polydactyl zinc finger proteins: software and hardware for
TRANSCRIPT
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 1
1
Polydactyl Zinc
Finger Proteins:
Software and Hardware
for Genomes
Carlos F. Barbas, III
Kellogg Professor
Departments of Chemistry
and Molecular Biology
The Scripps Research Institute
La Jolla, CA 92037
USA
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Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 2
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Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 3
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Polydactyl Zinc Finger proteins:
software and hardware for the genome
• Development of a universal system for gene regulation
• Targeted endogenous gene regulation in cells
and organisms
• Transcription factor l ibraries - a powerful new strategy
• The future: cutting and pasting genes and genomes –
zinc finger enzymes and designer epigenetic modifications
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A diversity of recognition domains
EcoRV
Leucine zipper
C4 class
Helix-turn-helix
C2H2 zinc finger protein
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The Zinc Finger domain
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 4
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N
C
Zif268
a Cys2-His2 Zinc Finger protein
11
-1 3
6
3'
mid
5'
12
Life in the major groove
Life in the major groove
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 5
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A complete Zinc Finger code
requires 64 defined domains
GNN
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Phage display:
an efficient format for directed molecular evolution
Phage display:
an efficient format for directed molecular evolution
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Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 6
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Results of phage selections of the finger 2 of Zif268
Stabilizing the fold
5’ 3’
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Molecular recognition in the GNN family of Zifs
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 7
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Exquisite sequence specificity of Zinc Finger
domains… only half from selections!
Refinement or De Novo design of Fingers is Required for ~50%
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Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 8
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3-finger proteins
are highly specific
A view of 80 proteins
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Three fingers… 9 bp recognition
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The specificity Issue and genome size
Humans
Corn
E. coli
3.5 x 109 bp
5 x 109 bp
4 x 106 bp
Number of zinc
finger domains
3
4
5
6
Recognition
sequence
9 bp
12 bp
15 bp
18 bp
Specificity
49, 2.6 x 105
412, 1.7 x 107
415, 1.1 x 109
418, 6.9 x 1010
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 9
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A 6-finger protein can specify a unique site
in the human genome
3 + 3 + 3 + 3 + 3 + 3 = 18 nucleotides
human genome = 3 x 109 nucleotides
418 = 7 x 1010 addressable sequences
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Genomic specificity requires polydactyl proteins
• A 3-finger zinc finger proteins binds ~10,000 genomic sites
• A 6-finger zinc finger proteins binds ~1 genomic site
273Fn16 (-289) 6Fn289 (-269)
Non-additive linkage effects
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 10
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Polydactyl proteins:
structure of the first 6-finger protein Aart
Nancy Horton, U. of Arizona; JMB 2006, 363, 405
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Frequency of 6xGNN and 6xRNN sites
in promoter sequences
►Transcription start
►Translation start
● 6xGNN: 11
● 6xRNN: 77
● 6xGNN: 7
● 6xRnn: 94
erbB-2 promoter
erbB-3 promoter
Frequency of 6xGNN and 6xRNN sites
in promoter sequences
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Alignment of erbB-2 target sequence
with erbB-3 5’-UTR
AGCCATGGGGCCGGAGCCGCAGTGAGCACC
GCAATCGGAGCCGGAGCCGGAGTCCGGGGA
-30 -1
-135 -164
erbB-2
erbB-3
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 11
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Bicistronic retroviral vector for co-expression
of Zinc Finger proteins and GFP
DNA
mRNA AAAAAA
5’ LTR 3’ LTRGFPZinc finger protein IRES
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Specific regulation of two oncogenes in A431 cells
surface protein levels
ErbB-1 ErbB-2 ErbB-3
KRAB
KRAB
VP16
VP16
ErbB-2 target site:
GGg GCC GGA GCC GcA GTg
Beerli et al., (2000) PNAS 97:1495
ErbB-3 target site: GGa GCC GGA GCC GgA GTc
Cell n
um
ber
Cell n
um
ber“functional
knockouts” !
Specific regulation of two oncogenes in A431 cells
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 12
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Prot ein Target Target Sequence Kd, nM
E2C e2c GGg GCC GGA GCC GcA GTg 0.75
E2C e3 GGa GCC GGA GCC GgA GTc 10
E3 e2c GGg GCC GGA GCC GcA GTc 10
E3 e3 GGa GCC GGA GCC GgA GTc 0.35
Affinities of E2C and E3 Six Finger Proteins
Ultra-high affinity will lead to transcriptional control of non-target genes
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Regulation of fetal hemoglobin
for sickle cell therapy
Goal: repress HbS and upregulate HbF
gg1 GTC AAG GCA AGG CTG GCC 0.7 nM
+ transcriptional activator
No regulation
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Efficient and specific γ-globin gene regulation
in retrovirally transduced K562 pools
Intra-cellular staining
FACS analysis
PE-Height
Counts
▬ K562 (unspecific IgG1-PE isotype AB)
▬ K562 (anti-HbF specific AB)
▬ Gg1-VP64 cells (GFP+)
▬ Gg1_KRAB cells (GFP+)
hemoglobin
Visualization
gg1-KRAB K562 gg1-VP64
Efficient and specific γ-globin gene regulation
in retrovirally transduced K562 pools
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 13
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Inhibition of HIV replication in primary
T lymphocytes by Zif-Krab
p24 (ng/m
l)
days post infection
Primary blood leukocytes (PBLs)
HIV strain 89.6(dual tropic)
Control
SKD-HLTR3
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Zinc Finger transcription factors in Planta
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Wild Type Flower
sepe
st
ca
AP1::VP64-AP3
Ap3 Activator
AP1::SID-AP3
Ap3 Repressor
Activation of Ap3 in sepals causes
partial sepal → petal transformation
Repression of Ap3 in petals causes
partial petal → sepal transformation
VP64-
SID-
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 14
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Creating cell and organism libraries
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Transcription factors libraries
• Activate or repress every gene within a genome
• Create diverse cellular l ibraries
• Modulate multiple pathways to create interesting phenotypes
• Create diverse libraries of organisms with gain of function
and loss of function mutations
Transcription factors libraries
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Transcription factor libraries:
every gene on or up, every gene off
Gene x
Gene y
Gene z
Gene x
Gene y
Gene z
Regulate genes Regulate genes
Assemble
into TF sZF
Assemble
into TF sZF
Repertoire of ZF
DNA-binding domains
3ZF proteins 6ZF proteins
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 15
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TFZF selections in vivo: looking for new genes
involved in oncogenesis and/or tumor suppression
and metastasis in mouse models
Infect C8161 cells with pMX-3ZF-VP library
Heterogeneous tumor cell population
(106 cells) Inject in mouse (I.V, sc)
Growth and selection occurs in the host
Remove primary tumor
and organs (metastasis)
PCR ZFs
from genomic
DNA & re-clone
In pMXVP64
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Cellular phenotypes induced by TF-ZF selected
from functional screens
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TFZF 20 promotes cell invasion in vitro and in vivo
Comparison of Cell Invasion in Vitro
0
20
40
60
80
100
120
140
160
Control Hela Hela+#20 ZF
# cells per field 6ZF–20–VP
HeLa
Number of lung metastasis
ControlpMXVP
#20VP0
10
15
20
25
30
1
2
3
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6
7
5
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 16
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Specific regulation of E48 antigen in TF-20VP
expressing cells and tumors
Flow cytometry
FL2-Height
counts
■ HeLa
▬ IRES
▬ 20-VP
▬ 20-SKD
▬ 20-VP-/PE
0
100
200
300
400
500
600
700
800
900
DNA Arrays
Specific regulation of E48 antigen in TF-20VP
expressing cells and tumors
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Transcription factors libraries:
opportunities in synthetic biology
• Create and screen complex phenotypes
• 3-finger transcriptions factors might be selected to regulate
multiple genes leading to interesting phenotypes
*3-finger transcription factors hit as many as 10,000 sites in the genome
• Reroute complex biosynthetic pathways: turn some genes
on and others off to create efficient syntheses
• Create novel productive cell types
• Select plants for disease resistance, salt tolerance, increased
yield, and other interesting phenotypes
48Plant J; 2006 Nov; 48 (3):475-83
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 17
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Chemical regulation of transgenes
and endogenous genes
• Tetracycline system- tetracycline analogs
• Estrogen receptor system- tamoxifen
• Progesterone receptor system- RU486
• Ecdysone receptor system- ecdysone and analogs
• Designed chemical complementation approach
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Induction of angiogenesis in a mouse model
using engineered transcription factors
Phase I clinical trial in August 2004 for the treatment of intermittent
claudication and a Phase I study in June 2005 for the treatment
of the more severe form of PAD, critical limb ischemia; A Phase Ib
clinical trial of in subjects with diabetic neuropathy in late 2005;
Currently in Phase II studies
Sangamo Biosciences Inc.
Nat Med; 2002 Dec;8(12):1427-32
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Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 18
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Zinc Finger nucleases: scissors for genome surgery
Applications:Applications:
• Gene correction (viaHR using a donor carrying a corrected gene)
• Gene disruption (viaHR using donor with a mutated gene or viamutagenesis NHEJ)
• Targeted integration (viaHR using donor with a inserted gene)
Zinc Finger replaced the DNA
binding domain of FokI
Figures from: Nature 2005 Jun
2;435(7042):646-51
Homology-directed repair
ZFN-driven homology-directed repair
X-ray induced DSB
ZFN-induced DSB
Sister chromatid Donor DNA (plasmid)
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Integration and gene disruptionSeamless gene exchange: recombinase
mediated cassette exchange (RMCE)
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Selected Serine recombinase crystal structures
• Yang W, Steitz TA; Crystal structure of the site-specific recombi nase
gamma delta resolvase complexed with a 34 bp cleavag e site
Cell; 1995 Jul 28;82(2):193- 207
• Li W, Kamtekar S, Xiong Y, Sarkis GJ, Grindley ND, Steitz TA; Structure
of a synaptic gamma delta resolvase tetramer covalentl y linked to two
cleaved DNAs
Science; 2005 Aug 19;309( 5738):1210- 5
• Kamtekar S, Ho RS, Cocco MJ, Li W, Wenwieser SV, Boocock MR,
Grindley ND, Steitz TA; Implicati ons of structures of synaptic tetramer s
of gamma delta resolvase for the mechanism of recombi nati on
Proc Natl Acad Sci U S A; 2006 Jul 11;103(28):10642- 7
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 19
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Recombinases: a powerful enzyme class
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4321098765432112345678901234
TGTCTGATAATTTATAATATTTCGAACG
F1
F2
F3
F1 F2 F3 F3 F2 F1
987654321-9bp-11-9bp-123456789
CCCCGCCCC-----AT-----GGGGCGGGG
Short linker
F3 F2 F1
123456789
GGGGCGGGG
γδResolvase Zif268
Zinc Finger - recombinase (RecZF) design
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Zinc Finger - recombinase (RecZF) evolution
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 20
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Zinc Finger - recombinase (RecZF) evolution
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Selective recombination
ZFs target catalytic function to corresponding sites
H1 = GGA GGC GTG
P2 = GCA GTG GCG
Inv ersion assay Resolution assay
F1 F2 F3 F3 F2 F1987654321-9bp-11-9bp-123456789CACGCCTCC-----AT-----GGAGGCGTGGTGCGGAGG-----TA-----CCTCCGCAC
F1 F2 F3 F3 F2 F1987654321-9bp-11-9bp-123456789CGCCACTGC-----AT-----GCAGTGGCGGCGGTGACG-----TA-----CGTCACCGC
ZFs
Sub H1 Sub P2
P2P2
H1H1
H1P2
P2H1
ZF:ZF:
BB
P2P2
H1H1
H1P2
P2H1
ZF:ZF:
BB
Selective recombination
ZFs target catalytic function to corresponding sites
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Selective gene excision in human cells
Gordley et al., J Mol Biol (2007)
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 21
•• Limitations of gene deliveryLimitations of gene delivery
–– Immunog eni c viral vectorsImmunog eni c viral vectors
–– Inefficient gene targetingInefficient gene targeting
•• Nonviral gene deliveryNonviral gene delivery
–– Phage integrasesPhage integrases
–– TransposasesTransposases
•• Gene targetingGene targeting
–– Homologous recombinati onHomologous recombinati on
–– SiteSite--specific recombinasesspecific recombinases
•• Cre/LoxPCre/LoxP
•• Flp/FRTFlp/FRT
•• Programmable recombinases to edit Programmable recombinases to edit
the endogenous human genomethe endogenous human genome
Gene delivery vehicles
++
Targeted integration into ErbB2 locus
IntegrationIntegration--specificspecific
PCR of genomic DNAPCR of genomic DNA
• Targeted integration into a natural site
in the human genome
• Ongoing work
– Quantificati on of ErbB2 protein levels
– Phenotypi c assays
– Cancer model & therapy
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Designer recombinase applications
• Gene Therapy
• Knockout of harmful genes (by excision or integration)
• Regulation of endogenous proteins (promoter modification)
• Allele replacement
• Enable Personalized Stem Cell Therapies
• Biotechnology
• Facilitated generation of transgenic organisms (ES cells)
• Marker excision (from plants)
• Genome modification without exogenous DNA
• Research
• Experiment al genetics in model organisms
• Synthetic biology
• Tools for molecular biology
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 22
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Epigenetic control: heritable repression of gene
expression by CpG methylation
Razin A, (1998) EMBO J. 17, 4905-8
Transient repression Stable repression
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Reassembly of M.HhaI
Split C-domain Split N-domain
Intact
Choe, W. et al., Biochem. Biophys. Res. Commun. (2005)
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In vivo site-specific DNA methylation
with a designed sequence-enabled DNA
methylase
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 23
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Targeted methylation at only 1 of 19 HhaI sites by the sequence assembled methylase
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Sequence-enabled methylase provides
for exquisite site-specificity as compared
to simple M.HhaI-ZF fusions
HS2 zf HS1 zf
HS2 zfIntact
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N
N NH2
O
H
Site-specific DNA modification by DNA methyltransferase:
sequence specific organic chemistry for pattern
generation and Nano-construction
MTase
cytosine
Lukinavicius, G. et al., J.Am. Chem. Soc. (2007)
N
N NH2
O
HN
O
H2N
N
NN
N
NH 2
O
HOHHH
S
CO OHH2 N
NH
O
H 2 N
H H
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 24
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Opportunities in Nano-construction
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Expression Vectors encoding DNA binding proteins
and transcription factors in under 2 w eeks…
as fast as you can make a synthetic gene
you can make a synthetic TF or enzyme
w ww.zincfingertools.org
Transcription factor and enzyme design
now automated
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Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 25
73
Zinc Finger technology
• Zinc finger proteins binding 9- and 18-bp DNA target sites with high
affinity and specificity can be rapidly prepared using predefined zinc
finger “modules”
• Domains binding 5’-GNN-3’ 5’-ANN-3’ and 5’-CNN-3’ sites prov ide
ready access to 466 or 9.5 billion nov el proteins that bind DNA
sequences with the structure 5’-VNN VNN VNN VNN VNN VNN-3’
• One 5’-([A/C/G]NN)6-3’ site should be present ev ery 3 nucleotides
therefore this technology can target any gene
• Any laboratory can build their own custom DNA-binding protein
today; endogenous genes can also be chemically regulated
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Zinc Finger technology… or RNAi?
Complementary approaches
Zifs provide:
• Activation - graded if desired
• Repression - graded if desired
• Wide variety of compatible chemistries
for regulated expression
• Linked transcription factors provide for the regulation
of multiple genes
• Opportunities to design networks of regulated genes
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• Roger R. Beerli
• Russell Gordley
• David Segal
• Pilar Blancafort
• Birgit Dreier
• Scott Eberhardy
• Karin Effertz
• Torbjorn Graslund
• Charles Gersbach
• Xuelin Li
• Caren V. Lund
• Laurent Magnenat
• Jeff Mandell
• Beatriz Gonzalez
• Justin T. Stege
• Beate Koksch
• Ulrich Schopfer
• Roberta Fuller
Acknowledgments
The Scripps
Research Institutecarlos@scripps. edu
Polydactyl Zinc Finger Proteins:
Software and Hardware for Genomes
Prof. Carlos F. Barbas, III
The screen versions of these slides have full details of copyright and acknowledgements 26
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Anti-HIV Strategies
Joao Goncal ves- URIA-Centro de Patogenese Molecular
Bruce Torbett- Scripps
Christina Swan- Scripps
Regulation of Plant Genes
Roger Beachy and Isabel Ordiz - Donald Danforth Center
Steve Briggs and Xuen Guan-Torr ey Mesa Research Institute, Syngenta
Directing HIV-1 Integrase
Samson Chow- UCLA School of Medicine
Structures of 6-finger Proteins
Nancy Horton- Arizona
David Segal- UC Davis
Our collaborators
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Sangamo Biosciences
Johnson & Johnson
Novartis
Syngenta/Diversa
Licensees of our Zif technology
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