crispr edits ice software tool  (Synthego Inc)

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 shows high editing efficiency in PGCs. (A) Phase-contrast images of cultured PGCs isolated from embryonic blood, showing typical colony morphology after 3 weeks in vitro . Left: male PGC colony; right: female PGC colony. Scale bar = 20 µm. (B) Immunofluorescence for germ cell markers in PGCs. These cells (male line shown) strongly express SSEA-1 (green, cell surface) and VASA/DDX4 (red, cytoplasm), even after long-term culture (>50 days). Nuclei are counterstained with DAPI (blue). Scale bar = 5 µm. (C) Representative fluorescence microscopy of EGFP + PGCs 5 days after co-electroporation with Cas9 and sgRNAs targeting EGFP (gEGFP1+2). Left: cells electroporated with Cas9 mRNA at 1 µg, 2 µg, or 3 µg (with constant gRNA amount). Right: cells electroporated with Cas9 protein (RNP complex) at equivalent molar doses (1:1.2 Cas9:sgRNA ratio). In both mRNA and protein conditions, higher Cas9 doses result in loss of EGFP fluorescence and reduced cell numbers (rounding and death) compared to lower doses. Scale bar = 20 µm. (D) Flow cytometry analysis of EGFP fluorescence and cell viability in edited versus control PGCs. Left: histogram overlays of EGFP intensity for control (untreated EGFP + PGCs, gray) vs. CRISPR-edited cells (green). Cas9-edited populations shift toward lower fluorescence, indicating EGFP knockout. Upper right: bar graph quantifying the percentage of EGFP + cells in each group (mean ± SEM, n = 3). Both Cas9 mRNA and Cas9 protein treatments caused a dose-dependent decrease in the fraction of EGFP-expressing cells compared to control (p-values are indicated in the figure by one-way ANOVA). Lower right: plot showing the percentage of live cells recovered during flow cytometry. Higher Cas9 doses correlate with reduced live-cell recovery, reflecting CRISPR-induced cytotoxicity in PGCs. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: The amplicons were subjected to Sanger sequencing, and sequencing traces were analyzed using the Inference of CRISPR Edits (ICE) software tool (v3.0, Synthego).

Techniques: CRISPR, Cell Culture, Isolation, In Vitro, Immunofluorescence, Fluorescence, Microscopy, Electroporation, Flow Cytometry, Control, Knock-Out, Expressing, Cell Recovery

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 induces DNA damage and apoptosis in PGCs. (A) Flow cytometry analysis 24 h after electroporation, quantifying the proportion of Annexin V + /PI + cells. The horizontal axis indicates PI and the vertical axis Annexin V. The upper-left quadrant (Annexin V + /PI + ) represents late apoptotic cells, and the lower-right quadrant (Annexin V + only) represents early apoptotic cells. Upper panels: results after electroporation with Cas9 + various gRNAs; lower panels: results with dCas9 + various gRNAs. (B) Bar graph of Annexin V + /PI + percentages across groups. Cas9 editing induced a highly significant increase in late apoptosis. (C) γ-H 2 AX foci (green) detected by immunofluorescence 24 h after electroporation. Foci appear as discrete nuclear puncta; nuclei are counterstained with DAPI (blue). Scale bar = 10 µm. (D) Quantification of γ-H 2 AX foci per cell. Cas9 targeting resulted in a significant increase in γ-H 2 AX foci per cell, whereas dCas9 with sgRNA did not. Statistical significance determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: The amplicons were subjected to Sanger sequencing, and sequencing traces were analyzed using the Inference of CRISPR Edits (ICE) software tool (v3.0, Synthego).

Techniques: CRISPR, Flow Cytometry, Electroporation, Immunofluorescence

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPRi has limited efficacy in gene knockdown in PGCs. (A) Schematic of the CRISPR interference (CRISPRi) system. (i) The PGK-CRISPRi-EGFP plasmid expresses dCas9-KRAB (catalytically inactive Cas9 fused to the KRAB repressor) and an EGFP marker under a constitutive PGK promoter. (ii) The gCAG-mCherry plasmid carries a U6.3 promoter–driven sgRNA targeting the CAG promoter and a CAG-driven mCherry reporter. (iii) Co-transfection strategy: dCas9-KRAB (plasmid i) is expressed in the cell, and the sgRNA (plasmid ii) guides it to the CAG promoter in the mCherry cassette, silencing mCherry transcription. (B) Summary of CRISPRi reporter knockdown efficacy in human 293T cells vs. chicken cells. Bars show the percentage of mCherry + cells in each condition (no sgRNA, mock control, +gCAG sgRNA). In 293T cells, introducing the CAG-targeting sgRNA significantly reduces the mCherry + fraction relative to controls, whereas in DF-1 cells the mCherry + percentage remains unchanged, and in PGCs only a slight decrease is observed. (C) Expression of the dCas9-KRAB-EGFP fusion protein in CRISPRi. Western blot confirmed that dCas9-KRAB-EGFP is only expressed in CRISPRi cells, indicating the successful construction of CRISPRi stable PGC cell lines. Blank: Untransfected cells served as the negative control. (D) Gene expression following CRISPRi-mediated knockdown in CRISPRi cells. qRT-PCR showed no significant reduction in expression of the target genes for which CRISPRi sgRNAs were designed. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: The amplicons were subjected to Sanger sequencing, and sequencing traces were analyzed using the Inference of CRISPR Edits (ICE) software tool (v3.0, Synthego).

Techniques: Knockdown, CRISPR, Plasmid Preparation, Marker, Cotransfection, Control, Expressing, Western Blot, Negative Control, Gene Expression, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: Autophagy modulates glioblastoma cell sensitivity to Selinexor-mediated XPO1 inhibition

doi: 10.1093/neuonc/noae280

Figure Lengend Snippet: Enhanced autophagic flux is an on-target effect of Selinexor in glioblastoma cells. ( A ) DNA sequence read from GBM43 cells ( bottom ) and GBM43 cells in which a CRISPR knock-in approach ( top ) encoding C528S mutant (mut) XPO1. ( B ) The visualization ( top ) and lowest bind energy ( bottom ) of molecular docking between Selinexor (Slx) and XPO1 wildtype/mut protein. ( C ) Representative immunofluorescence analysis ( top ) and quantitation ( bottom ) of GBM43 and GBM43mut cells incubated for 24 h with 0 or 1000 nM Selinexor, then analyzed for RanBP1 co-localization with nuclear DAPI staining (10–15 cells/group). ( D ) ( Left ) cell viability (normalized to control cells) of GBM43 and GBM43mut cell lines following 72 h continuous incubation with varying concentrations of Selinexor, and ( right ) IC50 values of Selinexor for GBM43 and GBM43mut cell lines. ( E ) Representative photos of clonogenic assays performed using GBM43 and GBM43mut cells continuously exposed to 0, 100, 200, or 400 nM Selinexor for 14 days. ( F ) Representative Western blot analysis of LC3B-I, LC3B-II, and ACTIN levels in GBM43 and GBM43mut cells lines incubated with DMSO, Selinexor (750 nM, 72 h) or rapamycin (500 nM, 16 h), or with non-targeted siRNA or a pool of siRNA targeting XPO1 (48 h after a 24-h siRNA incubation). ( G ) Viable number of GBM43 and GBM43mut cells (normalized to control cells) monitored daily after incubation with non-targeted siRNA or a pool of siRNA targeting XPO1 as in (F). Except where noted, all quantitated values listed are the means of 3 experiments. CYS, Cysteine; SER, Serine. ***, P ≤ .001.

Article Snippet: Complete replacement of wildtype (WT) XPO1 with C528S mutant (mut) XPO1 was confirmed using Synthego’s Inference of CRISPR Edits (ICE) software tool (Synthego Performance Analysis, ICE Analysis, v3.0).

Techniques: Sequencing, CRISPR, Knock-In, Mutagenesis, Immunofluorescence, Quantitation Assay, Incubation, Staining, Control, Western Blot

Journal: bioRxiv

Article Title: CRISPR screening reveals SYCP3 as a key driver of metastasis in prostate cancer

doi: 10.1101/2025.01.30.629925

Figure Lengend Snippet: A The Inference of CRISPR Edits (ICE) software output of the analyses of the Sanger sequencing data on the SYCP3 gene part flanking the exon 1 of the ORF. B Invasion analysis in DU145 cells using 10% FBS as a chemoattractant. Top panels, representative images of cells (bars: 100 µm); bottom panels, histograms showing the mean value ± S.E.M. of the percentage of invasive cells (n = 3). C Migration analysis of DU145 cells using a wound healing assay (n=3). D-F Adhesion/spreading analysis of DU145 cells. Representative images of adhered DU145 cells (C) and histograms showing the mean value ± S.E.M. of the percentage of adhered cells (D) or areas covered (E) of SYCP3 depleted cells referred to NTC cells (n = 3). Scale bars: 100 μm. G Immuno-fluorescence microscopy images of phalloidin staining ( red ) adhered NTC and SYCP3 depleted DU145 cells. Cell nuclei were stained with DAPI ( blue ). Scale bars: 100 μm. H-I Histograms showing the cytoskeleton (G) or nuclei area value ± S.E.M. of adhered DU145 indicated cells (n=2). Statistic tests: Student t test ( B, C ), two-way ANOVA ( E, F ) and U-Mann Whitney test ( H, I) . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: DNA sequencing chromatograms were analyzed using the Inference of CRISPR Edits (ICE) free software tool (synthego.com/products/bioinformatics/crispranalysis).

Techniques: CRISPR, Software, Sequencing, Migration, Wound Healing Assay, Fluorescence, Microscopy, Staining, MANN-WHITNEY