The Future of Agriculture: Grand Challenges and Technological Change

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1 The Future of Agriculture: Grand Challenges and Technological Change Moscow, March National Research University Higher School of Economics Molecular mutagenesis by genome editing Ervin Balázs MTA ATK Center for Agricultural Research H-2462 Martonvásár, Hungary

2 Since more and more primary structure of the genetic material of different organisms has been described, our understanding on the different genomes rapidly evolved. The first transgenic organism constructed by Paul Berg more than four decades ago, and the revolution of the molecular biology made it possible to extend that technology for almost all living organisms.

3 In the case of higher plants the first transgenic tobaccos were produced in the same time in two independent laboratories in the US lead by Mary Dell Chilton and in Belgium headed by Marc Van Montague and Jeff Schell.

4 These outstanding achievements attracted almost all molecular biology laboratories all over the world, and successively more and more transgenic organisms including plants have been reported. In the case of plants the first targets were the molecular use of biological control of plant diseases, and included the production of herbicide tolerant crops.

5 The series of insect resistant, herbicide tolerant and virus resistant plants commercial production started in Today their cultivation exceeded yearly 180 thousands of hectares in the world. The introduction of these crops is in the forefront of the debate among different stakeholders, and almost completely blocked in the European Union countries.

6 The major concerns of the opponents based on that fact that the genetic material contains foreign genes originating from other organisms which new combinations may not be formed in the nature. However the latest new methods of genome editing made it possible that even a single nucleotide addition organism not produced by these techniques. from different

7 These four molecular scissors are either directed by DNA, RNA or Proteins all just produced mutations on already existing genes in the organisms, just activating the silent gene of the organisms or blocking them. It is also possible with these techniques that a useful traits form the same species can be incorporate into a commercial varieties.

8 These mutants cannot be distinguished from naturally evolving mutants. These four techniques the oligo nucleotide directed mutagenesis, the zinc finger nucleases, the TALE nucleases and the CRISPR/Cas9 systems are efficient technologies

9 Specific gene editing methods CRISPR/Cas9 oligonucleotides

10 Oligonucleotide Targeted Nucleotide Exchange (OTNE) G T A Advantages: Not requiring transgene introduction Simple design Cheap synthesis T G A Major limitations: low frequency Identification of the non-selectable mutations Corrected sequence G C successful generation of herbicide tolerant variants of different crop species. Review by Breyer et al. (2009)

11 Oligonucleotide Targeted Nucleotide Exchange (OTNE) mgfp mutant Non-functional GFP gene Oligo mgfp OTNE GFP corrected GFP gene Correcting oligonucleotide: 5 -CCACC ATG GTG AGC AAG GGC GAG GAG CTG TTC ACC GGG GTG-3 (Dong et al., 2006)

12 Generation of mgfp transgenic maize cell lines Construction of vector: ubi mgfp ubi PAT Biolistic delivery PPT selection Sequencing mgfp transgenic maize cell lines are non-fluorescent!

13 Monitoring the nucleotide exchange events mgfp-transgenic maize cells (non-fluorescent) delivery of GFP-correcting oligos via particle bombardment GFP expressing fluorescent cells fluorescence microscopy flow cytometry The frequency of GFP positive cells is used as an indicator of gene repair efficiency

14 Restoration of GFP function a fluor. stereo microscope cell division Fluorescence stereo and confocal microscopy imaging of GFP positive cells

15 Number of GFP positive cell/10 6 events Quantification of GFP positive cells by flow cytometer wtgfp oligo wtgfp oligo wtgfp oligo GFP SDO GFP SDO GFP SDO Rep.1 Rep.2 Rep.3 Gene correction efficiency: GFP positive/million cells

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25 Genomic DNA complementer genome sequence Guide RNA Cas9 Donor DNA repair Gene therapy Targeted gene modification Transgenic animals cells

26 Negative regulator of muscle growing Inhibiting myoblast terminal differentiation and proliferation Protect myoblasts from apoptosis Lack of the protein causes hyperplasia/hypertrophia Fatty acid composition can be altered

27 Molecular work Cloning In vitro transcription

28 Streptococcus pyogenes Cas9 (SpCas9) NLS signal Purified mrna from Sigma (500 ng/ul, Sigma Ald.) Concentration 150 ng/ul 55 ng/ul

29 X X XX X No 5' UTR exon 7th Chromosome in rabbit 5 target was designed Possible off targets 78 In genes 0

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33 Acknowledgements Thanks are due to Dow AgroSciences, (Exact) Professor Dénes Dudits (ODM) Dr László Hiripi CRISPR/Cas9 for providing slides to this lecture