1. Lecture 16 The future of plant biotechnology: genome editing and concluding perspectives Neal 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

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 Xanthomonas bacteria via type III secretion system
Bind promoter sequences in the host plant
Recognize plant DNA sequences
Plant cell TF TF TF
Gene
Gene
Promoter
Nucleus

12. Structure and DNA binding code of TALEs
Central repeat domain AD
NLS
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 1 34 12 13 TS
Repeats (1.5 to 33.5)
Repeat variable diresidue (RVD)
NG = T
HD = C
NI = A
NN = G or A
DNA binding code 12 13

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

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

15. Why engineer TALEs? V X = or For targeted genome regulation TFs
TALE-TF
Modulator V X
mRNA transcripts 5’ 3’ 3’ 5’
Promoter
Target sequence
Gene
Modulator = or
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
First described in 1987
Streptococcus pyogenes CRISPR array
tracrRNA
Cas9
Cas1
Cas2
Csn2
spacers
direct repeats

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

19. CRISPR genome editing

20. CRISPR variants
DNA surgeon.With just a guide RNA and a protein called Cas9, researchers first showed that the CRISPR system can home in on and cut specific DNA, knocking out a gene or enabling part of it to be replaced by substitute DNA. More recently, Cas9 modifications have made possible the repression (lower left) or activation (lower right) of specific genes.
Published by AAAS
E Pennisi Science 2013;341:

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”
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

34. Manufactured in
Chicago
Full of patented IP
Last patent issued in
1957
Price (new) $22.50
Last sold (new) 1997
$85.00
$12.88
1950s Sunbeam T-20 radiant control toaster