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Genome Editing For the Future Food Feng Zhang Calyxt Inc July 13 th , 2017, UC Berkeley
Genome Editing For the Future Food
Feng Zhang
Calyxt Inc
July 13th , 2017, UC Berkeley
This communication expressly or implicitly contains certain forward-looking statements concerning Calyxt and its business. Such statements involve certain known and unknown risks, uncertainties and other factors, which could cause the actual results, financial condition, performance or achievements of Calyxt to be materially different from any future results, performance or achievements expressed or implied by such forward-looking statements. Calyxt is providing this communication as of this date and does not undertake to update any forward-looking statements contained herein as a result of new information, future events or otherwise. Calyxt proprietary information. Not to be copied, distributed or used without Calyxt prior written consent. Calyxt™ is a trademark owned by Calyxt Inc. and TALEN® a trademark owned by Cellectis S.A.
FORWARD LOOKING STATEMENTS
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Challenges : Agriculture in a Growing World
World population to reach 9.6 billion in 2050
World Population Prospects, UN, 2012
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20
25
7
2
Herbicide Tolerance
Insect Resistance
Other Farmer Traits
Modified Product Quality
Traditional GMO’s Have Revolutionized Agriculture but Largely Ignored Consumer Needs
Source: Adapted from FMI U.S. Grocery Shopper Trends, 2014-2015. Q: What health claims do you look for on the package when purchasing a food product?
10%
14%
19%
19%
20%
20%
22%
22%
26%
26%
28%
29%
29%
35%
36%
37%
38%
38%
41%
41%
Gluten-free
Calcium-fortified
Vitamin-enriched
Antioxidant-rich
Low carb
Certified organic
Heart healthy
Lowers cholesterol
Non-GMO
Natural
No HFCS
No/Low fat
Low calorie
No preservatives
No trans fats*
No artificial ingredients*
High fiber
Low sugar
Low sodium
Whole grain
Claims Most Sought by US Consumers-2015
Source: Adapted from Nature Biotechnology 34, 31-36 - 2016
Biotechnology Traits Commercially Cultivated in the US- 2014
≠
GMO trait R&D pipeline
Trait Discovery
$31.0 m
“Traditional” GM Trait Development Process
Farmer needs
Farmer needs
Construct Optimization
Construct Optimization
$28.3m
Commercial Event
Commercial Event
$13.6m
Regulatory Science
Regulatory Science
$17.9m + $28.0m
Regulatory Affairs
Regulatory Affairs
$17.2m
Breeding and TestingBreeding and TestingBroad Food
Industry Testing of GMO Quality
Traits
Broad Food Industry Testing of GMO Quality
Traits
Market LaunchMarket Launch
1-3 Years+ + + + + = $136M;13 years
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A Consultancy Study for Crop Life International, September 2011
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Reduce Waste
Abiotic Stress/Drought
Insect/Disease Resistance
Herbicide Tolerance
Reduce Inputs like nitrogen, phosphorus
Increase Yield
What if consumer and farmer needs were addressed in one product?
Gene Editing is a Paradigm Shift
MORE OF …
LESS OF …
Plant Proteins
Nutrients
Color, Flavor
Dietary Fibers
Good Fats
Vitamins
Chemical Additives
Saturated FatsTrans Fats
CarcinogensSugars
AllergensToxic
Compounds
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Major Genome Editing Platforms
®
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33Gene is repaired Improvement of gene function
Gene is inserted (Knock-in) insertion of a new trait
Gene is disabled (knock-out)removal of undesired traits
Gene Target
DNA at the f-interest site
Two TALENs recognize their
DNA target
The nuclease domains interact
and cut the DNA
Sequence-specific nucleases recognize
their DNA target
A DSB is induced in the gene-of-interest
Major Types of Targeted Modifications in Genome Editing
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Delivery methods need to be customized for different crops
Nu
cle
ase
sD
esi
gn &
Ass
em
bly Agrobacterium
Cereals, Other Crops
Particle Bombardment
Oilseeds, Cereals, Vegetables
Protoplast Potato, Alfalfa, Canola, Vegetables
Tissue Transformation Systems
Calli formation
Shoot and root regeneration
Plant with desired characteristic
1
2
3
4
5
2a
2c
2b
Seeds with desired characteristic
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More than 15 plant species have been modified through genome editing
Number of species (since 2009)
Number of targets
Overall 22 More than 70 genes individually and whole genome mutant library in rice
(12,802 genes)
Among all the cases, most of them are NHEJ-mediated mutations. Only less than 10 examples are through HDR.
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Gene is repaired Improvement of gene function
Gene is inserted (Knock-in) insertion of a new trait
Gene is disabled (knock-out)removal of undesired traits
Gene Target
DNA at the f-interest site
Two TALENs recognize their
DNA target
The nuclease domains interact
and cut the DNA
Major Types of Targeted Modifications in Genome Editing
Sequence-specific nucleases recognize
their DNA target
A DSB is induced in the gene-of-interest
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GOI
Transgenic plant that expresses sequence-
specific nuclease
In planta strategies for making targeted gene knockouts
GOI
Wild type plant
GOI = gene of interest
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GOI
Transgenic plant that expresses sequence-
specific nuclease goi
Wild type plant
In planta strategies for making targeted gene knockouts
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GOI
Transgenic that expresses sequence-
specific nuclease goi
Wild type plant
• After segregation away of the nuclease transgene, no foreign DNA remains in mutant plant
In planta strategies for making targeted gene knockouts
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Genome-edited crops enter the food supply
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Genome Editing in Glycine max (Soybean)
Soybean Production
• 320 million metric tons per year
• 297 million acres (~ area of South Africa)
• #1 source of protein for animal feed
• #2 source of oil for human consumption
Soybean61%
Rapeseed13%
Peanut8%
Other18%
Oilseed Production (World)
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Fatty Acid Composition of Plant Oils80%
50% 100%
Sunflower
23% 15%62%
Total Fatty Acids
82% 9% 9%
75% 10% 15%75%
31% 7%62%
10% 51%39%
58% 13%29%
19% 27%54%
16% 12%72%
Olive
Canola
Palm
Corn
Soybean
Cottonseed
Saffflower
= monounsaturated fats + = polyunsaturated fats
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US Government Mandates Healthier Oil
Hydrogenation Extends Shelf Life and Improves Heat Stability
Hydrogenation Trans-fat
Starting 2018
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Problems Facing Soybean Industry
81
% o
f o
il c
on
su
mp
tio
n
100
20
80
40
60
6981
7864
6261
55 5859
0
19 19 2231 36 38 39
45 41 42
Soybean oil Other vegetable oils
55 56
45 44
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Stacking High Oleic with Low Linolenic Traits
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Efficient Targeted Modifications in FAD2
Experiment IDTargeted
GenesGenotype
BackgroundNumber of TF
EventsNumber of
MutantsMuta.
Frequency
Gm004 FAD2-1a/1b WT 35 4 11%
Gm183/205 FAD3Afad2-1a/fad2-
1b33 10 30%
Gm184/206 FAD3A WT 39 6 15%
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OH
O
Palmitic acid
OH
O
Natural pathway
Oleic acid
OH
O
Linoleic acid
FAD2
OH
O
Palmitic acid
OH
O
Oleic acid
OH
O
Linoleic acid
FAD2
Modified pathway
High Oleic Soybean with Double Gene Knock-out
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Stacking High Oleic with Low Linolenic Traits
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Efficient Targeted Modification in FAD3
Experiment IDTargeted
GenesGenotype
BackgroundNumber of TF
EventsNumber of
MutantsMuta.
Frequency
Gm004 FAD2-1a/1b WT 35 4 11%
Gm183/205 FAD3Afad2-1a/fad2-
1b33 10 30%
Gm184/206 FAD3A WT 39 6 15%
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High Oleic Low Linolenic Oil Soybean
Demorest et al. BMC Plant 2016www.calyxt.com
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Fatty Acid Composition of Plant Oils
80%
50% 100%
Sunflower
23% 15%62%
Total Fatty Acids
82% 9% 9%
75% 10% 15%75%
31% 7%62%
10% 51%39%
58% 13%29%
19% 27%54%
16% 12%72%
Olive
Canola
Palm
Corn
Soybean
Cottonseed
Saffflower
= monounsaturated fats + = polyunsaturated fats
Calyxt HO 80% 9% 11%
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Opportunities for Gene Editing in Potato
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Opportunities and Challengesin Potato
Next generation potato
Improved storability: currently 15% wasted
Reduced browning: currently 9% wasted
Resistance to diseases
Challenges
Tetraploid and heterogenous genome
Vegetative propagation makes the integration/segregation strategy impractical
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Cold-induced sweetening results in loss of 15% of the potato crop annually
Improving storage and processing of potatoes by targeted knockout of a vacuolar invertase (VInv)
Clasen et al. 2016. Plant Biotech J. 14:169
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0
50
100
150
200
250
300
350
400
450
500
Atlantic Chips Ranger RussetFrench Fries
Russet BurbankFrench Fries
Acr
yla
mid
e (
pp
b)
Fried potato products exceed anticipated thresholds for acrylamide in food
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Efficient delivery method to transiently express nucleases
0102030405060708090
100
Atlantic RangerRusset
Shepody RussetBurbank
YukonGold
Tran
sfo
rmat
ion
Eff
icie
ncy
(%
)
Transformation efficiency can reach up to 90% in a number of potato commercial varieties.
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A series mutants obtained from transformed protoplast
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Transgene-free mutants Identified by PCR and Southern
Low Acrylamide Phenotype Confirmed in Mutants
34
Wisconsin Michigan
Potato field trials ongoing in the US…
35
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Sequence-specific nucleases typically make small deletions at the cleavage site that can disrupt gene function
Examples of targeted mutations made by TALENs
How will plants with targeted mutations be regulated?
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TALENs:Low acrylamide potato – Calyxt, November 10, 2014High oleic soybean – Calyxt, May 8, 2015Low linoleic soybean – Calyxt, May 21, 2015Disease resistant rice – Iowa State University, May 29, 2015Fungal resistant wheat – Calyxt, February 11, 2016
MeganucleasesUse of meganucleases for plant trait development – Calyxt, January 6, 2012Maize trait -- Agrivida, November 30, 2015
Zinc Finger Nucleases:Low phytate corn -- Dow AgroScience, May 26, 2010
CRISPR/Cas:Anti-browning mushroom – Penn State University, April 13, 2016Waxy corn – Dupont/Pioneer, April 18, 2016
Regulatory status of crop varieties created through targeted gene knockouts:
Source: USDA website
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Gene is repaired Improvement of gene function
Gene is inserted (Knock-in) insertion of a new trait
Gene is disabled (knock-out)removal of undesired traits
Gene Target
DNA at the f-interest site
Two TALENs recognize their
DNA target
The nuclease domains interact
and cut the DNA
Major Types of Targeted Modifications in Genome Editing
Sequence-specific nucleases recognize
their DNA target
A DSB is induced in the gene-of-interest
www.calyxt.com
Harnessing plant DNA viruses to achieve efficient
gene targeting
Gil-Humanes J, Plant J. 89:1251-1262 (2017)Nick Baltes, Plant Cell 26:151-163 (2014)
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Circular DNA genomes-Nuclease expression-Donor molecule delivery
Extrachromosomal replication-Transient-High-copy number
Harnessing plant DNA viruses to achieve efficient gene targeting
Geminiviruses
SIR
LIRMovement Protein
Coat Protein
Replicase Proteins BeYDV
SIR
LIRSequence-Specific Nuclease
Donor Molecule
Replicase Proteins BeYDV
Replicon
Baltes et al. 2014. Plant Cell. 26:151-63.Cermak et al. 2015. Genome Biology 16:232.
www.calyxt.com Plant Cell
Cytoplasm Nucleus
REP
ZFN TALEN
MeganucleaseCRISPR/Cas system
RecombinationAgrobacterium
VIR proteins
Genomic DNA
RB
LB
DONOR
NUCLEASE
RB
LB
DONOR
NUCLEASE
RB
LB
DONOR
NUCLEASE
DONOR
NUCLEASE
Using Agrobacterium to deliver geminivirus replicons to plant cells
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gus:nptII
GUS:NPTII
Zif268:FokI replicon
2x35S GUS NPTII
35S Zif268:FokILIR
SIR us:NPTII
1 kb 1 kb0.6 kb
2x35S gus nptII
Zif268 Target Site
Targeting a defective reporter gene in vivo
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At four different genomic loci in tobacco, geminivirus replicons enhance gene targeting
frequencies on average 25-foldRepliconStandard T-DNA
Baltes et al. 2014. Plant Cell. 26:151-63.
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Average ~25X enhancement
Replication
Rep/RepA
~5X enhancement
~5X enhancement(Replicating ZFN + Donor)
~3X enhancement(Replicating Donor)
DNA replicons increase gene targeting by providing more donor template and through
pleiotropic effects of Rep/RepA
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Average ~25X enhancement
Replication
Rep/RepA
~5X enhancement
~5X enhancement(Replicating ZFN + Donor)
~3X enhancement(Replicating Donor)
RepA binds Rb
S phase andelevated HR
DNA replicons increase gene targeting by providing more donor template and through
pleiotropic effects of Rep/RepA
Improved gene targeting with the geminivirussystem in tomato and wheat
Gene targeting frequency up to 11% by using both TALENs and CRISPRs;
>10 fold increase than non-viral method
Gene targeting frequency has 12 fold increase than non viral method.
Tomato Wheat
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Conferring resistance to DNA viruses using CRISPRs
Nicholas J. Baltes et al. Nature Plants (2015)
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CRISPR-Cas systems target DNA invaders
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Targeting the Bean Yellow Dwarf Virus with CRISPR/Cas9
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CRISPRs reduce proliferation of virus-based DNA replicons in plant cells
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0
1000
2000
3000
4000
5000
6000
Cas9 only gBRBS+
No
rma
lize
d v
iral lo
ad
Resistance cassette
gBM3+
a.
c.
sgRNA and target motif
Re
ad
s w
ith
in
de
ls (
%)
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
gBRBS+ gBM3+ gBM1- gB9nt+
sgRNA for BeYDV
gTM3+
d.
Plants expressing CRISPR/Cas9 reagents that target BeYDV have reduced viral loads
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193.25 (Cas9 only) 21 days 28 days 35 days
196.25 (gBM3 ) 21 days
28 days
35 days
Cas9 only (193.25) gBM3 (196.25)
Plants expressing CRISPR/Cas9 reagents that target BeYDV have reduced
symptoms of infection
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• Crop varieties with knockout mutations are
rapidly being created and tested.
• Achieving efficient gene replacement is the
major challenge in plant genome engineering
• Governmental authorities are determining how
plants made through genome engineering will be
regulated and enter the food supply
Summary
