Transcript New breeding technologies: GM and genome editing

Synthetic Biology,
‘New Breeding Techniques’
GM & Genome Editing
Ricarda A. Steinbrecher, PhD
Econexus
Oxford, 6 January 2016
[email protected]
Tools & technologies of Synthetic Biology
– BioCAD software,
– Robotic cloning,
– Metabolic modelling,
– Protein engineering,
– DNA databases and registries,
– Part and device libraries,
– Regulatory circuits,
– DNA synthesis,
– Gene and genome assembly,
– Genome editing: MAGE, CRISPR, TALENs, zinc fingers **
– Epigenetic engineering **
• Microscopy, Molecular profiling
Predictability
Approach
- designing and controlling
biological processes
- making organisms perform
according to design
of the outcome
of our doing & intervention
on all levels
?
Precision
the dream of the realisation
of precision
• Microorganisms
• Plants
• Animals
• Human
invites a mechanistic approach and
mindset
(as we have insufficient knowledge)
Princeton School of Engineering and Applied Science www.princeton.edu/cbe/people/data/j/javalos/profile/
New Breeding Techniques
under scrutiny by EC (are they GMOs??):
1) * Zinc Finger Nuclease Technology (ZFN-1/2/3)
2) * Oligonucleotide Directed Mutagenesis (ODM)
3) * Cisgenesis/Intragenesis
4) RNA-dependent DNA methylation (RdDM);
5) Grafting (onto a GMO rootstock);
6) Reverse Breeding (RB);
7) Agro-infiltration (both Agro-infiltration ‘sensu stricto’ & Agro-inoculation)
(8 Synthetic genomics )
plus other NBTs mentioned by industry and interest groups:
eg: MAS (marker assisted selection) - not GM !
distributed by EuropaBio
Metabolic pathways
Metabolism is the set of chemical reactions that occur in
a cell, which enable it to keep living, growing and dividing.
Metabolic processes are usually classified as:
catabolism - obtaining energy and reducing power from
nutrients.
anabolism - production of new cell components, usually
through processes that require energy and reducing
power obtained from nutrient catabolism.
ct: glycolysis - glucose oxidation in order to obtain ATP
cb: citiric acid cycle (Kreb’s cycle): acetyl-CoA oxidation in order to obtain GTP and
valuable intermediates.
bl: AA degradation
metabolic
map
Organism =
chemical factory
 making different
compounds according to
design
 utilise “precursors”
 Change plumbing (genes)
 “Petrol” in …. product out
cells
nucleus
chromosome
DNA
gene
A
A
C
T
C
G
T
Basepairs: A-T & C-G (nucleotides)
T
T
G
A
G
C
A
Genes and Genetics: The Fundamentals
“The New Genetics”
3. In many cases, more than one RNA/protein is produced
from a given gene.
DNA
transcription
RNA
processing (alterations, splicing)
mRNA
translation
protein
3D Protein
Gene & gene construct
promoter
Gene for trait (Bt toxin)
end
Regulatory sequence: on/off switch - often CaMV (virus)
Coding sequence of a gene - e.g. pat or bar gene for herbicide
resistance from soil bacteria
Regulatory sequence: Termination signal - e.g. from pea
Plasmid backbone DNA, superfluous genetic material
Transformation
?
Particle
bombardment
Agrobacterium
used as “shuttle”
No control of where the gene will insert itself:
• Random integration
• Imprecision (incl. superfluous DNA)
• 100 -1000s of Mutations (Sala et al. 2000, Wang et al. 1996, Labra et al 2001)
Special
enzyme
Targeted ‘break’ of
both strands of
DNA in a
chromsome
The special enzymes are called:
1. CRISPR/Cas
2. ZFN (zinc finger nucleases)
3. Tales/Talens (transcription activator–like effector
nucleases)
4. Meganucleases
9/35
Synthesis
ACGTTTTAACGTTTACGGGT … …
A
C
G
T
T
G
G
G
C
T
A
C
G
T
T
T
C
T
A
A
C
G
A
T
A
“Computer, give me 1000 variants” – put into 1000 organisms
Material
‘deleted’
Two separate
breaks result in
large deletion of
material
6/35
the double-stranded break results in degradation of both strands by
endogenous enzymes, enlarging the area of damage
the damage can be repaired by rejoining (leaving a deletion or other
change), by a template-directed repair (that may change function) or a
template-directed insertion
Unpredictabilities and risks
- Off-target effects (due to non-specific binding to nontarget DNA): off-target mutations in the genome. These
mutations can a) if in the coding sequence, result in
changes of function of proteins, or b) if in regulatory
sequences, result in changes in the expression of genes,
such as increased presence of plant toxins, or absence of
proteins important for nutrition, plant defence or disease
resistance, increased presence of allergens.
- Integration of added DNA or oligonucelotides into the
genome
- Impacts of the genetic engineering processes
(mutations)
Steinbrecher, R (2015). Genetic Engineering in Plants and the “New Breeding
Techniques” (NBTs) - Inherent risks and the need to regulate.
The idea of Precision
The idea of precision is based on that one knows
what one is doing – here this is not the case.
Knowledge & precision at the level of nucleotides is only the
bottom layer.
What is missing is the contextualisation into
• the genome
• the epigenetic landscape
• the organism
• the ecosystems
• the socio-economic conditions that differ around the world
Precision around nucleotides gives a false sense of
predictability and safety – there is no data to support such
extrapolations.
thank you
Push Pull Technology – East
Africa
Problem for maize yield:
Stem borer (eg chilo partellus moth) effect a third
of the region’s maize crop.
Scientists of the Mbita Point Research Station,
Lake Victoria, Kenya came up with a simple
solution:
• Pull: Napier grass (up to 70% yield increase).
• Push: Desmodium, a common legume “weed”
species, repels the stem borer
Secondary benefits:
Desmodium enriches soil with nitrates, prevents
soil erosion during rains and suppresses the
growth of Striga weed, a parasitic plant causing
US$ 10 billion yield loss/year (effecting 100
million Africans).
• Napier grass can be used and sold as fodder.
GE vs. Alternatives in Developing Countries
Locally researched Solutions already Exist
GE vs. Alternatives in Developing Countries
“Biomass doesn’t cut
it.. Carbohydrates are
not a substitute for oil. I
was wrong in that and I
admit it. That will never
replace oil because the
economics don’t work.
You can’t take
carbohydrates and
convert them into
hydrocarbons
economically”
Alan Shaw (formerly
Codexis)