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Supplementary Information Titles
Journal: Nature Medicine
Article Title: Modelling colorectal cancer using CRISPR-Cas9-mediated
engineering of human intestinal organoids
Corresponding
Author:
Toshiro Sato
Supplementary Item & Number
Title or Caption
Supplementary Figure 1 Establishment of CRISPR-Cas9-engineered organoids.
Supplementary Figure 2 CRISPR-Cas9-mediated introduction of mutations in driver
pathways.
Supplementary Figure 3 Heat maps of differentially expressed genes in engineered
organoids.
Supplementary Figure 4 Establishment of lentivirally engineered organoids.
Supplementary Figure 5 Tumour grafts of lentivirally engineered organoids
Supplementary Figure 6 Tumour grafts of CRISPR-Cas9-engineered organoids and
CRC organoids.
Supplementary Figure 7 Global gene expression of adenoma, engineered and CRC
organoids.
Supplementary Figure 8 Sequence analysis of engineered organoids.
Supplementary Figure 9 Establishment of engineered adenoma organoids.
Supplementary Table 1 Summary of clinical information
Supplementary Table 2 Gene targeting efficiency of CRISPR-Cas9 in human colonic
organoids.
Supplementary Table 3 Sequence analysis of predicted off-target loci of CRISPR/Cas-
9 edited organoids.
Supplementary Table 4 Oligonucleotides used in this study.
Nature Medicine: doi:10.1038/nm.3802
Supplementary Figure 1
Supplementary Figure 1: Establishment of CRISPR-Cas9-Engineered Organoids. (a) Summary of strategy for
CRISPR-Cas9-based organoid engineering. (b) Summary of clinical information for organoids used in this study. R:
Rectum, FAP: familial adenomatous polyposis. (c) Generation of second and third line of CRISPR-Cas9-engineered
organoids. Sequences of del (deletions) and ins (insertions) are shown in Supplementary Fig. 8b,c.
aSelectionCRISPR
Trypsinization
Cloning
SURVEYOR
assay
Sequencing
Organoids
Lentivirus-
engineering
Normal
organoids
Adenoma
organoids
CRC
organoids
CRISPR-Cas9
engineering
Normal1 Normal2 Ade1 Ade2 Ade3 Ade4 Ade5 CRC1 CRC2 CRC3 CRC4 CRC5 CRC6 CRC7 CRC8 CRC9
Lentivirus-
engineering
CRISPR-
engineering
Normal3
b
c
(WR ENA)
AS’ AST’ AKST’
AST AKST AKSTP
AKSTP’
(E+TGF) (E+Nut) (None) (MEKi)
(MEKi)(E+TGF+Nut) (None)
Normal2A: 1 bp del(8/8)
S: 2 bp ins (4/8), 51 bp ins (4/8)
T: 1 bp del (8/8)
K: V12G (direct), P: E545K (3/8)
A: 3 bp (SD) del (5/8), 11bp del (3/8)
S: 3 bp (V370) del, large ins
T: 2 bp del (4/8), 2 bp del (4/8)
K: V12G (direct), P: E545K (5/8)(ENA)
A
(S.Fig.8c)
(S.Fig.8b)(WR ENA)
Normal1
A’
(ENA)
Nature Medicine: doi:10.1038/nm.3802
b-actin
b-catenin
AWT
SMAD4
a b
b-actin
c d
TP53
b-actin
e f
100
200
300
100
200
300
100
200
300
400
400
(bp)
(bp)
(bp)
Supplementary Figure 2
pERK
KI
WT
ERK
g h
pERK
2
3
5
8
(kb)
i
AKT
pAKT
*
*
A-
SWT S-
AWT A-
SWT S-
TWT T- TWT T-
Supplementary Figure 2: CRISPR-Cas9-mediated introduction of mutations in driver pathways. (a, c, e)
SURVEYOR assay to detect CRISPR-Cas9-mediated cleavage at the APC-targeted (A-), SMAD4-targeted (S-) and
TP53-targeted (T-) organoids. WT: Wild type. (b, d, f) Western blot analysis of engineered organoids. Expression
of b-catenin (a consequence of Wnt activation), SMAD4 and TP53 are indicated. b-actin, loading control. (g)
Southern blot analysis of DNA isolated from control and KRASG12V organoid clones. Probes for the Southern blot
analyses are indicated in (Fig. 1g). (h) Western blot for phospho-ERK and total ERK in KRASWT or KRASG12V
organoids. (i) Western blot analysis of phospho-AKT and total AKT in KRASG12V and KRASG12V PIK3CAE545K
organoids. *, non-specific bands.
Nature Medicine: doi:10.1038/nm.3802
Supplementary Figure 3
Supplementary Figure 3. Heat maps of differentially expressed genes in engineered organoids. Gene
expression data of adenoma organoids is added on the heat maps shown in Fig. 2g.
TRPM2EREGKRT23
CA1REG4GCGLYZCHGAPLA2G2ATFF1TFF2SPINK4MUC2
Adenoma CRCEngineered
-2 0 2
ETV4
Nature Medicine: doi:10.1038/nm.3802
ENA NA TGFb Nutlin3 MEKi
BK
ST
P
b
pErk1/2
Erk1/2
TP53
- + - + Nutlin-3
Control TP53KD
b-actin
b-actin
b-catenin
Cont BS33Y Cont KG12V
SMAD4
Cont SKD
b-actin
pAkt
Akt
Cont PE454K
c d
e f
g
Normal Organoid 2
WR ENA Hygro G418 Puro
B BK BKS BKST
Lentivirus CTNNB1S33Y KRASG12V
PGK-Hygror
SMAD4KD
PGK-Neor
TP53KD
PGK-Puror
BKSTP
Zeo
PIK3CAE545K
PGK-Zeor
ENA
Supplementary Figure 4
Supplementary Figure 4: Establishment of Lentivirally Engineered Organoids. (a) Selection strategy for
lentivirus-based engineered organoids. Abbreviations for overexpressed or knockdown genes were as follows: B,
constitutive active form of b-catenin; K, KRASG12V; S, knockdown (KD) of SMAD4; T, KD of TP53; P,
PIK3CAE545K. (b-f). Validation of lentivirus-based engineered organoids. (b) Western blot analysis illustrates
upregulation of the active form ofb-catenin in B organoids (BS33Y). Cont: wild-type control organoids. (c)
Downstream ERK activation in control wild-type KRAS or KRASG12V-transduced (KG12V) organoids under EGF
removed culture conditions. Western blot analyses of the phosphorylated form of ERK1/2 (pERK) and total ERK
protein. (d) Knockdown of TP53 protein. Nutlin-3 treatment induced TP53 protein in wild-type organoids. The
effect of Nutlin-3 was abolished in TP53 knockdown (TP53KD) organoids. (e) Knockdown of SMAD4 protein in
SMAD4-shRNA transduced organoids (SKD). (f) Downstream PI3 kinase activation in control wild-type PIK3CA or
PIK3CAE545K-transduced organoids under EGF removed culture conditions. Western blot analyses of the
phosphorylated form of Akt (pAkt) and total Akt protein. (g) Response to niche modulated culture conditions (See
Figure 2B for the abbreviations) in BKSTP-organoids.
a
Nature Medicine: doi:10.1038/nm.3802
Supplementary Figure 5
Supplementary Figure 5: Tumour Grafts of Lentivirally Engineered Organoids
(a) Photographs of representative tumours derived from indicated organoids in kidney subcapsules of NOG mice.
Xenotransplanted tumours are labelled by EGFP. Tumours were isolated 1 month (1M) or 2 months (2M) after
transplantation. Scale bar = 5 mm. (b) The average surface area of xenografts derived from organoids indicates the
tumour size at 1 or 2 months after transplantation (n=4). Error bars indicate s.e.m. (c) Histochemical analysis of
BKSTP-organoids isolated 1 month after the transplantation. Indicated staining was shown. KRT: cytokeratin 20.
0
5
10
15
20
25
B BK BS BT BKS BKST BKSTP
1M
2M
GF
P a
rea
(m
m2)
HE Ki67 KRT20PAS
2M1M
BB
KB
KS
BK
ST
a b
c
BK
ST
P
Nature Medicine: doi:10.1038/nm.3802
Supplementary Figure 6
Supplementary Figure 6: Tumour Grafts of CRISPR-Cas9-Engineered Organoids and CRC Organoids.
Representative histochemical analysis of kidney subcapsule xenografts derived from AKST-organoids (a) or CRC-
organoids (b) isolated 1 month after transplantation. Indicated staining was shown. KRT20: cytokeratin 20.
Scale bar = 500 m
Ki67 PAS CK20HE
AK
ST 1
M
HE
CR
C 1
M
b-catenin CK20
a
b
Nature Medicine: doi:10.1038/nm.3802
Supplementary Figure 7
Supplementary Figure 7: Global gene expression of adenoma, engineered and CRC organoids. PCA analysis
including gene expression of AdeCIN TSK- and AdeCIN TSP- organoids (grey). Engineered organoids from normal
epithelium (blue), Adenoma organoids (red) and CRC organoids (brown) were indicated. Arrows indicate engineering
from parental adenoma organoids.
AdeCINTSK
Ade
AdeCIN TSP
CRC
Adenoma/Engineered
0 1000-1000 2000 3000
-1000
0
1000
2000
AdeCIN TS
PC1
PC
2
Nature Medicine: doi:10.1038/nm.3802
Supplementary Figure 8
Supplementary Figure 8. Sequence analysis of engineered organoids. (a-c) Sequences of AKSTP organoids shown
in Fig. 2a and Supplementary Fig.1c. (d, e) Sequences of Ade ST organoids (d) or AdeCIN TSK organoids (e) shown in
Fig. 4a. SD: splicing donor sites, del: deletion, ins: insertion (underlined). Target regions were amplified by PCR and
subcloned to E. Coli. Indicated number of subclones were sequenced. Note that SMAD4 V370 is in a conserved
loop/helix region of MH2 domain and critical for TGF-b/BMP signalling (Shi, Y., Hata, A., Lo, R.S., Massague, J. &
Pavletich, N.P. A structural basis for mutational inactivation of the tumour suppressor Smad4. Nature 388, 87-93
(1997))
APC
SMAD4
TP53
7 bp (SD) del (8/8)
3 bp (V371) del (3/6)
2 bp del (3/6)
1 bp ins (4/8)
18 bp del (4/8)
2 bp ins (4/8)
51 bp ins (4/8)
1 bp del (8/8)
3 bp (SD) del (5/8)
11 bp del (3/8)
3 bp (V370) del (8/8)
2 bp del (4/8)
2 bp del (4/8)
SMAD4
TP53
APC
SMAD4
TP53
SMAD4
TP53
SMAD4
TP53
1 bp del (3/6)
2 bp del (3/6)
N369K/V370 del (8/8)
1 bp del (8/8)
3 bp del NV369N (4/8)1 bp del (4/8)
a
b
c
d
e
APC 1 bp del (8/8)
……..…….. 168 bp ins (8/8)
Nature Medicine: doi:10.1038/nm.3802
(ENA)
Adenoma 2 Ade2 TSK(EGFRi+TGF+Nut)
(ENA)
Adenoma1(Puro)
Ade1 T
CRISPR
Lentivirus
(Neo)
Ade1 TS(Hygro)
Ade1 TSK
Adenoma1
Ade1 TSK
Ade2 TSK Are
a o
f m
eta
sta
ses lo
g (
mm
2)
Adenom
a
Ade T
Ade S
Ade C
INT
S
Ade
CIN
TS
P
Ade
CIN
TS
K
CR
C
AT
KS
P
Ade S
T
Adenom
a1
Ade
1T
SK
Ade
2T
SK
a
b c
Supplementary Figure 9
Supplementary Figure 9. Establishment of engineered adenoma organoids. (a) Selection strategy of second and
third line of engineered adenoma organoids. (b) Photographs of representative tumours derived from indicated
organoids in kidney subcapsules of NOG mice. Xenotransplanted tumours are labelled by EGFP. Tumours were
isolated 1 month (1M) after transplantation. Scale bar = 5 mm. (c) Data of metastasized lesions from Adenoma1,
Ade1TSK and Ade2TSK organoids is added on the scatter plot shown in Fig. 4e. Plots represent EGFP+ area of
metastasized lesions in liver. n=4 mice per genotype.
Nature Medicine: doi:10.1038/nm.3802
Supplementary Table 1
Supplementary Table 1. Summary of Clinical Information. NS: not specified. F: female, M: Male.
Patient
IDOrganoid ID
Tumor
Location
Patient
Disease
Clinical
Stage
Tumor
Histology
Patient
Sex
Prior
treatment
#1 CRC1 R CRC Stage IV Moderate F None
#1 CRC2 R CRC Stage IV Moderate F None
#1 CRC3 Liver CRC Stage IV Moderate F None
#2 CRC4 Liver CRC Stage IV Moderate M None
#3 CRC5 R CRC Stage IV Moderate F None
#4 CRC6 Distal CRC Stage IIIA Well M None
#5 CRC7 Proximal CRC Stage IIA Moderate F None
#6 CRC8 Proximal CRC Stage II Moderate M None
#7 CRC9 Distal CRC Stage I Well M None
#8 Adenoma1 (Ade1) Colon NS FAP F None
#9 Adenoma2 (Ade2) Colon NS FAP F None
#10 Adenoma3 (Ade3) Colon NS FAP M None
#11 Adenoma4 (Ade4) Colon NS FAP M None
#11 Adenoma5 (Ade5) Colon NS FAP M None
#12 Normal1 Proximal CRC Stage 0 F None
#13 Normal2 Distal CRC Stage 0 M None
#10 Normal3 Colon NS FAP M None
Nature Medicine: doi:10.1038/nm.3802
Supplementary Table 2
Supplementary Table 2. Gene Targeting efficiency of CRISPR-Cas9 in human colonic organoids. Summary of
the genome engineering efficiency. Indicated number of dissociated cells from Normal, A-organoids or AKST-
organoids were electroporated with indicated sgRNA. The experiments were performed in triplicate and average
number (Ave. No.) of recovered organoids± s.e.m is shown. After electroporation, around 1-5 % of electroporated
cells were recovered. In some experiments, Piggy-Bac GFP-Puro vectors were co-electroporated to determine the
efficiency of gene introduction. The antibiotics - or niche factor- selected organoids were verified by Sanger
sequencing analysis.
Recipient Cells CRISPR/Cas9
No. of
electroporated
cells
Ave. no. of
recovered
organoids (n=3)
Ave. no. of
selected
organoids
Correctly targeted
clones
Normal PiggyBac-GFP-Puro/APC 25000 785 73.0 (+Puro) N.A.
Normal PiggyBac-GFP-Puro/APC 25000 785 6.3 (-Wnt) 3/3
Normal APC 10000 319.3 2.7(-Wnt) 3/3
Normal APC/SMAD4 1x105 N.A. 2 (-Wnt/+BMP4) 2/2
A PiggyBac-GFP-Puro/TP53 5000 175.3 2.3 (+Puro) N.A.
A PiggyBac-GFP-Puro/TP53 5000 175.3 2.0 (+Nutlin) 2/2
A KRAS (Knock-in) 1x105 N.A. 3 (+EGFRi) 3/3
AKST PIK3CA (Knock-in) 1x105 N.A. 3(+EGFRi/MEKi) 2/3
Nature Medicine: doi:10.1038/nm.3802
Supplementary Table 4
Supplementary Table 4: Oligonucleotides used in this study.
miRNA target sequence
TP53 miRNA #1 TCCACTACAACTACATGTGTA
TP53 miRNA #2 AGTCTGTGACTTGCACGTACT
SMAD4 miRNA #1 GGTGTGCAGTTGGAATGTAAA
SMAD4 miRNA #2 GGTGGAGAGAGTGAAACATTT
CRISPR target sequence
APC CRISPR GGCAACTTCTGGTAATGGTC AGG
SMAD4 CRISPR #1 GCTGAAGATGGCCGTTTTGG TGG
SMAD4 CRISPR #2 GTCAACTCTCCAATGTCCAC AGG
TP53 CRISPR GAATGAGGCCTTGGAACTCA AGG
KRAS CRISPR GCATTTTTCTTAAGCGTCGA TGG
PIK3CA CRISPR TCTCTCTGAAATCACTGAGC AGG
Donor ssOligo
PIK3CA Donor ssOligo
(anti-sense)AGCACTTACCTGTGACTCCATAGAAAATCTTTCTCtTGCTtgGTGATTTC
Primers for SURVEYOR Assay
APC SURVEYOR-F CGACCGCCAATCGTACTGGAGG
APC SURVEYOR-R GACAGCACATTGGTACTGAATGC
SMAD4 #1 SURVEYOR-F TATAAAAGCAAATTAACCCATGT
SMAD4 #1 SURVEYOR-R TTACAAAAAATACTGAAAAGCAC
SMAD4 #2 SURVEYOR-F GAGAGACATTTAAGGTTCC
SMAD4 #2 SURVEYOR-R ACAATTAAGATGGAGTGCT
TP53 SURVEYOR-F CAGCTGTATAGGTACTTGAAGTGCAGTTT
TP53 SURVEYOR-R CAATGAGATGGGGTCAGCTGCCTTTG
Nature Medicine: doi:10.1038/nm.3802