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Lentiviral Design for Delivery of Homing Endonucleases

R. R. U5. U5. RRE. sinU3. RSV. Repair Template. Cppt. hPGK. HA-NLS-HE. mCherry. Lentiviral Design for Delivery of Homing Endonucleases. RRL Lentiviral Backbone. untreated. 1:10. 1:50. 1:100. 1:500. 1:1000. psPAX2. D64V. Integrase Deficient Lenti Virus. DAY 2. DAY 12.

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Lentiviral Design for Delivery of Homing Endonucleases

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  1. R R U5 U5 RRE sinU3 RSV Repair Template Cppt hPGK HA-NLS-HE mCherry Lentiviral Design for Delivery of Homing Endonucleases RRL Lentiviral Backbone

  2. untreated 1:10 1:50 1:100 1:500 1:1000 psPAX2 D64V Integrase Deficient Lenti Virus DAY 2 DAY 12 DAY 8 DAY 5 Day 19 post transduction: 0.62% of cells remain GFP+

  3. Directions • Effect of local chromatin structure on the efficiency of cutting and repair • Role of repair template topology on conversion frequency

  4. HEs for Targeted Integration Can an HE induced double stranded break facilitate targeted transgene integration? Genomic -7C+11C Sce Site in Canine

  5. recSce cleaves +11C miscognante linearized 0.2units 5units 0.5units -7C +11C -7C +11C -7C +11C -7C +11C Substrate: Ani Ani WT -7C +11C WT -7C +11C Ani Ani WT -7C +11C WT -7C +11C

  6. 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

  7. 70 bp S H G Intron eBFP HE Substrate eBFP1-52 eBFP53-238 Asc1 Age1 Swappable HE substrate T Y G Fluorophore “Switch” Repair Template T Y G eGFP Repair Product eGFP53-238 Asc1 Age1 Exogenous Template HDR Blue-Green Reporter System • 2 nucleotide changes switch fluorophores • Swappable HE cleavage site 70bp from target, does not affect reporter • Intron allows use of >1Kb repair template

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