Lecture 16
The future of plant biotechnology:
genome editing and concluding
perspectives
Neal Stewart & Agnieska Piatek
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?
Problems in plant biotechnology:
might be addressed with new
technologies
• Agrobacterium- and especially biolisticsmediated 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?
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 contextdependent, 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.
Figure
17.8
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?
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
Zinc finger nucleases
www.bmb.psu.edu, www.wpclipart.com, www.faculty.ucr.edu
Figure
17.5
Figure 17.5 Engineered Zinc-finger nucleases (ZFNs; a) and transcription activatorlike 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 640 bp in length, respectively.
ZFNs in gene therapy
Nature 435:577
Transcription activator-like effectors (TALEs)
Transcription factors
Secreted by Xanthomonas bacteria via type III secretion system
Bind promoter sequences in the host plant
Plant cell
Recognize plant DNA sequences
Promoter
Gene
Nucleus
www.plantwise.org
11
Structure and DNA binding code of TALEs
Repeats (1.5 to 33.5)
TS
NLS AD
N’
C’
Central repeat domain
1
34
12 13
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG
Repeat variable diresidue (RVD)
DNA binding code
12 13
NG
HD
NI
NN
=
=
=
=
T
C
A
G or A
12
How to engineer TALEs?
Engineered central repeat domain
TS
NLS AD
N’
HD HD HD NI NI NI NG NG NG NK NI NG HD NK
DNA Target
HD
= C
NI
= A
NG
= T
NK
= G
C’
TCCCAAATTTGATCG
Prerequisite of T nucleotide preceeding
the DNA target sequence
13
Why engineer TALEs?
For targeted genome mutagenesis and editing:
TALEN B
TALEN A
5’
3’
Target
sequence A
Target
sequence B
FokI dimer
3’
5’
Outcome:
Chromosomal deletion
Point mutation
Insertion
Deletion
AACGT
TTGCA
14
Why engineer TALEs?
TFs
For targeted genome regulation
TALE-TF
Modulator
V X
5’
3’
Promoter
Target
sequence
Modulator
mRNA transcripts
3’
5’
Gene
=
or
Repressor
Activator
15
RNA based genome
engineering platform
PAM
16
CRISPR – Bacterial Immunity
Clustered Regularly Interspaced Short
Palindromic Repeats
Acquired adaptive immunity
in bacteria against viruses
First described in 1987
Streptococcus pyogenes CRISPR array
direct repeats
tracrRNA
Cas9
Cas1 Cas2 Csn2
spacers
17
Mechanism of CRISPR-mediated immunity in
bacteria
Virus
DNA
Plasmid
DNA
1. Acquisition
leader
1
2
3
4
CRISPR array
n
Cas
Cas locus
2. Expression
Pre-crRNA
3. Interference
Cas
proteins
crRNA
18
CRISPR genome editing
CRISPR variants
Published by AAAS
E Pennisi Science 2013;341:833-836
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?
“Ordinary tomatoes do not contain genes,
while genetically modified ones do”
52
Canada
33
45
United States
45
34
Austria
22
32
France
36
Italy
35
44
29
20
44
44
21
51
Netherlands
27
24
30
48
21
31
40
0
22
46
Switzerland
United Kingdom
10
39
Germany
Sweden
15
38
20
40
22
60
80
100
Percent Response
1996 - 1998
False (Correct)
Don't Know
True
People in different countries have varied knowledge about the facts of genetics and
biotechnology.
Slide courtesy of Tom Hoban
American consumers’ trust in
biotechnology information sources
41
American Medical Association
46
32
Food and Drug Administration
51
28
University Scientists
Registered Dietitians
20
Farmers
20
Food Manufacturers
7
Chefs
6
Activist Groups 4
0%
14
61
19
54
30
52
37
52
41
48
46
43
20%
52
40%
A Lot
Slide courtesy of Tom Hoban
12
66
11
Biotechnology Companies
17
59
16
TV News Reporters
13
60%
Some
80%
None
100%
Source of information trusted most to
tell the truth about biotechnology
(includes all European countries)
26
Consumer Organizations
20
14
Environmental Groups
17
21
Medical Profession
16
7
Universities
9
7
Government
8
4
4
Mass Media
1999
1996
6
Don't Know
9
6
None (Spontaneous)
7
0
Slide courtesy of Tom Hoban
5
10
15
20
Percent Response
25
30
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
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
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