NGEC Applications Meeting

05-06-08 Mike Certo Scharenberg Lab

Transient DR-GFP Assay

In-Vitro recSce Cleavage of Canine Site linearized Substrate: WT -7C +11C -7C +11C Ani 5units WT -7C +11C -7C +11C Ani 0.5units

WT -7C +11C -7C +11C Ani 0.2units

WT -7C +11C -7C +11C Ani An endogenous intergenic 2-off Sce site (-7C +11C) in the Canine genome is a potential safe harbor site for transgenes. Linearized plasmid DNA containing the indicated targets was digested with recombinant I-SceI to assess cleavability.

Transient DR-GFP Assay for In-vivo Enzyme/Target Discrimination Untreated WT-DR WT-DR WT-DR GFP control Sce DR alone Sce DR + I-SceI

WT-Sce

-7C-DR -7C-DR

untreated I-AniI vs Y2 In-Vivo Ani-DR Ani-DR + Ani Ani-DR + Ani-Y2

I-SceI is Intolerant of C-terminal Fluorescent Protein Fusions I-SceI Sce DR-GFP I-SceI -mCherry I-SceI-3xG4S -mCherry I-SceI-IRES -mCherry Transient DR assay to assess functionality of Sce-Cterm fusions

Conclusions: Transient DR-GFP

Assay

• Can be used to quickly discern activity between enzyme variants and targets.

• Signal can be adjusted by varying DNA input.

• Provides information in the context of mammalian gene conversion.

Experimental

• -7C + 11C Canine target will likely require enzyme optimization • WT I-AniI induces gene conversion at suboptimal rates • Y2 variant performs similar to I-SceI • I-SceI is non-permissive of C terminal fluorescent fusions, but IRES allows for enzyme function and detection

Blue-Green Color Change

Trans-HDR Blue-Green Reporter eBFP 1-52 70 bp Intron Asc1 Swappable HE substrate Age1 S H T Y Asc1 Age1 T Y eBFP 53-238 eGFP 53-238 • 2 nucleotide changes switch fluorophores • Swappable HE cleavage site • Intron allows use of >1Kb repair template eBFP HE Substrate Fluorophore “Switch” Repair Template eGFP Repair Product

Lenti Viral Vector for Color Change Reporter

AmpR

9215 bp

F1 ORI SV40 pA/ORI PolyA U3*RU5 Nef* WPRE PuroR RSV RU5 PBS (SL123) Gag (SL4) RRE cPPT IRES eBFP N-term SA SD mouse IL2Rgamma Intron 4 YTRAY Branch point I-Sce target site S65 H66 eBFP

Color Change Reporter Transduction 100,000 293T cells transduced with either RRL eGFPsce reporter or RRL eBFPsce reporter. Based on eGFP positive cells at 1uL of viral supernatant, 7 titer is estimated at ~ 2 x10 I.U./ml giving M.O.I ~ 0.5 . This should yeild a population averaging 1 integration per cell.

Single Cell Sorting We gated on increasingly stringent populations (3% to 0.5%) and single cell sorted into 96 wells plates. As clones come up, we will re-analyze by flow for expression, and conduct southern blots to determine single cell integrants.

Integrated Reporter Is Unspliced I-SceI Digestion of gDNA PCR Product PCR of Genomic DNA Following Transduction Across Intron Non-spliced product yeilds: 1300bp Spliced product yeilds: 720bp Within Intron Non-spliced product yeilds: 1140bp Spliced product yeilds: no band The PCR product of the integrated reporter was digested with recombinant Sce to ensure the presence of a functional target 1500bp 1000bp No unspliced products were detected 1500bp 1000bp I-SceI site is cleavable

untreated Gene Conversion repair template alone sce expression alone Sce + repair template Polyclonal population of puro selected cells were transfected with a plasmid containing I-Sce expression and an eGFP repair template. Cells were FACS 5 days post transfection.

Effect of Homology and Track Length on Conversion in Trans untreated repair Sce Sce + repair 1uG Sce + repair 2uG Sce + repair 5uG untreated repair Sce Sce + repair 1uG Sce + repair 2uG Sce + repair 5uG

Disparity of Gene Conversion at Distinct Loci Single cell clones from the polyclonal population were subjected to gene conversion assay 293T polyclonal Single Cell Clones

IDLV Gene Conversion ~ 60,000nG p24 untreated repair alone Sce alone Sce + repair

Conclusions: Blue-Green Color Change

Assay

• eBFP likely detectable as single integrant at particular loci • Conversion rates of ~ 3% • Blue to Green? Fluorescent protein stability an issue?

Experiments

• Single cell clones exhibit differential conversion ability • Low expression off IDLV contributes to inefficient repair