New Plant Breeding Techniques: Zn Finger Nucleases and Transcription Factors

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1 New Plant Breeding Techniques: Zn Finger Nucleases and Transcription Factors Andrew F. Roberts, Ph.D. Deputy Director, CERA September 19, 2013

2 Contents of the talk Old Plant Breeding Techniques and Biosafety Zn Finger Nucleases What is a Zn Finger? What is a Zn Finger Nuclease? How can this be used in plant breeding? What does this mean for biosafety regulation? Transcription factors What are they? How can this be used in plant breeding What does this mean for biosafety regulation?

3 Part I: Old Plant Breeding Techniques Crossing of populations of the same species Crossing of closely related species Crossing of distantly related species Pollen-mediated Embryo Rescue or Somatic Hybridization Mutation Breeding Ionizing radiation Chemical mutagenesis Traditional Genetic Engineering Agrobacterium mediated transformation Biolistic transformation

4 Relative likelihood of unintended genetic effects associated with various methods of plant genetic modification. (NRC 2004)

5 Part II: Zn Finger Nucleases

6 What is a Zinc (Zn) Finger? A protein motif Contains a bound Zn ion And a protein finger First discovered in X. laevis As the DNA binding domain of a transcription factor Classified in different ways Numbers of cysteines and histidines Structure

7 Fig. from Krishna et al, 2003 Classification of Zn Fingers by Structure Can be structurally divided into 8 classes These have different binding properties Both within and between classes Bind to DNA, protein, small molecules Just about anything Many are coordinated by Zn Ions, but not all

8 What is the utility for genetic engineering? Zn fingers bind DNA in a sequence specific manner Usually specific to a 3 nucleotide codon The small motif can easily be incorporated as units in a larger protein These can be engineered to bind to different sequences Of varying length Can be used to target other protein functional domains to a specific DNA sequence

9 This can be done to order

10 Zn Finger Nucleases A series of Zn finger motifs, bound to a DNA nuclease The Zn fingers bind a specific DNA sequence The nuclease induces a double strand break at the site Method typically uses a nuclease dimer and two different Zn finger protein motifs Heterodimeric

11 What does this look like?

12 Genetic Engineering with Zn Finger Nucleases Two General Methods Both are site specific Induce double stranded DNA breaks Non-sequence specific repair Makes use of non-homologous end joining machinery Error prone Produces point mutations, small insertions or deletions Sequence specific repair Homologous repair machinery Using a homologous DNA template This template can be quite large (multiple Kb)

13 What does this look like?

14 Fig. from Carrol, 2008 More detailed figure showing nonhomology of Zn Finger nucleases

15 Advantages of Zn Finger Nucleases in Genetic Engineering High fidelity, site specific insertion You know exactly where your insert will be Gene editing Point mutations Induced sequence changes in native genes Very few (if any) extraneous DNA sequences in the final product

16 Fig. from Carrol, 2008 Potential Concerns for Genetic Engineering

17 Potential concerns Off target cleavage Sequence similarity within the genome homodimer sequence match Could lead to unintended mutations These are being addressed in a number of ways Increasing the number of Zn finger binding domains Engineering nucleases that do not function as homodimers Concerns are important for gene therapy; less so in the context of plant breeding

18 What does this mean for plant breeding? Highly site-specific, targeted mutation or gene insertion Uses endogenous cellular machinery The trans elements for making the change can be removed from the final product Zn Finger Nuclease protein is not found in the resulting plant Very little extraneous material backbone, construct, marker

19 Unintended Effects to the Genome are Virtually None

20 Biosafety Considerations for GE plants Created Using Zn Finger Nucleases Unintended Genomic Effects are limited Very low potential for multiple copies or insertion sites Potential risks from the intended change will depend on the case Point mutations, small insertions or deletions Very similar to traditional breeding Difficult to detect Inserted genes Just like traditional genetic engineering

21 Part III: Transcription Factors

22 What is a transcription factor? A protein that binds to DNA and influences gene transcription The most common transcription factors contain Zn finger DNA binding domains Many transcription factors control multiple target genes With related functions Transcription factors may interact with other transcription factors In concert In opposition In complex interactions

23 What does this mean for plant breeding? Transcription factors are not new A variety of traits important to modern agricultural varieties can be linked to changes in transcription factors Our knowledge of how they work is improving Process specific transcription factors have been identified Linked to abiotic stress tolerance Drought and salinity Transcription factors are increasingly targeted in Genetic Engineering to change complex traits

24 Transcription Factor Traits under Development Drought Tolerance Salinity Tolerance Secondary metabolism Improved nutrient use efficiency Nitrogen Phosphorous For medically important metabolites

25 What are the implications for biosafety? In traditional GE plants, a single protein or gene directly confers a single trait Herbicide tolerance Insect resistence Global effects on gene transcription are considered unintentional Comparative risk assessment is often misunderstood to mean any differences are necessarily harmful At the very least differences must be explained

26 Transcription factors induce lots of gene regulation changes That s why they re interesting targets of engineering Many of the target genes will be of uncertain function Most genes in the genome are still not well characterized How will regulators deal with a large number of changes in gene expression?

27 Two theoretical ways to approach biosafety Identify all potential changes Analyze the potential for each change to affect safety Identify potentially harmful changes Look to see if any of those changes have occurred If they have, determine their actual impact on safety

28 Acknowledgements and References The Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects, Committee on Identifying and Assessing Unintentional Effects of Geneticlly Engineered Foods on Human Health, National Research Council (2004) Carroll, D., Zinc Finger Nucleases as Gene Therapy Agents, Gene Ther. November 2008; 15(22): Gupta, M., et al., Transcriptional activation of Brassica napus -ketoacyl- ACP synthase II with an engineered zinc finger protein transcription factor. Plant Biotechnology Journal, 2012: p Strange T.L., Petolino J.F., Targeting DNA to a previously integrated transgenic locus using zinc finger nucleases. Methods Mol Biol, : p Petolino, J.F., et al., Zinc finger nuclease-mediated transgene deletion. Plant Mol Biol, (6): p Cai, C.Q., et al., Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol, (6): p Shukla, V.K., et al., Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, (7245): p U156.

29 Thank You!