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.