Plant Science & Physiology

Biography

Biography: Denes Dudits

Abstract

Advancements in moving from random towards directed mutagenesis can revolutionize plant research and crop improvement.  In parallel with the rapid evolution of CRISPR/Cas9 tools with myriad of functionalities and capabilities, the chemically synthesized single stranded DNA oligonucleotides (SDOs) gain increasing significance as vectors to produce oligonucleotide-targeted nucleotide exchange (OTNE) at a specific site of plant genomic DNA. Furthermore, the combinatorial use of oligonucleotides with programmable nucleases became a routine in plant gene editing. In this overview, we analyze several major factors that can improve the efficiency of oligo-directed mutagenesis (ODM). For phenotypic assay of ODM, we use transgenic maize cell lines expressing the non-functional Green Fluorescent Protein (mGFP) gene carrying a TAG stop codon. In the ODM treated cells, nucleotide exchange in the stop codon (TAG) from T to G nucleotide that resulted in the restoration of GFP function. This test system allowed to compare SDO molecules with different size and chemical modifications. The ODM efficiency primarily depends on the uptake protocol. We use bombardment of SDO^GFP into maize cells, in addition, we have tested novel delivery methods to stimulate uptake into protoplasts. Experiments are in progress to compare ODM and CRISPR/Cas9 technologies in correction efficiency. As we showed, the open chromatin structure of recipient cells can increase the number of ODM events (Tiricz et.al 2017). Presently editing protocols are dependent on the in vitro tissue culture systems, where selection of edited cells with altered agronomic traits can be limited. Therefore, we test different co-editing systems with one selectable and one non-selectable marker by ODM and CRISPR/Cas9 technologies.  Based on the presented experimental findings and the published data one can conclude that DNA oligonucleotides (SDOs) play a central role in editing of plant genomes.