Supplementary Material

Abstract

Supplementary material

Full text

Supplementary Figure 1. Generation of C2C12-knockout cell clones with CRISPR-Cas9
technique. (a) Electrophoresis of PCR product from C2C12Cas9 myoblasts transfected with or
without gRNAs. (b) DNA sequence maps around the targeting locus of control and selected
MyoDKO cell clones. (c) Detection off-targets of MyoD sgRNA. (d) Immunostaining of MF20
(Red) and DAPI (Cyan) in control and MyoDKO C2C12 myoblasts after 3 days induction of
differentiation.
Supplementary Figure 2. Overexpression of miR-133 inhibits adipogenesis of MyoDKO
C2C12 myoblasts. (a and b) qPCR analysis of miR-133a (a) and adipogenic marker genes (b).
N=3 different passages of myoblasts. Error bars represent SD, * indicates p < 0.01.
Supplementary Figure 3. Loss of MyoD facilitates adipogenic transdifferentiation of
myoblasts in vivo. (a) Immunostaining of BODIPY (green) and Dystrophin (red) in TA muscles
of mdx mice injected with control or MyoDKO C2C12 cells. (b) Quantification of Dystrophin+
myofibers. (c) Triglyceride concentrations in TA muscles injected with control or MyoDKO
C2C12 cells. N=3 independent biological samples. Four sections were analyzed for each sample.
Error bars represent SD, * indicates p < 0.05.
Supplementary Figure 4. MyoD transdifferentiates brown preadipocytes into myocytes. (a)
Immunostaining of BODIPY in MyoDOE brown preadipocytes. (b) Immunostaining of MF20
(red) in control and MyoDOE brown adipocytes. (c) Ratio of MF20 signal in elongated GFP+ cells.
N=3 different passages of myoblasts. Error bars represent SD, * indicates p < 0.01.

Supplementary Table 1 List of top 5 putative off-targets homologous to MyoD-sgRNA
Position
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
N
G
G
Locus
MyoD
C
C
A
G
G
T
C
C
T
C
A
A
A
A
A
A
G
C
G
C
A
G
G
Chro.:Start position
OT1
C
C
T
A
G
T
C
C
T
C
A
A
A
A
A
A
G
C
T
C
A
G
G
chr1:-106859905
OT2
C
T
A
A
G
T
C
A
T
C
A
A
A
A
A
A
G
C
A
C
T
G
G
chr13:+78086880
OT3
C
C
A
T
C
T
C
A
T
C
A
A
A
A
A
A
G
C
T
C
A
G
G
chr19:+50585297
OT4
A
C
A
C
G
T
G
C
T
C
A
A
A
A
A
A
G
C
A
C
G
A
G
chr1:-39340833
OT5
C
C
A
G
G
T
G
C
T
C
C
A
A
A
A
A
G
C
A
C
A
G
G
chr12:-113574844
PAM is in grey. Bases different from sgRNA are in red.

Supplementary Table 2 Primer sequences (5′-3′)
Gene
Sequence
OT1
GGCTGAAAGGCTTGTACTCG
GGAGCATTTCAGGCATCAAT
OT2
GGTGGAAAGGCTGTTGATGT
GCCAGAGTGATCGGAATGAT
OT3
TCCCTGGCTATGCAAAATTC
CATTCATGGAGCCTGTGTGT
OT4
ACATGTATGCCCAGATGCAA
GTGCTCCAACGAGTGAGACA
OT5
CCCAAGTTCCATGTCCCTAA
TGAGCAAGGTTTGCAGTTTG
MyoD-f
AAGCGGCCGCATGGAGCTTCTATCGCCGC
MyoD-r
TTTCTAGATCAAAGCACCTGATAAATC

gRNA PAM
Control
G A CC T G G AC C C G CG C 11 bp deletion Stop at 72 AA
C T T T T T T GA G G A C C T 1 bp deletion Stop at 55 AA
MyoDKO1
G C G A CC T G G A C C C 11 bp deletion Stop at 72 AA
MyoDKO2
Control gRNA
a
300bp
b
c
Supplementary Figure 1
300bp
400bp
300bp
400bp
OT2
OT5 400bp
500bp
OT4
650bp
850bp
OT1
500bp
650bp
OT3
C KO1 KO2
d Control MyoDKO1 MyoDKO1
MF20/DAPI

0
1
2
3
4
5Fabp4
mRNA levels
mRNA levels
0
1
2
3
4Fasn
0
2
4
6
8Cebpα
C KO2
KO1 C KO2
KO1
- - - + + +
*
*
* *
* *
*
*
Supplementary Figure 2
C KO2
KO1 C KO2
KO1
miR133a - - - + + +
0
100
200
300
400
500
600
700
800
900
1000
miR133a - - - + + +
C KO1 KO2 C KO1 KO2
miRNA levels
miR-133a
a b
Induction 2 days
miRNA-133
0
1
2
3
4
5Igf1r

MyoDKO Control
Dystrophin/BODIPY/DAPI Dystrophin BODIPY
0
5
10
15
20
25
30
Dystrophin+ fibers
per field
Control KO
b
*
0
0.5
1
1.5
2
2.5
Control KO
*
TG (mg/g)
c
a
Supplementary Figure 3
CTX Myoblasts
2 8
Sample
Days

GFP BODIPY
GFP/BODIPY
MyoDOE
Supplementary Figure 4
a
MF20
Control
MyoDOE
GFP/MF20 GFP
b
% elongated GFP+ cells with MF20
0
20
40
60
80
100
Control MyoDOE
*
c