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