Nature - USA (2019-07-18)

reSeArCH Letter

a

Control Notum KO 2

gRNA 2

site

Exon 3

294 bp

188 bp

Exon 123456789101112

gRNA 1

sequenceSignal gRNA 2

300

200

bp Scr1 Scr2 NotumKO 2

400

500

CRISPR targeting of Notum

Ratio 1.15 0.84 0.23

gRNA 1

site

Exon 2

174 bp

84 bp

b

c

d

Notum KO 1

1

2

3

4

Notum mRNA

relative to control (Log

) 2

Exp1

5

Exp2Exp3

0 dA To m

dA To mdA Notum

CD24

Side Scatter

CD24med

300

200

bp Scr1 Scr2 NotumKO 1

100

Ratio 1.21 0.79 0.08

e

0.0

0.5

2

3

4

Neg

cntrl

Not

KO 1

Not

KO 2

Scr

1

Scr

2

# Crypts per organoid

0.0490.0040

1.2×10

-4

0.0430.00610.0012

Notum WT

Notum KO

Notum WT

Notum KO

orthotopic

transplantation

g

Organoid mixture

1:1 ratio

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Relative mRNAexpression (Log

) 2

dA To m

Axin2Lgr5

0.043

0.048

Analysis

8 weeks

Colonoscopy Necroscopy Histology

tdTo

mato

DNA

bright field bright field tdTo

mato

tdTo

mato

Rag2(-/-)

Rag2(-/-)

Notum KO

Young Old

Scr control

Young Old

# Colonies per CD24

med

SSC

lo cell

dA

To m

dA

Notum

0.00

0.01

0.02

0.03

0.04

f 0.05 0.045

Extended Data Fig. 7 | Notum regulates intestinal stem cell function.
a, Notum gene targeting. Schematic represents sites of genome editing.
Gene editing was confirmed by PCR with primers flanking the editing site
(174 bp product for Notum KO1 and 294  bp product for Notum KO2) and
hitting the edited site (84 bp product for Notum KO1 and 188  bp product
for Notum KO2). Representative agarose gel images from two independent
experiments with similar results are shown. b, Regenerative growth of
Notum knockout organoids. De novo crypt domains were quantified two
days after subculture (n = 5 repeated experiments with the same organoid
lines). Representative images of organoids two days after subculture are
shown. Scale bar, 100  μm. c, Schematic presenting in vivo competition
assay of gene-edited organoid growth by orthotopic transplantation to
immunodeficient Rag2−/− mice. Representative colonoscopy, necroscopy
and histology images used for assay quantification (n = 8 mice
transplanted). Scale bars, 1  mm for necroscopy and 200  μm for histology.
d, Representative images of CRISPR-targeted young and old organoids

two days after subculturing (n = 4 mice per group). Scale bar, 100  μm.

e, Relative Notum expression in organoids with synergistic activator

mediator complex (SAM) targeted to Notum promoter (dA Notum) grown

for two days in ENR medium. Three independent experiments; relative

to control (dA Tom). f, Quantification and representative images of day-5

colonies formed by isolated CD24medSSClo cells from Notum activator

(dA Notum) and control (dA Tom) organoids. Scale bar, 100  μm. n =  4

repeated experiments with the same organoid line. g, RT–qPCR analysis

of relative Axin2 and Lgr5 expression in CD24medSSClo cells sorted

from Notum activator (dA Notum) organoids. Values show fold change

(expressed in log 2 ) in comparison to control (dATom) (n = 3 replicate

wells per organoid line). In box plots, unless otherwise indicated, the

line represents median, the box shows interquartile range and whiskers

represent the range. All other data are mean ± s.e.m.; two-tailed unpaired

Student’s t-test; exact P values shown in corresponding panels. For gel

source data see Supplementary Fig. 3.