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EXZACTTM is a Toolkit for Precise Genome Modification
Genome Modification: several modalities
Targeted mutagenesis of an endogenous gene
Targeted gene addition at an endogenous locus
Targeted genome editing at an endogenous locus
• Based on proprietary zinc-finger
engineering.
• Precisely targets any DNA sequence.
• Accurately modify the genome
• Mode of action based upon natural DNA
repair processes and promoter activity.
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EXZACTTM is Based on Zinc-Finger Proteins
• ZFPs are the most abundant class of transcription factors occurring in nature
— DNA binding proteins - αββ
— sequence specificity is determined by key residues in the protein
structure
Residues “-1” to “+6” of the alpha helix
H
COOH
C
C
H
6
3
5’
2
-1
NH2
Finger “Design”
-1 2 3 6
Triplet
Specificity
RSDELTR
RSDHLTR
QSSDLSR
QSGSLTR
RSDALTQ
RSDHLTQ
GCG
GGG
GCT
GCA
ATG
AGG
etc…
3’
Tupler R, Perini G, Green MR (2001). Nature 409: 832-833.
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Designing Custom ZFPs Linked to Functional Domains
fingers
Recognition Domain
Functional
Domain
triplets
3-finger ZFP
Zinc Finger DNA
Binding Protein
(ZFP)
• Activation
• Repression
• Cleavage
9bp target site
A deep archive of “finger”
modules allow designs that bind
to almost any sequence
Sequence-specific recognition domains
coupled to functional domains mediate
effects at the intended target
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ZFP Design in a Nutshell
ZFsare
areprotein
proteindomains
domainswhich
which
ZFs
bindto
tospecific
specific33base
basepair
pairDNA
DNA
bind
sequences. Altering the amino acid sequence
sequences.
Altering the amino acid sequence
inthe
theZF
ZFchanges
changesits
itsDNA
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binding
in
specificity.
specificity.
seriesof
ofzinc
zincfinger
fingerprotein
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AAseries
domainsare
arecustom
customengineered
engineered
domains
tobind
bindto
toaatarget
targetDNA
DNAsequence.
sequence.
to
Theaddition
additionof
ofaafunctional
functional
The
domainprovides
providesspecific
specificDNA
DNA
domain
modificationfeatures.
features.
modification
Functional Domain
Recognition Domain
Cleavage
Cleavage
Activation
Activation
Repression
Repression
6-finger protein creates an 18 base pair recognition module
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Designed ZF-Nucleases Mediate Sequence Modification
Dimeric FokI nuclease domain induces
double stranded breaks
DNA repair mechanisms are invoked
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Example : Targeted Gene Disruption in Corn
Targeted Mutagenesis of ZmIPK1
Direct DNA delivery
Cell culture
gDNA amplification
Ultra-deep sequencing
yeast cell SSA MelI
Shukla et al. Nature 2009
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Example : Targeted Gene Addition in Corn
Direct DNA co-delivery
ZFN plus donor
Cell culture
Selection
Genotyping/Sequencing
Shukla et al. Nature 2009
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Example > Stable, Heritable Trait Engineering
T1/F1 Segregating Plants
1
1 event
T0
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19
20
TI ZmIPK1
WT ZmIPK1
Herbicide (PAT)
selection comes
from the donor
418-8
Multiple lines
T0
I
2
X
418-6 X 5XH751
T2/F2 Segregating Seeds
I
I
I
Self or outcross
T1 / F1
Herbicide tolerance and reduced phytate
Shukla et al. Nature 2009
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EXZACTTM Add: Precision Insertion > > Trait Stacking
AAD
DHT
HT
•
•
•
gene 1
cen
DHT
HT
gene 2
“molecular stacks” at a single
locus
gene 3
cen
DHT
HT
•Endogenous loci act as “landing pads” for
targeted insertion of multiple trait genes
•Loci may be
modified via KO
neutral (potential safe harbors)
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Research Program Key Findings
• The ZFP design platform is robust
and highly specific. ZFPs can be
designed and validated to bind to
almost any sequence.
• Because plant genomes are
complex and highly redundant, a
priori knowledge of target gene
sequence and genome
representation is critical.
• Expression of ZFNs is necessary
and sufficient to drive highfrequency targeted gene disruption,
editing and gene addition.
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Applications of EXZACTTM: the Toolkit
EXZACT  Technology provides multiple methods to
improve crops through precise genome modification:
EXZACT  Add
EXZACT  Delete
EXZACT  Edit
Targets a specific DNA sequence and precisely adds genes/traits
Single-gene traits, stacking, pathway engineering
Improves crops by removing undesirable plant traits.
Gene/sequence removal or functional knockout
Rewrites gene sequences to produce novel crops and products
Point mutations, small deletions / additions
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Represents a New Way to Develop Biotech Products
Targeted Mutagenesis / non-GMO
Targeted Trait Insertion / GMO
• End-product is
indistinguishable from mutation
breeding
• Target trait/gene to a specific
genetic locus
• Insert multiple traits/genes at
one locus
• More efficient generation of
desired GMO events
• Target DNA to location of
current de-regulated event or
‘safe’ locus
• GMO events with no disruption
of native gene function
• Desired mutations at intended,
specific sequences
• More efficient screening and
breeding
• No foreign DNA will be present
in the genome (considered a
conventional variety)
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Shaping the Future of Agriculture with EXZACTTM
Technology
Deployment
DAS Traits
Early-Stage
Discovery
Acceleration
Stacking
Trait CoDev
Partnerships
New Trait
Opportunities
Novel
Gene/Trait
Discovery
Accelerated Trait Development
Expanded Range of Traits
Novel Traits
Technology
Enhancement
Ongoing DAS
Research
Program
Sponsored
Academic
Research
Continuing
Sangamo
Research
Continuous Improvement
Push Technical Limits
Novel Applications
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