Download Engineering Programmable Nucleases: Applications in the Study of

Engineering Programmable
Nucleases: Applications in the
Study of Gene Function and
eventually Gene Therapy
Scot Wolfe
Molecular, Cell and Cancer Biology
UMass Medical School
Frontiers in Science Presentation
March 18th, 2015
One of our fundamental areas of exploration is the
development of tools for the targeted engineering
of genomes.
One of our fundamental areas of exploration is the
development of tools for the targeted engineering
of genomes.
In analogy with surgical tools - we endeavor to
make genomic scalpels that permit the precise
modification of the genome.
One of our fundamental areas of exploration is the
development of tools for the targeted engineering
of genomes.
In analogy with surgical tools - we endeavor to
make genomic scalpels that permit the precise
modification of the genome.
These tools have utility for:
1) The study of gene function during vertebrate
development
2) The correction of disease-causing genetic
lesions
How do we locate a specific “address” within the
genome?
Gene X
How do we locate a specific “address” within the
genome?
Gene X
How do we locate a specific “address” within the
genome?
Gene X
-TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA-AGCAAGCCGAGCGCAATTCGTTCACGTCTT-
How do we locate a specific “address” within the
genome?
Gene X
-TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA-AGCAAGCCGAGCGCAATTCGTTCACGTCTTSequence-specific
DNA recognition module
How do we locate a specific “address” within the
genome?
Gene X
-TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA-AGCAAGCCGAGCGCAATTCGTTCACGTCTTSequence-specific
DNA recognition module
Where do we begin? - By understanding how
naturally-occurring DNA-binding proteins locate their
sequences.
Cys2His2 Zinc Finger proteins (ZFPs) are the most
diverse family of DNA-binding domains in
vertebrates
Cys2His2 Zinc Finger proteins (ZFPs) are the most
diverse family of DNA-binding domains in
vertebrates
How can we
understand
DNA
recognition
by these
proteins?
A bacterial one-hybrid system can be used to
determine DNA-binding specificity
RNA polymerase
Zinc Finger Protein
ZFP
Weak
Promoter
Reporter
Vector
Bacterial
Selection
Strain
ΔrpoZ
ΔhisB
ΔpyrF
A bacterial one-hybrid system can be used to
determine DNA-binding specificity
RNA polymerase
Zinc Finger Protein
ZFP
Match
Reporter
Vector
Bacterial
Selection
Strain
ΔrpoZ
ΔhisB
ΔpyrF
Weak
Promoter
A bacterial one-hybrid system can be used to
determine DNA-binding specificity
RNA polymerase
Zinc Finger Protein
ZFP
Match
Weak
Promoter
Reporter
Vector
Bacterial
Selection
Strain
ΔrpoZ
ΔhisB
ΔpyrF
ZFP
ZFP
ZFP
ZFP
Noyes et al. Nucleic Acids Research, 2008, 36, 2547-2560.
DNA binding site selections are performed in a single round to yield
interacting partners
Overview of a B1H binding site selection
pol-ZFP
DNA binding site selections are performed in a single round to yield
interacting partners
Overview of a B1H binding site selection
pol-ZFP
DNA binding site selections are performed in a single round to yield
interacting partners
Overview of a B1H binding site selection
pol-ZFP
DNA binding site selections are performed in a single round to yield
interacting partners
Overview of a B1H binding site selection
pol-ZFP
SP &
KLF
M4
ZFA
sr
lo
l
p h a -P
O
o
CG3065 F1-5
1
4
ph
6
8
ol
lol
PL
a- A
P
2
al
78
lo
31
G
C
19
3
8
CG
9B
D1
f
vl
PN
lolaY
lola-P
71
CG110
lola-PK
Cf2-PB
kr
ttk-PA
pad
lolaPD
crolF716
CG
736
8
l(3)
neo
CG
38
sa 1 2 2 3
lr
6 -P
ttk -F3B
5
ab -PF
-P
B
5-7
peb-F
o vo
fru
PE
r
bPC
b rPL
b rhb
C
ZI
A
SN
T
CR
&S
br
-P
A
-P
F
od
d
so
b
bo
w
l
lola
-PU
lola
-PT
la
lola-PJ
lola-PW
chinmo
peb-F1-3
36-PA
lola-PQ
CG122
CG14
her
962
lo
wor
7181
CG1
sn a
SP
AL
T
d
lm g
su a
op
3 -5
ci
g F 05
es
6
12
CG
t
scr
E
-P 7
ab 26
10
CG
&
O
S D
k
ipp D
ed
T
Cf2-PA
lola-PP
HN
OVO
ZEP
CG7928
shn-F1-2
Aef1
LI
G
DIS
CO
p-
FEZ
PC
2
ke
P
P I
D1 G
9A
se
ns
2
sen
disc
s
o -rF12
disc
o
CG3
407
CG31
670
CG4854
CG4360-F1-3
a-
gl
n
GF
I
a-
a-
sq z
rn
52
20
C G 1 -9
-F
jim
lol
lol
lol
0
YY
1
CG5669
Sp1
btd
bteb2
hkb
CG
120
29
CG
9
lun 895
a
klu
BL
IM
P1
EG
R
im
Bl
We have
determined
the DNAbinding
specificity of
more than
100 naturallyoccurring
zinc finger
proteins
(ZFPs) and
many more
synthetic
ZFPs
Enuameh, et al. Genome Research 2013
SP &
KLF
M4
ZFA
sr
lo
l
p h a -P
O
o
CG3065 F1-5
3
2 1
lola-PJ
lola-PW
chinmo
peb-F1-3
36-PA
lola-PQ
CG122
CG14
wor
7181
CG1
sn a
3’
her
962
lo
la
M 3
DNA triplet
3’
SP
AL
T
br
-P
A
-P
F
od
d
so
b
bo
w
l
lola
-PU
lola
-PT
5’ 5
kr
ttk-PA
pad
lolaPD
crolF716
CG
736
8
l(3)
neo
CG
38
sa 1 2 2 3
lr
6 -P
ttk -F3B
5
ab -PF
-P
B
d
lm g
su a
op
3 -5
ci
g F 05
es
6
12
CG
t
scr
E
-P 7
ab 26
10
CG
PL
a- A
P
2
al
78
lo
31
G
C
19
3
8
CG
9B
D1
f
vl
PN
lolaY
lola-P
71
CG110
lola-PK
CG7928
shn-F1-2
5’
T
CR
&S
Cf2-PA
lola-PP
5-7
peb-F
o vo
fru
PE
r
bPC
b rPL
b rhb
-1
lol
ZEP
6
5
ol
A
SN
8
Cf2-PB
O
S D
k
ipp D
ed
T
ph
6
Aef1
HN
OVO
CG5669
Sp1
btd
bteb2
hkb
CG
120
29
CG
9
lun 895
a
klu
4
C
ZI
GF
I
2
1
DIS
CO
p-
n
FEZ
PC
ke
P
P I
D1 G
9A
se
ns
2
sen
disc
s
o -rF12
disc
o
CG3
407
CG31
670
CG4854
CG4360-F1-3
a-
gl
Zn
&
a-
a-
sq z
rn
52
20
C G 1 -9
-F
jim
lol
lol
lol
0
YY
1
LI
G
BL
IM
P1
EG
R
im
Bl
We have
determined
the DNAbinding
specificity of
more than
100 naturallyoccurring
zinc finger
proteins
(ZFPs) and
many more
synthetic
ZFPs
Enuameh, et al. Genome Research 2013
We have constructed an archive of
characterized zinc finger modules can be
assembled into ZFPs to recognize a specific
“address” within the genome
Two Finger
modules)
ZincArchive
Finger (194
Nucleases
OneC Finger Archive (27 modules)
We have constructed an archive of
characterized zinc finger modules can be
assembled into ZFPs to recognize a specific
“address” within the genome
Two Finger
modules)
ZincArchive
Finger (194
Nucleases
OneC Finger Archive (27 modules)
-TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA1
2
3
1
2
3
-AGCAAGCCGAGCGCAATTCGTTCACGTCTT-
We have constructed an archive of
characterized zinc finger modules can be
assembled into ZFPs to recognize a specific
“address” within the genome
Two Finger
modules)
ZincArchive
Finger (194
Nucleases
OneC Finger Archive (27 modules)
aZFP
-TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA1
2
3
1
2
3
-AGCAAGCCGAGCGCAATTCGTTCACGTCTT-
ZFPs or other platforms can be used to create
artificial nucleases that allow targeted gene editing
ZFPs or other platforms can be used to create
artificial nucleases that allow targeted gene editing
ZFPs or other platforms can be used to create
artificial nucleases that allow targeted gene editing
ZFPs or other platforms can be used to create
artificial nucleases that allow targeted gene editing
Zinc Finger Nuclease construction of
disease models in zebrafish
Diabetes: leptin receptor (db)
miR-375 (1 & 2)
Obesity: melanocortin receptor 4 (mc4r)
melanocortin receptor 3 (mc3r)
leptin (A & B) (ob)
Ankit Gupta, Victoria Hall, Dana DeSantis
Zinc Finger Nuclease construction of
disease models in zebrafish
Diabetes: leptin receptor (db)
miR-375 (1 & 2)
Obesity: melanocortin receptor 4 (mc4r)
melanocortin receptor 3 (mc3r)
leptin (A & B) (ob)
Ankit Gupta, Victoria Hall, Dana DeSantis
Pancreatic islet development is largely conserved
between humans and zebrafish
6 hpf
10 hpf
11 hpf
12 hpf
14 hpf
15 hpf
Tiso et al., Mol. Cell Endocrinology 312, 24-30, 2009
miRNAs can inhibit translation and destabilize their target mRNAs
miR-375
Winter et. al., Nature Cell Bio., 2009,11(3):228-34
Fo
kI
Two Copies of miR-375 are present in the zebrafish genome
Spacer (6bp)
~
~
genomic
target site
(mir-375-2)
Fo
kI
Fo
kI
Spacer (6bp)
zebrafish miR-375-2 pri-miRNA hairpin
I
Fo
k
donor
plasmid
60
50
inverted
A
A
G A G G G U U site
U U G U U C G U U C G G C U C
U
U A C
80
G C G U
G C A G A U G CA
GACGU C C G U A A C A C G C A C A C C G A G U U G C A
U G U U C A U GU
30
NHEJ-mediated
ligation
Fo
kI
ZFN target site
C U C
5’ ZFP site
10
3’ ZFP site
Spacer (6bp)
Spacer (6bp)
Gal4FF
miR375-2 TCGTTCGGCTCGcgttacGCAGATGCAGAC
CGAGCCGAACGA GCAGATGCAGAC
Fo
kI
CGAGCCGAACGA GCAAGTGCAGAA
Fo
kI
TCGTTCGGCTCGcgttaaGCAAGTGCAGAA
~
~
genomic
target site
miR375-1
(mir-375-2)
20
Fo
kI
A
70
Gal4FF
Ankit Gupta
Injecting nucleases into zebrafish embryos
cap
pCS-HA-ZFN
SP6
NLS
HA
1
2
3
Fok I DD
cap
pCS-Flag-ZFN
SP6
polyA
signal
polyA
signal
NLS
inject both ZFN mRNAs
into 1-cell stage embryos
FL
1
2
3
Fok I RR
Fo
kI
ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA
sequences within the genome
Spacer (6bp)
~
~
genomic
target site
(mir-375-2)
Fo
kI
Fo
kI
Spacer (6bp)
Gal4FF
I
Fo
k
donor
plasmid
inverted
site
Fo
kI
Spacer (6bp)
Spacer (6bp)
Fo
kI
Fo
kI
Maresca, et. al. (2013). Genome Research, 23(3), 539.
~
Gal4FF
~
genomic
target site
(mir-375-2)
Fo
kI
NHEJ-mediated
ligation
Fo
kI
ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA
sequences within the genome
Spacer (6bp)
~
~
genomic
target site
(mir-375-2)
Fo
kI
Fo
kI
Spacer (6bp)
Gal4FF
I
Fo
k
donor
plasmid
inverted
site
Fo
kI
Spacer (6bp)
Spacer (6bp)
Fo
kI
Fo
kI
Maresca, et. al. (2013). Genome Research, 23(3), 539.
~
Gal4FF
~
genomic
target site
(mir-375-2)
Fo
kI
NHEJ-mediated
ligation
Fo
kI
ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA
sequences within the genome
Spacer (6bp)
~
~
genomic
target site
(mir-375-2)
Fo
kI
Fo
kI
Spacer (6bp)
Gal4FF
I
Fo
k
donor
plasmid
inverted
site
Fo
kI
Spacer (6bp)
Spacer (6bp)
Fo
kI
Fo
kI
Maresca, et. al. (2013). Genome Research, 23(3), 539.
~
Gal4FF
~
genomic
target site
(mir-375-2)
Fo
kI
NHEJ-mediated
ligation
Fo
kI
ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA
sequences within the genome
Spacer (6bp)
~
~
genomic
target site
(mir-375-2)
Fo
kI
Fo
kI
Spacer (6bp)
Gal4FF
I
Fo
k
donor
plasmid
inverted
site
Fo
kI
Spacer (6bp)
Spacer (6bp)
Fo
kI
Fo
kI
Maresca, et. al. (2013). Genome Research, 23(3), 539.
~
Gal4FF
~
genomic
target site
(mir-375-2)
Fo
kI
NHEJ-mediated
ligation
ObLiGaRe insertion into mir-375-2 locus
Fo
kI
Fo
kI
5’ primer
~
Gal4FF
Fo
kI
Fo
kI
3’ primer
embryos (N/D)
N
N
N
N
D
N
D
ZFNs (pg)
0
63
0
63
63
125
125
Donor (pg)
0
0
25
25
25
25
25
ladder
~
genomic
target site
(mir-375-2)
1.3%
Ankit Gupta
miR-375-2 knockin fish crossed to insulin:mcherry
UAS:EGFP
Pancreas
Pituitary
ins:mcherry
overlay
miR-375-2 knockin fish allows pancreatic progenitor cells to be followed during
development (23 to 30 hpf)
Ankit Gupta
miR-375-2 knockin fish allows pancreatic progenitor cells to be followed during
development (23 to 30 hpf)
Ankit Gupta
A subset of miR-375 positive cells express insulin at 2 dpf
miR-375-2 Gal4FF;
UAS: EGFP
ins: mcherry
overlay
miR-375-1-/- miR-375-2 -/- animals display defects
in pancreatic islet formation
3 dpf - insulin:mCherry
WT
double mutant
miR-375 null animals have reduced β-cell numbers at 3dpf
-cell number at 3 dpf
30
p<0.001
27
16
20
10
N
ul
ls
0
W
T
insulin:mCherry cells
40
miR-375 Genotype
Ankit Gupta
miR-375 nulls show elevated glucose levels at 6 dpf
p<0.001
36 M
40
20
18 M
ul
ls
N
T
0
W
whole embryo glucose
concentration
60
miR-375 Genotype
Ankit Gupta
miR-375 positive cells can be sorted from wild-type and mutant
animals to identify genes that are potentially responsible for the
observed phenotypes
miR-375-2 Gal4FF;
UAS: EGFP
ins: mcherry
overlay
Fold Change relative to WT
Upregulated genes that are potential miR-375 targets
1000
104.0
100
147.0
10
4.3
beta cell
alpha cell
6.1
5.3
3.5
1
ELAVL4
ELAVL3
Ebf3
The CRISPR/Cas9 system provides a simple
system for genome editing
CRISPR/Cas9 system provides RNA guided
nuclease activity
-3’
-5’
S. pyogenes Cas9 (SpCas9) : nGG >> nAG; nGA >> others
The primary constraint on target recognition is the
PAM specificity of the Cas9 protein
Jinek et al. Science. 2012; 337(6096):816-21
Cong et al. Science. 2013; 339(6121):819-23
Development of chimeric Cas9 - DNAbinding domain (DBD) fusion
Hypothesis: Fusion of Cas9 to a programmable DNA-binding domain will
provide improved genome editing precision and broader targeting range.
Target site
-3’
-5’
DBD
Development of chimeric Cas9 - DNAbinding domain (DBD) fusion
Hypothesis: Fusion of Cas9 to a programmable DNA-binding domain will
provide improved genome editing precision and broader targeting range.
Target site
-3’
-5’
DBD
Goals:1) Identify parameters to create functional Cas9-DBD framework
2) Attenuate Cas9 to make nuclease activity DBD dependent
GFP reporter assay to monitor nuclease
activity in cell culture
mammalian cell
homology driven repair
with functional
nuclease
Wilson et al. MTNA (2013) 2, e87
GFP reporter assay to monitor nuclease
activity in cell culture
mammalian cell
homology driven repair
with functional
nuclease
Wilson et al. MTNA (2013) 2, e87
GFP reporter assay to monitor nuclease
activity in cell culture
mammalian cell
homology driven repair
with functional
nuclease
Wilson et al. MTNA (2013) 2, e87
Identifying parameters to create a functional Cas9-DBD framework
Identifying parameters to create a functional Cas9-DBD framework
Identifying parameters to create a functional Cas9-DBD framework
A DBD fusion expands the targeting repertoire of
spCas9
Cas9-ZFP is functional on genomic targets
with suboptimal PAMs
Cas9-ZFP = SpCas9-Zif268
T7 Endonuclease I (T7EI) assay
(11bp)
Cas9-ZFP is functional on genomic targets
with suboptimal PAMs
Cas9-ZFP = SpCas9-Zif268
T7 Endonuclease I (T7EI) assay
(11bp)
To attenuate independent Cas9 activity mutagenesis
was performed on the PAM contact residues
PAM recognition by Arg 1333 and Arg 1335
Arg1335
Arg1333
Anders et al. Nature (2014) 513, 569–573
Arg1333 and Arg1335 mutants cause loss of
function which can be restored by a ZFP fusion
Arg1333
Lysine
Serine
Arg1335
Lysine
Serine
Arg1333 and Arg1335 mutants cause loss of
function which can be restored by a ZFP fusion
Arg1333
Lysine
Serine
Arg1335
Lysine
Serine
Arg1333 and Arg1335 mutants cause loss of
function which can be restored by a ZFP fusion
Arg1333
Lysine
Serine
Arg1335
Lysine
Serine
Can Cas9-ZFPs improve precision?
GTGGGTGAGTGAGTGTGTGCGTGTGGGGTTGAGGGCGTTGGAGCGGGG
VEGF-A Cas9 TS3
ZFP target site
Fu et al. Nat Biotechnol. 2014; 32(3):279-84
Can Cas9-ZFPs improve precision?
GTGGGTGAGTGAGTGTGTGCGTGTGGGGTTGAGGGCGTTGGAGCGGGG
VEGF-A Cas9 TS3
ZFP target site
Fu et al. Nat Biotechnol. 2014; 32(3):279-84
TS3
OT3-2
Control
Cas9-MT3-ZFTS3
sgRNA-TS3
Cas9-MT3
sgRNA-TS3
Cas9-WT
sgRNA-TS3
Control
Cas9-MT3-ZFTS4
sgRNA-TS3
Cas9-MT3-ZFTS3
sgRNA-TS4
Cas9-MT3-ZFTS3
sgRNA-TS3
Cas9-MT3
sgRNA-TS3
Cas9-WT
sgRNA-TS3
Cas9-ZFPs increase the precision of genome
editing
TS3
OT3-2
Control
Cas9-MT3-ZFTS3
sgRNA-TS3
Cas9-MT3
sgRNA-TS3
Cas9-WT
sgRNA-TS3
Control
Cas9-MT3-ZFTS4
sgRNA-TS3
Cas9-MT3-ZFTS3
sgRNA-TS4
Cas9-MT3-ZFTS3
sgRNA-TS3
Cas9-MT3
sgRNA-TS3
Cas9-WT
sgRNA-TS3
Cas9-ZFPs increase the precision of genome
editing
TS3
OT3-2
Control
Cas9-MT3-ZFTS3
sgRNA-TS3
Cas9-MT3
sgRNA-TS3
Cas9-WT
sgRNA-TS3
Control
Cas9-MT3-ZFTS4
sgRNA-TS3
Cas9-MT3-ZFTS3
sgRNA-TS4
Cas9-MT3-ZFTS3
sgRNA-TS3
Cas9-MT3
sgRNA-TS3
Cas9-WT
sgRNA-TS3
Cas9-ZFPs increase the precision of genome
editing
Artificial nucleases are powerful tools for
manipulating vertebrate genomes in a
directed manner
Artificial nucleases are powerful tools for
manipulating vertebrate genomes in a
directed manner
1) Artificial nuclease platforms have been developed that can
target the majority of sequences with a vertebrate genome.
Artificial nucleases are powerful tools for
manipulating vertebrate genomes in a
directed manner
1) Artificial nuclease platforms have been developed that can
target the majority of sequences with a vertebrate genome.
2) These tools allow the study of gene function in model
organisms and the creation of disease models to understand
dysfunction at the systemic and molecular level
Artificial nucleases are powerful tools for
manipulating vertebrate genomes in a
directed manner
1) Artificial nuclease platforms have been developed that can
target the majority of sequences with a vertebrate genome.
2) These tools allow the study of gene function in model
organisms and the creation of disease models to understand
dysfunction at the systemic and molecular level
3) More precise nucleases are being developed that will permit
the realization of genetic correction of aberrant loci for the
treatment of disease.
Acknowledgements
UMass Medical School (UMMS)
Molecular, Cell and Cancer Biology (MCCB)
Wolfe Lab
Fatih Bolukbasi
Ankit Gupta
Dana DeSantis
Victoria Hall
Selase Enuameh
Cong Zhu
Yuna Asriyan
Amy Rayla
Stephanie Chu
Heather Bell
Marcus Noyes
Brodsky Lab
•Michael Brodsky
Sarah Oiekemus
Bioinformatics MCCB
Julie Zhu
Jianhong Ou
Maehr Lab (DCOE)
René Maehr
Nicola Kearns
Ryan Genga
Hannah Pham
Bioinformatics Core (UMMS)
Manuel Garber
Alper Kucukural
Pederson Lab (BMP)
Thoru Pederson
Hanhui Ma
Nathan Lawson (MCCB)
Nick Rhind (BMP)
Michael Parsons (Johns Hopkins)
Funding:
NIGMS, NHLBI, NIAID & NHGRI
Jack Leonard
Karl Simin
Erik Sontheimer
Laura Alonso
Jeremy Luban