Using Zinc Finger Nucleases to
Manipulate the Mammalian Genome
Matthew Porteus
UT Southwestern Medical Center
Depts. of Pediatrics and Biochemistry
Dallas, TX
Gene Targeting is a Precise
Recombination Event
Definition: Gene targeting is the replacement of
genomic DNA with exogenous DNA by homologous
recombination.
Commonly Used For Experimental Purposes in
certain cell types (yeast, chicken DT40 cells, murine
embryonic stem cells)
In addition to its usefulness for mammalian somatic cell
genetics, it could also be an ideal way to treat genetic
diseases.
GFP Gene Targeting System
37GFP-IRES-CD8
CMV-Sce
37GFP-IRES-CD8
CMV/CBA-GFP*-IRES-CD8a-PGK-Neo
Stop-Sc
Stop-SceeSite
Site
DSB-Induced Gene Targeting
CMV/CBA-GFP-IRES-CD8a-PGK-Neo
Optimized Rate=3-5%
(30-50,000 events per million transfected cells)
Porteus and Baltimore (2003)
Two Components for
DSB-Induced Homologous
Recombination
1. Repair Substrate: Fragment of DNA that serves as
template for repair of DSB by homologous
recombination.
2. Nuclease: Enzyme to create DSB in target gene.
Schema of DSB-Induced Gene Conversion
S
Undamaged DNA (Allele S)
DSB Created (spontaneous
or induced, e.g. by ZFN )
Strand Invasion into Undamaged
Homologous DNA (Allele A)
A
In gene targeting exogenous DNA serves as
homologous DNA donor.
Repairing of original strands of DNA. Gaps
filled by DNA polymerase and nicks sealed
by DNA ligase.
A
Conversion of Blue Allele(“S”) into Red
Allele (“A”) in region of DSB
Endogenous Genes Do Not
have Recognition Sites for
Homing Endonucleases
1. Modify Homing Endonucleases to Recognize
new target sites.
2. Use Zinc Finger Nucleases
Zinc Finger Nucleases as Potential Reagents to
Create Double-Strand Breaks in Normal Genes
FokI nuclease
domain (Fn)
FokI nuclease
domain (Fn)
Initially developed by labs of Srinivasan Chandrasegaran (Johns Hopkins)
and Dana Carroll (Univ. Utah)
ZFN Full Site/Sce Site
GFP*
CMV/CBA
IRES
tGFP
G
WRE
Sce or ZFN
Expression
Plasmid
GFP Donor
CMV/CBA
CD8
FP
IRES
CD8
WRE
tGFP
CMV/CBA
GFP
IRES
CD8
WRE
Model Zinc Finger Nucleases
Stimulate Gene Targeting
7000
6000
5000
4000
3000
2000
1000
0
6
Zif Site
QQR Site
4150
4785
Sce
Porteus and Baltimore (2003)
Sce Site
14
53
QQRL0
Zif
QQRL0/Zif
Continuous Expression of ZFNs
causes Cytotoxicity
Relative Rate of
Gene Targeting
Time Course of Gene Targeting Using Sce
2.0
1.5
1.0
0.5
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Time Course of Gene Targeting Using Zinc Finger Nucleases
6000
5000
4000
3000
2000
1000
0
QQRLO-CN/Zif-CN with
Target QQR/Zif
QQRLO-CN with Target
QQR6
0
5
10
15
Can we design a pair of zinc
finger nucleases to stimulate
gene targeting in a real gene
in human somatic cells?
Zinc Fingers Bind Triplets
FokI nuclease
domain (Fn)
FokI nuclease
domain (Fn)
How to assemble a new zinc
finger protein?
1. By altering the contact residues one can alter the target
triplet.
2. By mixing different fingers one can assemble a zinc finger
protein with new target site specificity.
3. Theoretically if one had zinc fingers for all 64 possible
triplets one could assemble a zinc finger protein to
recognize any sequence.

Zinc fingers have been published that recognize all 16
GNN,ANN, CNN triplets.

But, the GNN fingers are best.
Can we assemble a pair of zinc
finger nucleases to stimulate
gene targeting?
Full Site Consensus Sequence
5’ nnCnnCnnCnnnnnnGnnGnnGnn 3’
(GNNGNNGNN inverted repeat separated by 6 bp)
Such a sequence occurs in both GFP (twice) and CD8
Lucky, eh?
Empiric Design of
Zinc Finger
Nucleases
(assembly
approach)
From Liu et al. (2002)
Gene Targeting with Zinc Finger
Nucleases to GFP
Fn
GFPZF2
5’ acC atC ttC ttc aag Gac Gac Ggc aac stop-Sce site tac
3’ tgG taG aaG aag ttc Cgc Ctg Ccg ttc
GFPZF1
Fn
Finger1
Finger2
Finger3
GFPZFN-1
QSSHLTR
(ggt)
TRGNLVR
(gat)
QSGNLAR
(gaa)
GFPZFN-2
DRSHLTR
(ggc)
DRSNLTR
(gac)
DRSNLTR
(gac)
GFP ZFN Site
Sce Site
GFP*
CMV/CBA
IRES
tGFP
G
WRE
Sce or ZFN
Expression
Plasmid
GFP Donor
CMV/CBA
CD8
FP
IRES
CD8
WRE
tGFP
CMV/CBA
GFP
IRES
CD8
WRE
Gene Targeting with Zinc
Finger Nucleases to GFP
GFP Positive Cells per
Million Transfected Cells
6000
4543
5000
4000
3000
2000
1071
1000
0
Sce
Sce
GFP-CN
GFPZF1-Fn
GFPZF2-Fn
CD8 Knockout Using Zinc
Finger Nucleases
Fn
CD8ZF2
bp 441 5’acc ggCgcCcaC catcgc GtcGcaGcc ctg 3’ bp 471
tgg ccGcgGgtG gtagcg CagCgtCgg gac
CD8ZF1
Fn
Knockout of CD8 transgene Using
CD8 Zinc Finger Nucleases
Cell Line
CD8
Knockout
Plasmid
CD8
Knockout
Plasmid
+
CD8 ZFNs
CMV/CBA
GFP*
IRES
CD8
16% CD8 Negative
M1
M1
80% CD8 Negative
85% CD8
Positive
10/10 clones
CD8+
0/12 clones
CD8+
Demonstrates in Principle
1. Can make somatic cell knock-outs with
ZFNs
2. Can do targeted transgenesis with ZFNs.
i.e. Substitute gene of interest for
selectable marker (or both. . .) and
insert into pre-selected, “safe” and
“permissive” genomic location.
How far from the site of the
break can you get targeting?
Co-conversion of Markers by Gene Targeting
37GFP-IRES-Puro-IRES-CD8
CMV-Sce
37GFP-IRES-Puro-IRES-CD8
CMV/CBA-GFP*-IRES-CD8a-PGK-Neo
Stop-Sc
Stop-SceeSite
Site
Frequency of Co-Conversion using DSB Mediated
Gene Targeting
CMV/CBA-GFP-IRES-Puro-IRES-CD8a-PGK-Neo
% GFP + Cells
Total GFP + Cells
Day 3
(no puro)
0.041%
1200
% GFP + Cells
Total GFP + Cells
0.013%
405
Day17
(puro selection)
58%
66
Fold Change
17%
2
1200
(-200)
1400
(-18)
Demonstrates:
1. Can get targeting at a distance (up to 400
bp) from site of DSB (at a price).
2. Can do co-conversion
i.e. Correct mutation at one location and
insert gene that confers selective
advantage nearby.
Can we design zinc finger
nucleases to stimulate gene
targeting in a gene that causes
human disease?
Collaboration with Sangamo Biosciences (Richmond, CA)
Human Interleukin-2 Receptor
Common Gamma Chain Deficiency
(IL2RG)
1.
2.
3.
4.
5.
Part of Receptor Complex for IL-2, IL-4, IL-7, IL-9, IL-15, IL-21. . .
On X-chromosome
Mutations in which are the most common cause of SCID (severe combined
immunodeficiency)
-25% of mutations lie in Exon 5.
Selective Advantage for corrected cells.
Treatment
-Bone Marrow Transplantation
: Allogeneic (sibling)
: Haploidentical (parent)
-Gene Therapy
: Alain Fischer trial in France
: Ooops, leukemia.
ZFN Gene Correction
at the IL2RG gene
IL2RG ZFN-R
5’CTACACGTTTCGTGTTCGGAGCCGCTTTAACCCACTCTGTGGAAGTGCTC 3’
3’GATGTGCAAAGCACAAGCCTCGGCGAAATTGGGTGAGACACCTTCACGAG 5’
IL2RG ZFN-L
GFP Gene Targeting Reporter for IL2RG ZFNs
5’ GFP
IL2RG site
Target site of GFP ZFNs
Sce site
3’ GFP
GFP Positive Cells per
Million Transfected Cells
Stimulation of Gene Targeting Using ZFNs
for the IL2RG Gene
2500
1968
2000
1500
715
1000
500
0
5-8L0/5-9L0
IL2RG
ZFN-L
IL2RG ZFN-R
M16/M17
GFP
ZFNs
GFP Positive Cells per
Million Transfected Cells
Optimization of IL2RG ZFN-L
5000
3892
4500
4000
2897
3500
3000
2500
2000
1276
1500
1000
500
0
IL2RG ZFN-R
IL2RG ZFN-L
IL2RG ZFN-R
IL2RG ZFN-R
IL2RG ZFN-L(D) IL2RG ZFN-L(G)
GFP Positive Cells per
Million Transfected Cells
Optimization of cgc ZFN-R
5000
4420
4500
4000
3500
3000
2943
2940
2689
2656
2500
1937
2000
1500
1000
500
0
1
2
cgc ZFN-LG
cgc ZFN-LG
cgc ZFN-R cgc ZFN-R
(A)
(B)
3
cgc ZFN-LG
cgc ZFN-R
(C)
4
cgc ZFN-LG
cgc ZFN-R
(D)
cgc 5ZFN-LG
cgc ZFN-R
(E)
cgc6ZFN-LG
cgc ZFN-R
GFP Positive Cells per
Million Transfected Cells
4-Finger Zinc Finger Nucleases Seem
to Have Less Cytotoxicity
6000
5000
4000
3000
2000
1000
0
Day3
Day5
Day7
Experimental Design to
Detect Targeting at
Endogenous IL2RG Locus
1. Transfect K562 cells with IL2RG ZFNs with
repair substrate that contains BsrBI
polymorphism.
2. Isolate individual clones (no selection).
3. Expand individual clones (no selection).
4. Harvest genomic DNA from individual clones.
5. Analyze genomic DNA for BsrBI
polymorphism.
Bi-Allelic Targeting in Human Somatic Cells
(K562)
bB BB
bB
bB
bB bB
BBBB
bB
Total clones:
Corrected:
bB:
BB:
bB
BB
BB
bBbB
76
14 (18%)
9 (11.5%)
5 (6.5%)
Future Directions
1. Design ZFNs to other target genes.
2. Develop efficient method to make specific
ZFNs that recognize a broad range of
sequences.
3. Refine ZFNs for use in primary cells, including
stem cells.
4. Assess possible induction of genomic
rearrangements by ZFNs.
I.
Eliminate
II. Use as a tool to study sequence specific
DSBs in genetic instability.
5. Develop as a therapeutic tool.
Potential Applications to
Aging Research
1. Audience will be more clever than I.
2. Use as an experimental tool to study genetics
of aging in mammalian cells.
3. Create allele specific gene variants in stem
cells that are associated with slower “aging.”
Thank You
UT Southwestern
Patrick Connelly
Ruth Ebangit
Brian Ellis
Shondra Pruett
Kimberly Wilson
Sangamo Biosciences
Fyodor Urnov
Michael Holmes
Jeff Miller
Philip Gregory
Casey Case
Funding
Burroughs-Wellcome Fund Career Development Award
NIH Career Development Award
UT Southwestern Medical Center