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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)


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)


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.



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


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: 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



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



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



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



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)


(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. 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.


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 CRC3 Liver CRC Stage IV Moderate F None

#2 CRC4 Liver CRC Stage IV Moderate M 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



#12 Normal1 Proximal CRC Stage 0 F None

#13 Normal2 Distal CRC Stage 0 M None

#10 Normal3 Colon NS FAP M None



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



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


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