design a generic cassette containing the GFP gene, the best CPF1 enzyme, gRNAs against the cassette borders and an additional gRNA against the CRTISO gene (which gives the orange color of tomato wen mutate). Obtaining orange fruit in a non-fluorescent plant will prove the editing and the excision of the cassette.

פרופ' אברהם לוי

Prof. Avraham Levy 
Project researcher

Faculty of biochemistry,
Weizmann institude of science.

Research areas:

  • The connection between epigenetic modifications and the maintenance of genome integrity.
  • In addition, we are harnessing DNA repair mechanisms for developing new approaches for precise genome editing for plant breeding.

Prof. Martin Goldway
Project researcher

MIGAL, Dep. of Biotechnology Tel-Hai College, Kiryat Shmona.

Research areas:

  • Pollination and fertilization in vardenii – apple, pear, apricot, peach and plum.
  • Ripening of climacteric fruits.
  • Development of transformation protocols for fruit trees.

Dr. Amir Raz
Project researcher

MIGAL, Dep. of Biotechnology Tel-Hai College, Kiryat Shmona.

Research areas:

  • Pollination and fertilization in vardenii – apple, pear, apricot, peach and plum.
  • Ripening of climacteric fruits.
  • Development of transformation protocols for fruit trees.
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Research partners:

Rida Habshi
PhD student

Institution:
Plant Sciences and Genetics in Agriculture, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem.