1. gfp - Gene correction in trans: a model for gene therapy Luther Davis Jason Cummings Nancy Maizels

2. GFP + Gene correction in trans: a model for gene therapy Luther Davis Jason Cummings Nancy Maizels

3. Gene correction in trans PuroeGFPPoly-lacO Cut sites trGFP nuclease PuroeGFPPoly-lacO

4. Gene correction in trans PuroeGFPPoly-lacO Cut sites trGFP nuclease PuroeGFPPoly-lacO

5. I-AniI Y2 promotes correction in trans EndoDonor% GFP + I-SceI- 0.002  0.0004 Ani Y2 - 0.001  0.0006 -+ 0.002  0.0005 I-Sce+ 0.15  0.03 (75) Ani Y2 +0.06  0.01 (30) GFP + donor + I-SceI + donor + Ani Y2 FL2-H

6. I-AniI Y2 “nickase” promotes correction in trans I-AniIDonor% GFP + Y2- 0.002  0.001 Y2-nick- 0.001  0.0007 -+ 0.002  0.001 Y2+ 0.06  0.006 (30) Y2-nick+0.01  0.003 (5) + donor + Ani Y2 + donor + Ani Y2-nick GFP FL2-H

7. Modulation of gene correction in trans: tethering factors at recipient PuroeGFPPoly-lacO Cut sites trGFP nuclease tethered factor tethered factor PuroeGFPPoly-lacO

8. Recipient chromatin structure regulates gene correction in trans Tethered Protein % GFP + (fold reduction) * I-SceI----0.007 I-SceI + donor ----0.24 I-SceI + donor LacI0.20 I-SceI + donor HP1-LacI0.017 (11) * Average of two experiments + HP1-LacI GFP FL2-H + LacI

9. Modulation of gene correction in trans: tethering factors at donor PuroeGFP Cut sites trGFP nuclease tethered factor tethered factor PuroeGFP Poly-lacO

10. 10 0 10 1 10 2 10 4 10 3 10 0 10 1 10 2 0.24% HP1 tethered at donor blocks correction in trans LacO donor + ISceI LacO donor + ISceI + HP1-LacI LacO donor + ISceI + LacI 10 4 10 0 10 1 10 2 10 4 10 3 10 0 10 1 10 2 10 4 10 0 10 1 10 2 10 4 10 3 10 0 10 1 10 2 10 4 0.005% 0.22% GFP FL2 Height 40-fold decrease in recombination Jason Cummings