super piggybac transposase expression vector  (Addgene inc)

Journal: bioRxiv

Article Title: Image-based, pooled phenotyping reveals multidimensional, disease-specific variant effects

doi: 10.1101/2025.07.03.663081

Figure Lengend Snippet: (A) VIS-seq uses fluorescent in situ sequencing of abundant circular RNA barcodes to genotype cells expressing protein variants. (1) A variant library in the VIS-seq expression cassette is integrated into cells via piggyBac -ase. (2) Cells are fixed; barcodes are reverse transcribed, captured with a padlock probe and amplified; (3) cells are stained and imaged; (4) barcode is sequenced in situ ; (5) single cell phenotype-genotype pairs are determined using STARCall; and (6) features for each cell are extracted using CellProfiler. (B) Schematic of VIS-seq expression cassette. PB = piggyBac ; PD = padlock probe binding site; UCOE = universal chromatin opening element ; HBB IVS2 = hemoglobin subunit beta intervening sequence 2 , ; IRES = internal ribosome entry site ; WPRE = woodchuck hepatitis virus post-transcriptional regulatory element ; cHS4 = chicken beta globin locus control region hypersensitive site 4 . (C) Time-series plot of mEGFP positivity of human WTC11 iPS cells and derived cardiomyocytes. mEGFP-tagged WT histone H1.4 is expressed via the VIS-seq cassette (blue) or lentivirus (red). Two replicates are plotted. (D) Violin plot of the number of rolling circle amplification colonies per cell after one cycle of sequencing for VIS-seq expression cassette (blue) and lentivirus (red) cells from 1F at day 14. Silenced cells were excluded. Two replicates are shown. (E) Images of VIS-seq library of mEGFP-tagged lamin A variants in U2OS cells, stained with DAPI, phalloidin, WGA, and mitoprobe (left). First base in situ sequencing of the same field-of-view; magenta=guanine, blue=thymidine, yellow=adenine, and red=cytosine (right). Some cells express lamin A variants that mislocalize into aggregates. Scale bar indicates 20 μm. (F) Images of libraries of mEGFP-tagged PTEN in human WTC11 PTEN -KO inducible-NGN2 iPS cells and mEGFP-tagged H1.4 and RPS19 in human WTC11 iPS cells, stained with DAPI and phalloidin-CF568 (left). Imaging of mEGFP-tagged PTEN library in derived neurons, seven days after induction of NGN2, stained with DAPI and anti-MAP2 AF568 (right). Some cells express variants with localization defects (PTEN=nucleocytoplasmic, HIST1H1E=chromatin binding, RPS19=nucleolar-cytoplasmic). Scale bar indicates 20 μm. (G) Imaging after first base incorporation of VIS-seq libraries from (F). PTEN library contains two 8bp barcodes per variant in each cell, whereas other libraries have one 12bp barcode per cell. Nucleobase coloring identical to (C). Scale bar indicates 20 μm.

Article Snippet: pHSG299 (Takara Bio, 3299) pHR-UCOE-SFFV-Zim3-dCas9-P2A-EGFP (Addgene, 188899) piggyFlex (Addgene, 218234) lentiGuide-Puro (Addgene, 52963) pCAG-NLS-HA-Bxb1 (Addgene, 51271) Hyperactive piggyBac transposase expression vector (hyPBase; gift from Jay Shendure lab) pMD2.G (Addgene, 12259) psPAX2 (Addgene, 12260)

Techniques: In Situ, Sequencing, Expressing, Variant Assay, Reverse Transcription, Amplification, Staining, Binding Assay, Virus, Control, Derivative Assay, Imaging

Journal: bioRxiv

Article Title: Image-based, pooled phenotyping reveals multidimensional, disease-specific variant effects

doi: 10.1101/2025.07.03.663081

Figure Lengend Snippet: (A) Fraction of cells with 1, 2, or 3 integrations, determined by 4-base in situ sequencing, after co-transfection of LMNA VIS-seq library DNA at different quantities (in nanograms) with plasmid encoding Piggybac-ase (at a 4-fold lower mass). (B) Imaging of day-7 neurons derived from clonal NGN2-inducible PTEN knockout lines stained for DAPI (blue) and NCAM1 (red). Scale bar indicates 20 μm. (C) Western blot of U2OS cells showing parental line (left) and LMNA knockout lines (right) stained for lamin A protein. (D) Western blot of NGN2-inducible iPS cells showing parental line (left) and PTEN knockout lines (right) stained for PTEN and phospho-AKT protein. Clonal lines C2 and C18 were used for PTEN VIS-seq experiments.

Article Snippet: pHSG299 (Takara Bio, 3299) pHR-UCOE-SFFV-Zim3-dCas9-P2A-EGFP (Addgene, 188899) piggyFlex (Addgene, 218234) lentiGuide-Puro (Addgene, 52963) pCAG-NLS-HA-Bxb1 (Addgene, 51271) Hyperactive piggyBac transposase expression vector (hyPBase; gift from Jay Shendure lab) pMD2.G (Addgene, 12259) psPAX2 (Addgene, 12260)

Techniques: In Situ, Sequencing, Cotransfection, Plasmid Preparation, Imaging, Derivative Assay, Knock-Out, Staining, Western Blot