NGEC Applications Meeting Mike Certo Scharenberg Lab

Transient DR-GFP Assay

WT-7C+11C -7C +11C Ani WT-7C+11C -7C +11C Ani WT-7C+11C -7C +11C Ani WT-7C+11C -7C +11C Ani Substrate: In-Vitro recSce Cleavage of Canine Site linearized 5units0.5units 0.2units 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 GFP control Sce DR alone Sce DR + I-SceI Untreated

I-AniI vs Y2 In-Vivo untreatedAni-DRAni-DR + AniAni-DR + Ani-Y2

I-SceI is Intolerant of C-terminal Fluorescent Protein Fusions - I-SceI -mCherry I-SceI-3xG4S -mCherry I-SceI-IRES -mCherry Sce DR-GFP 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

2 nucleotide changes switch fluorophores Trans-HDR Blue-Green Reporter Swappable HE cleavage site Intron allows use of >1Kb repair template T Y Fluorophore “Switch” Repair Template Asc1Age1 eGFP T Y eGFP Repair Product S H Asc1Age1 eBFP 1-52 eBFP Intron 70 bp eBFP HE Substrate Swappable HE substrate

Lenti Viral Vector for Color Change Reporter pRRL SFFV eBFP IRES Puro Sce WPRE 9215 bp AmpR Gag (SL4) PuroR eBFP N-term eBFP mouse IL2Rgamma Intron 4 RU5 U3*RU5 SFFV PBS (SL123) RRE cPPT WPRE PolyA IRES SD YTRAY Branch point SA I-Sce target site RSV F1 ORI SV40 pA/ORI Nef* S65 H66

Color Change Reporter Transduction 100, T cells transduced with either RRL eGFPsce reporter or RRL eBFPsce reporter. Based on eGFP positive cells at 1uL of viral supernatant, 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. 7

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

untreated repair template alonesce expression aloneSce + repair template Gene Conversion 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 untreatedrepairSce Sce + repair 1uG Sce + repair 2uG Sce + repair 5uG untreatedrepairSce Sce + repair 1uG Sce + repair 2uG Sce + repair 5uG eBFP azBFP

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

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

Assay eBFP likely detectable as single integrant at particular loci Conversion rates of ~ 3% Blue to Green? Fluorescent protein stability an issue? Conclusions: Blue-Green Color Change Experiments Single cell clones exhibit differential conversion ability Low expression off IDLV contributes to inefficient repair