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Modeling human hereditary
syndromes using CRISPR/Cas9 mediated genome editing in
Xenopus tropicalis
Kris Vleminckx
Ghent University
Belgium
Nov - 2017
- High number offspring (> 1000 eggs)
- embryogenesis proceeds rapidly
- Vertebrate – tetrapod (aquatic)
- embryos have a large size
Xenopus – main advantages
- External embryonic development
- Xenopus tropicalis is true diploid
- High synteny of genome
- Targeted injections are possible >< X. laevis and zebrafish
= X. laevis and zebrafish
- They don’t smell (>< the mouse)
Injections Cas9 and CRISPR guide RNAs (2–8 cell stage)
Phenotypic Readout
Xenopus – injections CRISPR/Cas9
Design gRNA (CRISPRscan)
Primer ligation + direct T7 RNA synthesis (cloning free)
Inject gRNA + Cas9 protein
Assess efficiency (NGS – MiSeq) Mostly > 70%
Raise or Adjust protocol
Analysis F0 mosaic (e.g. cancer models)
F0 F0
F1 phenotyping
< 2
we
eks
~
10
day
s
~ 6
mo
nth
s
Experimental Pipeline
< 2
mo
nth
s
> 80 genes targeted
Methods for genotyping
F0 mosaics • Heteroduplex Mobility Assay (HMA) • TA-cloning and Sanger sequences • NGS + Batch-GE analysis
F1 offspring • High Resolution Melting Analysis (HRMA)
• Sanger Sequencing - Tracking of Indels by DEcomposition (TIDE) analysis
https://tide-calculator.nki.nl/
CRISPR/Cas efficiency analysis by NGS
Decreasing cost
Speed High throughput
Availability
PCR NGS (MiSeq) Data analysis
Slides courtesy of Annekatrien Boel (CMGG)
Decreasing cost
Speed High throughput
Availability
PCR NGS (MiSeq) Data analysis
BATCH-GE
CRISPR/Cas efficiency analysis by NGS
Methods for genotyping
F0 mosaics • Heteroduplex Mobility Assay (HMA) • TA-cloning and Sanger sequences • NGS + Batch-GE analysis
F1 offspring • High Resolution Melting Analysis (HRMA)
• Sanger Sequencing - Tracking of Indels by DEcomposition (TIDE) analysis
https://tide-calculator.nki.nl/
IPL INL
PRL ONL
RPE
OPL
RCBTB1-mosaic
Left eye
Right eye
IPL
PRL
OPL INL
ONL
GCL
Inherited Retinal Dystrophy (collab. E. De Baere)
Rcbtb1 (mosaic KO) – Unilateral injection
Visel et al., Nature 2009
Regulation Shh expression (limb enhancer (ZRS) in LMBR1 intron)
Targeting Anterior-Posterior polarity – Shh
ZRS deletion
ZRS point mutations
ZRS = ZPA (Zone of Polarizing Activity) Regulation Sequences
Smith et al., Nat. Genet. 2013
Regulation Shh expression (limb enhancer (ZRS) in LMBR1 intron)
Targeting Anterior-Posterior polarity – Shh
HD MT
binding EB1
binding
Arm MCR
15 AA repeat (β-catenin) 20 AA repeat (β-catenin) SAMP repeat (Axin)
Apc
TCTTCAGTACACCATACACGGACTAAAAACAACAGACTTCAAACATCAAA TCTTCAGTACACCATACACGGACT -AAAACAACAGACTTCAAACATCAAA (∆ 1) x2 TCTTCAGTACACCATACA - - - - - - - - - - - - - -ACAGACTTCAAACATCAAA (∆ 13) x2 TCTTCAGTACACCATACACGGACT - - - - - - - - - - - - - - -TCAAACATCAAA (∆14)
Targeting APC MCR in X. tropicalis (TALEN)
Familial Adenomatous Polyposis in Xenopus (targeting APC tumor suppressor gene)
Van Nieuwenhuysen et al., Oncoscience 2015
• Extra-colonic manifestations:
– Congenital Hypertrophy of Retinal Pigment Epithelium (CHRPE) √
– Desmoid tumors √
– Brain tumors (medulloblastoma) √
– Abnormal dentition
– Osteomas
– Epidermal cysts √
Familial Adenomatosis Polyposis in Xenopus (targeting APC tumor suppressor gene)
– Medulloblastoma (Wnt, Shh, other) √
– Neurofibromatosis type 1 (NF1) /
– Neurofibromatosis type 2 (NF2) (√)
– Bladder cancer (NF1, PTEN) √
– Leukemia (Notch1, FBXW7, PTEN) √
cancer models under development?
Add. KOs : p53, Rag2
BC-2059 : inhibitor of TBL1 dependent β-catenin signaling
application 1 : Desmoid tumor preclinical compound testing
Dissect tumor Isolate DNA Sequence Gene X
Gene X disrupted In tumor?
YES
Gene X not important for tumorigenesis
NEVER
Gene X REQUIRED for tumorigenesis
Gene X = therapeutic target
Genome modification of APC and
gene x
application 3 : Desmoid tumor therapeutic target identification via gRNA multiplexing
workflow
Midkine (MDK)
0
10
20
30
40
50
60
70
80
90
100
DT1 DT2 DT3 DT4 DT5 DT6 DT7 DT8 DT9
APC
MDK
bi-allelic mutations in MDK detected in desmoid tumors (7); MDK is not essential for in vivo desmoid tumor initation
CRISPR/Cas9 editing efficiencies
Naert et al. unpublished
apc + ptt CRISPR/Cas9
Targeted deep sequencing of apc and ptt loci Dissect tumor
Identification of genes essential for desmoid tumorigenesis
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
60,00%
Percentage of tumors exhibiting bi-allelic out-of-frame mutations
n= 3 12 6 3 5 9 6 22 9 12
Domain 1 does not drive negative selection and can tolerate in-frame mutations
0
10
20
30
40
50
60
70
80
90
DT1 DT2 DT3 DT4 DT5 DT6 DT7 DT8 DT9 DT10DT11DT120
10
20
30
40
50
60
70
80
90
DT1 DT2 DT3 DT4 DT5 DT6 DT7 DT8 DT9 DT10 DT11 DT12
APC
EZH2
Raw targeted deep sequencing data In frames removed
Per
cen
tage
of
mu
tan
t se
qu
enci
ng
read
s
Perc
en
tage
of
mu
tan
t se
qu
enci
ng
read
s A
fter
rem
ovi
ng
in f
ram
es m
uta
nt
read
s
User CRISPR for Domain Mapping Negative selection to small in frames within functional domain reveals this domain as essential for tumorigenesis
D1 D2 D3 D5 D4
User CRISPR for Domain Mapping Negative selection to small in frames within functional domain reveals this domain as essential for tumorigenesis
Domain 5 targeting results in desmoid tumors that retain one fully wild type allele
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
DT1 DT3 DT5 DT7 DT9 DT11DT13DT15DT17DT19DT21DT23DT25
Raw targeted deep sequencing data
Per
cen
tage
of
mu
tan
t se
qu
enci
ng
read
s
D1 D2 D3 D5 D4
• Extremely simple, cheap and fast (>< mouse)
• Diploidy required (>< zebrafish)
• Targeted injections are great benefit (>< zebrafish)
Why do this in Xenopus tropicalis?
• So far primarily KO experiments
• Can we do KI ??
– HDR with repair template – using oocyte transfer
– Microhomology Mediated Endjoining (PITCh)
generation KO generation KI using host transfer
Generation Xenopus knock-in models
Slide courtesy of Marjolein Carron
Acknowledgements
Unit Developmental Biology Tom Van Nieuwenhuysen Hong Thi Tran Thomas Naert Suzan Demuynck Rivka Noelanders Dionysia Dimitrakopoulou UZ Gent pathology department Dr. David Creytens Stanford Clinical pathology Prof. Matt van de Rijn Joanna Przybyl
Collaborations: Center Medical Genetics Ghent Paul Coucke Annekatrien Boel Wouter Steyaert Andy Willaert Elfride De Baere Pieter Van Vlierberghe Gnommix Marnik Vuylsteke