lentiviral design for delivery of homing endonucleases mcherry ha-nls-he rsvr u5 hpgk r u5sinu3...
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Lentiviral Design for Delivery of Homing Endonucleases
mCherry
HA-NLS-HERSV R U5 hPGK R U5sinU3Repair Template CpptRRE
RRL Lentiviral Backbone
0 102 103 104 105
Texas Red PE-A
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f Ma
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0 102 103 104 105
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102
103
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Texa
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PE
-A
0.47 0
1.4298.1
0 102 103 104 105
FITC-A
0
102
103
104
105
Texa
s R
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PE
-A
89.1 3.29
0.0837.54
0 102 103 104 105
FITC-A
0
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C-A
0.052
0 102 103 104 105
FITC-A
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SS
C-A
96.4
0 102 103 104 105
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C-A
59.8
0 102 103 104 105
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SS
C-A
33.1
0 102 103 104 105
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C-A
11.8
0 102 103 104 105
FITC-A
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C-A
5.96
0 102 103 104 105
FITC-A
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C-A
0.1
0 102 103 104 105
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SS
C-A
62
0 102 103 104 105
FITC-A
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C-A
10.9
0 102 103 104 105
FITC-A
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C-A
5.17
0 102 103 104 105
FITC-A
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0.86
0 102 103 104 105
FITC-A
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SS
C-A
0.94
0 102 103 104 105
FITC-A
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SS
C-A
0.16
0 102 103 104 105
FITC-A
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SS
C-A
0.29
0 102 103 104 105
FITC-A
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SS
C-A
0.066
0 102 103 104 105
FITC-A
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SS
C-A
0.074
0 102 103 104 105
FITC-A
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SS
C-A
0.051
0 102 103 104 105
FITC-A
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SS
C-A
0.039
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SS
C-A
0.051
0 10 2 10 3 10 4 10 5
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SS
C-A
94.4
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SS
C-A
50.4
0 10 2 10 3 10 4 10 5
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SS
C-A
29.1
0 10 2 10 3 10 4 10 5
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SS
C-A
10.2
0 10 2 10 3 10 4 10 5
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C-A
5.13
0 10 2 10 3 10 4 10 5
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C-A
0.039
0 10 2 10 3 10 4 10 5
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C-A
20.1
0 10 2 10 3 10 4 10 5
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SS
C-A
2.19
0 10 2 10 3 10 4 10 5
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SS
C-A
0.92
0 10 2 10 3 10 4 10 5
FITC-A
0
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SS
C-A
0.17
0 10 2 10 3 10 4 10 5
FITC-A
0
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SS
C-A
0.21
0 10 2 10 3 10 4 10 5
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SS
C-A
0.1
0 10 2 10 3 10 4 10 5
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SS
C-A
0.24
0 10 2 10 3 10 4 10 5
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SS
C-A
0.08
0 10 2 10 3 10 4 10 5
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SS
C-A
0.053
0 10 2 10 3 10 4 10 5
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SS
C-A
0.039
0 10 2 10 3 10 4 10 5
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C-A
0.055
0 10 2 10 3 10 4 10 5
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SS
C-A
0.14
0 10 2 10 3 10 4 10 5
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SS
C-A
93.5
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C-A
48.8
0 10 2 10 3 10 4 10 5
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27.1
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8.98
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4.82
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0
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3.14
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0.46
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0.16
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0.042
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0.06
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0.028
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0.058
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0.11
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0.042
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0.028
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0.015
Integrase Deficient Lenti Virus
0 10 2 10 3 10 4 10 5
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0.11
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96.8
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64.4
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40.2
0 10 2 10 3 10 4 10 5
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16.6
0 10 2 10 3 10 4 10 5
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10.3
0 10 2 10 3 10 4 10 5
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0.15
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93.6
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48.5
0 10 2 10 3 10 4 10 5
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0
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27.5
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C-A
5.55
0 10 2 10 3 10 4 10 5
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6.55
0 10 2 10 3 10 4 10 5
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2.04
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14.7
0 10 2 10 3 10 4 10 5
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1.76
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1.5
0 10 2 10 3 10 4 10 5
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1.24
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3.04
psP
AX
2D
64V
untreated 1:10 1:50 1:100 1:500 1:1000
DAY 2DAY 12DAY 8DAY 5
Day 19 post transduction:0.62% of cells remain GFP+
Directions
• Effect of local chromatin structure on the efficiency of cutting and repair
• Role of repair template topology on conversion frequency
HEs for Targeted Integration
A G A T A G G T C A G G G A T A A C A G G G T A A C G C G T A A C A G T
A G A T A G G T C A G G G A T A A C A G G G T A A C G C G T A A C A G T207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242
1696
0
Fragmentmc9_B03
Can an HE induced double stranded break facilitate targeted transgene integration?
Genomic -7C+11C Sce Site in Canine
WT -7C +11C-7C
+11CAni WT -7C +11C
-7C+11C
Ani WT -7C +11C-7C
+11CAni WT -7C +11C
-7C+11C
AniSubstrate:
recSce cleaves +11C miscognante
linearized 5units 0.5units 0.2units
Directions
• Characterize -7C-Sce cleavage of the target site using DR-reporter
• Assess cleavage of the genomic site in-vivo
• Attempt to target reporter to the locus
• 2 nucleotide changes switch fluorophores
Exogenous Template HDR Blue-Green Reporter System
• Swappable HE cleavage site 70bp from target, does not affect reporter • Intron allows use of >1Kb repair template
T Y G
Fluorophore “Switch” Repair Template
Asc1 Age1
eGFP53-238
T Y G eGFP Repair
Product
S H G
Asc1
Age1
eBFP1-52 eBFP53-238Intron
70 bp
eBFP HE Substrate
Swappable HE substrate
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