Discussion questions

1. Lecture 16 The future of plant biotechnology:genome editing and concluding perspectivesNeal Stewart & Agnieska Piatek

2. Discussion questions • What is the main dichotomy between innovation and caution (or risk, or the perception of risk)? • What is genome editing? What are the current tools for genome editing? • What are zinc-finger nucleases, TALENs, and CRISPR? How might they alter the future of plant biotechnology? • How do feelings and trust influence plant biotechnology?

3. Problems in plant biotechnology:might be addressed with new technologies • Agrobacterium- and especially biolistics-mediated transformation are imprecise: many transgenic events must be produced. • Transgenic plants are regulated because they are transgenic- is there another way? Hint: genome editing. • What is synthetic biology? Will it help solve problems or complicate them?

4. Figure 17.3 An abstraction hierarchy that supports the engineering of genetic systems. The parts [such as promoters, ribosome binding sites (RBS) in prokaryotes, coding regions and terminators] are DNA sequence-based and sometimes context-dependent, but can be engineered rationally to produce different devices such as inputs, logic gates and outputs, which permit assembly into artificial systems for further practical, desirable applications.

5. Figure 17.8

6. Genome editing • Altering the sequence of DNA “in situ” • Targeted mutagenesis • Knock-outs • Point mutations • Gene insertions or “trait landing pads” • Ideally leaving no transgene footprint • Is genome engineering plant breeding, genetic engineering or both?

7. Genome editing tools in plants • Meganucleases: 1990s • Oligonucleotide-directed mutagenesis: late 1990s • Zinc finger nucleases (ZFNs): mid 2000s • Transcription activator like effector nucleases (TALENs): early 2010s • Clustered regularly spaced short palindromic repeats (CRISPR): 2013

8. Zinc finger nucleases www.bmb.psu.edu, www.wpclipart.com, www.faculty.ucr.edu

9. Figure 17.5 Figure 17.5 Engineered Zinc-finger nucleases (ZFNs; a) and transcription activator-like effector nucleases (TALENs; b) for targeted genome modification (Reprinted from Liu et al., 2013b). Each nuclease contains a custom-designed DNA binding domain and the non-specific DNA-cleavage domain of the FokI endonuclease which has to dimerize for DNA cleavage within the spacer regions between the two binding sites. The spacer regions between the monomers of ZFNs and TALENs are 5-7 bp and 6-40 bp in length, respectively.

10. ZFNs in gene therapy Nature 435:577

11. Transcription activator-like effectors (TALEs) • Transcription factors • Secreted by Xanthomonasbacteria via type III secretion system • Bind promoter sequences in the host plant • Recognize plant DNA sequences Plant cell TF Gene TF TF Gene Promoter Nucleus www.plantwise.org

12. Structure and DNA binding code of TALEs Repeats (1.5 to 33.5) TS AD NLS N’ C’ Central repeat domain 1 34 12 13 L T P E Q V V A I A S H D G G K Q A L E T V Q R L L P V L C Q A H G Repeat variable diresidue (RVD) 12 13 DNA binding code NG = T HD = C NI = A NN = G or A

13. How to engineer TALEs? Engineered central repeat domain TS AD NLS C’ N’ NI NI NI NI Prerequisite of T nucleotide preceeding the DNA target sequence = C = A DNA Target HD = T NI NG NG NG NG NK NK NG NK = G TCCCAAATTTGATCG HD HD HD HD

14. Why engineer TALEs? For targeted genome mutagenesis and editing: TALEN A TALEN B 3’ 5’ 3’ 5’ Target sequence A Target sequence B FokI dimer Outcome: 3’ 5’ 3’ 5’ Chromosomal deletion Point mutation Insertion Deletion AACGT TTGCA

15. Why engineer TALEs? For targeted genome regulation TFs mRNA transcripts 5’ 3’ 3’ 5’ Target sequence Promoter Gene X V TALE-TF Modulator = or Modulator Repressor Activator

16. RNA based genome engineering platform PAM

17. CRISPR – Bacterial Immunity Clustered Regularly Interspaced Short Palindromic Repeats Acquired adaptive immunity in bacteria against viruses direct repeats First described in 1987 tracrRNA Cas9 Cas1 Cas2 Csn2 spacers Streptococcus pyogenes CRISPR array

18. Mechanism of CRISPR-mediated immunity in bacteria Plasmid DNA Virus DNA 1. Acquisition Cas n leader 1 2 3 4 CRISPR array Cas locus 2. Expression 3. Interference Cas proteins Pre-crRNA crRNA

19. CRISPR genome editing

20. CRISPR variants Published by AAAS E Pennisi Science 2013;341:833-836

21. Last questions of the semester Is food too hot (emotionally) to be addressed by biotechnology? Where on earth? What is the scientist’s role here? What about non-food plant biotechnology such as bioenergy? What about genome editing?

22. “Ordinary tomatoes do not contain genes, while genetically modified ones do” 1996 - 1998 People in different countries have varied knowledge about the facts of genetics and biotechnology. Slide courtesy of Tom Hoban

23. American consumers’ trust in biotechnology information sources Slide courtesy of Tom Hoban

24. Source of information trusted most to tell the truth about biotechnology(includes all European countries) Slide courtesy of Tom Hoban

26. Way forward • Technological innovations will continue • Until disaster strikes (think Fukushima) • And then innovations will resume • But they will be (more) regulated • So there is a balance between innovationpotential risks and regulationensure safety

27. Trends • Plant biotech plant synthetic biology • Designed components • More precise gene integration and regulation • Building a crop from scratch? • Transgenicsgenome editing • Regulations? What about a tiered approach? • Increasing gap between public’s knowledge of science and technology and science and technology advances • But…patents expire and economics shift…times change

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