CRISPR Gene Editing in Stem Cells

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noneforsknoneforsknoneforsknoneforsknoneforsknoneforsknoneforsknoneforsk
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LGHGLGHGLGHGLGHGLGHGLGHGLGHGLGHG
A
JUNB FOS FOSB
** * *
wt CDKAL1-/-
EGR3
FOSB
EGR2
KRT5
FOS
LHX5
EGR1
CXCL14
DUSP1
CYP26C1
NR4A1
TWIST2
CYP26A1
HIST4H4
CRYBA2
G6PC2
JUN
1 3 5 7 9
E
22.2 23.9 60.5
Annexin V
wt CDKAL1 -/-
ctrl-p palmitate ctrl-p palmitate
40.9
% of Annexin V
+ cells
in INS-GFP
+ cells
Relative mRNA
expression
Fold Change
wt CDKAL1-/- B
Fold Change
*
**
n.s.
n.s.
**
n.s.
wt CDKAL1-/-
J K
wt CDKAL1-/-
scramble sgRNA
sgFOS sgRNA sgFOS sgRNA sgFOS
scramble scamble
F
FOS
GAPDH
wt CDKAL1 -/-
41 kDa
37 kDa Color
−1 0 0.5 1 Value
Key
0
0.5
1
1.5
2
2.5
3
3.5
4
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Systemic lupus erythematosus
GnRH signaling pathway
ECM-receptor interaction
Pathways in cancer
B cell receptor signaling pathway
Neuroactive ligand-receptor interaction
Epithelial cell signaling inHelicobacter
pylori infection
Vibrio cholerae infection
MAPK signaling pathway
Complementand coagulation cascades
Focal adhesion
cFOS/JUN
- log P-value
D
1 2 1 2 1 2 1 2
*
**
** **
n.s.
Human c-peptide (% of content)
1 2 1 2 1 2 1 2
Fold Change
**
**
n.s.
n.s.
***
n.s.
wt CDKAL1-/-
wt CDKAL1-/-
scramble sgRNA
sgFOS sgRNA sgFOS sgRNA sgFOS
scramble scamble
1 2 1 2 1 2 1 2
**
**
** **
n.s.
Human c-peptide (% of content)
1 2 1 2 1 2 1 2
H
sgFOS2 sgFOS1 scramble sgRNA2 scramble sgRNA1
wt CDKAL1-/-
7AAD
ctrl-g high glucose ctrl-g high glucose
19.2 40.1 18.3 55.3
18.2 39.6 18.5 42.7
18.0 40.8 18.4 55.2
18.9 40.6 18.0 44.5
0
10
20
30
40
50
60
70
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
ctrl-g glu ctrl-g glu ctrl-p palm ctrl-p palm
wt CDKAL1-/- wt CDKAL1 -/-
scramble sgRNA sgFOS
n.s.
n.s.
n.s.
**
n.s.
n.s.
n.s.
***
42.3 24.2 46.0 22.9
19.1 40.4 20.5 61.9
19.2 40.9 20.2 44.5
wt CDKAL1-/- C
G I
0
5
10
15
20
25
30
35
40
45
% of PI
+INS
+cells
in INS
+cells
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
ctrl-g glu ctrl-g glu ctrl-p palm ctrl-p palm
wt CDKAL1-/- wt CDKAL1-/-
scramble sgRNA sgFOS
n.s.
n.s.
n.s.
**
n.s.
n.s.
n.s.
**
Focal Adhesion
cFOS/JUN
ACTG2
(legend on next page)
Cell Stem Cell 19, 326–340, September 1, 2016 335