Optimizing efficiency and safety of targeted gene correction
Homology-directed repair
NHEJ religationin situ
Cleave or nick
-
I-AniI Cleavase
I-AniINickase
-H2AX Merge/DAPI
-H2AX: cleavase/nickase
0.4 21 1
25
20
15
10
5
0
Fociper
nucleus
21-fold
-H2AX foci induced by cleavase but not nickase
Reporter:
Cleavage-associatedgenomic instability
Products:
Inversion following cleavage at both sites
Deletion following cleavage at both sites
Processing and NHEJ of a single cleavage event
polyAP
LHE LHE
H2Kd+, CD4-H2-Kd CD4
CD8
P
H2Kd-, CD4+CD4
polyAP
LHE
H2Kd-, CD4-2-Kd CD4
CD8
P
LHE LHE
CD8+, H2Kd-, CD4-
polyA
H2-Kd
CD8 CD4
A non-homologous end joining (NHEJ) reporter
Guirouilh-Barbat et.al. 2004
-
Gene correction and NHEJ:a model for optimization
gfp-
-
NHEJ
-
-
Gene correction and NHEJ:a model for optimization
gfp-
-
NHEJ
-
GFP
donor templateLHE
transfect
-GFP+
-
NHEJ
-x
Gene correction and NHEJ:a model for optimization
100 101 102 103 104
100
101
102
103
104
100 101 102 103 104
100
101
102
103
104
100 101 102 103 104
100
101
102
103
104
2.4% 14.5%
0.23% 0.11%
0.37%
97.3% 84.4%
0.42%
0.29% 0.003%
0.004%
H2-Kd (PE)
GFP
Cell line Targeting (%) H2-Kd loss (%)
among
uncorrected corrected
#1 0.42 12.1 67.6
#1 1.04 29.3 69.9
#2 1.96 1.3 50.9
#2 4.11 3.4 50.5
no I-SceIH2-Kd stained
+ I-SceIstained H2-Kd
+ I-SceIunstained
Correction @ A Mutation @ B
100 101 102 103 104
100
101
102
103
104
100 101 102 103 104
100
101
102
103
104
100 101 102 103 104
100
101
102
103
104
9.7% 10.9%
0.77% 0.67%
1.62%
89.4% 86.6%
1.96%
1.34% 0%
0.001%
H2-Kd (PE)
GFP
Cell line Targeting (%) H2-Kd loss (%)
among
uncorrected corrected
#1 0.42 12.1 67.6
#1 1.04 29.3 69.9
#2 1.96 1.3 50.9
#2 4.11 3.4 50.5
no I-SceIH2-Kd stained
+ I-SceIstained H2-Kd
+ I-SceIunstained
Correction @ A Mutation @ B
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