Precision engineering of the plant genome. Sjef Smeekens Institute of Environmental Biology Utrecht University

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1 Precision engineering of the plant genome Sjef Smeekens Institute of Environmental Biology Utrecht University

2 Mutation breeding Molecular basis of traits, the Zea mays TGA1 example Mutation breeding using random mutagenesis Mutation breeding using precision mutagenesis Examples Regulatory issues: product vs. process

3 The origin of the naked grains of maize Teosinte Glume Architecture 1 (TGA1) Doebley lab, UWM

4 Teosinte Glume Architecture 1 (TGA1) Teosinte GTA (Lysine, TGA1 active) Maize CTA (Asparagine, TGA1 inactive) Doebley lab, UWM, Nature 2005

5 Traits selection and cultivar development Kale, Broccoli, Brussels Sprouts, and Cabbage are all the Same Species

6 Induced mutations for plant improvement Chemical: alkylating agents e.g. EMS, DMS Physical: ionizing radiation, e.g. gamma rays (Co-60, Cs-137), Röntgen The Atomic Garden Society Biological : transposons Massive DNA damage: re-arragangements, mutations, deletions, insertions A garden show in 1961 (Frank Scherschel for Life)

7 Japan's Gamma Field, at the Institute of Radiation Breeding Google Earth, 2015

8 What has changed? Rapidly expanding body of knowledge on genes and their variants (alleles) that specify desired traits New Plant Breeding Techniques: precision engineering of the genome Desired trait Gene selection & precision mutation Novel cultivar development

9 Precision engineering of the genome Zinc-Finger Nucleases (ZFN) Transcription Activator-Like Effector Nuclease (TALEN) RNA guided nuclease (CRISPR/CAS) Oligonucleotide-directed mutagenesis

10 Precision engineering of the genome Osakabe & Osakabe PCP, 2015

11 Nuclease-induced genome editing Sander&Joung Nature Biotech 2014

12 Precision engineering: deletions, insertions, mutations NHEJ: Insertions and deletion Arabidopsis PDS gene Li, et al, Nature Biotech 2013 HDR: Precise point mutations Rice ALS gene Endo et al, Plant Physiol 2016

13 Delivery of engineered nucleases CRISPR-Cas9 1) Delivering the engineered nuclease to cells Transient or stable DNA or RNA vector Pre-assembled CRISPR-Cas9 2) Regenerate plants 3) Identify plants with induced mutation 4) Develop new cultivar Pre-assembled CRISPR-Cas9

14 DMR genes for disease resistance breeding in crops van den Ackerveken Lab, Utrecht University downy mildew resistant (dmr) mutants DMR6 encodes a 2-oxoglutarate-Fe(II)-oxygenase, present in all flowering plants Inactivation of DMR6 leads to broad spectrum disease resistance in Arabidopsis and all crops tested crops Van Damme et al, MPMI 2005, Plant J. 2008

15 CRISPR-Cas9 mediated mutagenesis of a DMR6 ortholog in tomato confers broad-spectrum disease resistance Inactive protein Inactive protein Staskawicz lab, BIORXIV 2016

16 CRISPR-Cas9 mediated mutagenesis of a DMR6 ortholog in tomato confers broad-spectrum disease resistance Staskawicz lab, BIORXIV 2016

17 CRISPR/Cas9-mutagenesis of ripening-regulating transcription factor genes in tomato Ruud de Maagd Rufang Wang Gerco Angenent wild type nor/+ nor/+ rin/rin rin/+ nor/nor nor/+ rin/+ nor/? nor/+ rin/rin

18 CRISPR/Cas9-mutagenesis of ripening-regulating transcription factor genes in tomato wild type ap2a/ap2a wild type ful1/ful1 ful2/ful2 homozygous mutant without the CRISPR-Cas9 construct Pictures: Rufang Wang

19 Azolla as a novel quality protein crop UU Molecular Plant Physiology UU Geochemistry/NIOZ Wageningen UR, PRI: Henriette Schluepmann, Paul Brouwer Klaas Nierop, Gert-Jan Reichart Adrie van der Werf

20 A. filiculoides genes required for tannin biosynthesis AfAH10-like AfANS AfANR AfCHS AfCHS: Chalcone synthase AfANS: Anthocyanidin synthase AfANR: Anthocyanidin reductase AfAH10-like: Autoinhibited atpase

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22 European Academies Science Advisory Committee Recommendation, July 2015 We ask that EU regulators confirm that the products of new breeding techniques, when they do not contain foreign DNA, do not fall within the scope of GMO legislation. Joint position on New Techniques of Genetic Engineering Greenpeace, March 2016 EU GMO law must be fully applied to the so-called New Plant Breeding Techniques

23 Spot the difference Natural Variant Selection Precision Engineering N N N N N G N N N N N N N N N G N N N N N N N N N C N N N N N N N N N C N N N N

24 New Plant Breeding Techniques Regulatory system: Process based novelty and risks of the production process is considered, not of the resulting product Product based novelty and risks of the individual trait is considered, not the process by which the trait was produced.

25 An inclusive technology Easy Efficient Fast Cheap

26 The sustainability of our agricultural enterprise is at stake To create crops that are flexible and adaptable to the challenges of environment and population, we must increase the predictive and synthetic abilities of plant scientists Unleashing a Decade of Innovation in Plant Science A V I S I O N F O R , American Society of Plant Biology

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