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: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: A) Induction of embryonic calli from male flowers of Bhimkol after 6 months of culture. B) A magnified view at 40x of embryogenic calli. C) Establishment of ECS after 8 months of culture was used for genetic transformation mediated through Agrobacterium harboring pSS03. D) Embryonic calli in selection medium with 40 mg/L hygromycin, and 250 mg/L timentin. E) A magnified view of putative transformed mature somatic embryos at 40x magnification. F) Shoots regeneration in M4 medium containing 40 mg/L hygromycin, and 250 mg/L timentin after one month of culture. G) Rooting from i n vitro regenerated shoots in the R1 rooting medium containing 40 mg/L hygromycin, and 250 mg/L timentin after 14 days of culture. H) Putative CRISPR-edited Bhimkol plantlets with healthy shoots and roots, ready for hardening.

Article Snippet: Utilizing ICE (Inference of CRISPR Edits) software from Synthego provides a high level of bioinformatic confidence.

Techniques: Transformation Assay, Selection, CRISPR

Journal: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: CRISPR-edited Bhimkol plants transferred to the transgenic net house, A) 03_01, B) 03_05, C) 03_06, D) 03_17 and E) Wild Type (Control)

Article Snippet: Utilizing ICE (Inference of CRISPR Edits) software from Synthego provides a high level of bioinformatic confidence.

Techniques: CRISPR, Transgenic Assay, Control

Journal: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: Sequence analysis of the INO gene in CRISPR/Cas12a-edited lines. Amino acid sequences from lines 03_01, 03_05, 03_06, and 03_17 were aligned with the wild type sequence. Red asterisks indicate stop codons.

Article Snippet: Utilizing ICE (Inference of CRISPR Edits) software from Synthego provides a high level of bioinformatic confidence.

Techniques: Sequencing, CRISPR

Journal: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: Analysis of YABBY and HMG-box_2 domain in the WT and CRISPR-edited lines. The CRISPR-edited Bhimkol lines exhibit a complete absence of the HMG_box_2 and truncated YABBY domains.

Article Snippet: Utilizing ICE (Inference of CRISPR Edits) software from Synthego provides a high level of bioinformatic confidence.

Techniques: CRISPR

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production

doi: 10.1016/j.omtm.2025.101598

Figure Lengend Snippet: Testing of two different gRNAs to induce double-strand breaks in the IGH locus, in the absence of a donor template (A) Apheresis RM WBCs were thawed, and enriched B cells were stimulated for 2 days in culture. At day 3, the cells were electroporated in the presence of a CRISPR-Cas9 gRNA complex targeting the IGH locus. Genomic DNA was isolated 2 and 5 days post-editing, PCR-amplified, and sequenced. (B) The Sanger sequencing was analyzed by ICE to quantify the indels. Two gRNAs targeting the IGH locus were tested (gRNA 1 and gRNA 2) in B cells from healthy donor animals (uninfected) or from SHIV-infected and ART-treated animals (SHIV + ART-suppressed). Two-tailed paired t tests were performed. p values are shown in the figure. (C) The MMEJ and NHEJ indel signatures were quantified at 2 days and 5 days post-editing. The data shown are representative of three different experiments in three different donors. Paired t tests were performed. p values are shown in the figure. (A) was generated using BioRender. Statistical analyses were performed using Prism software.

Article Snippet: The analyses were performed using Inference of CRISPR Edits (ICE) software by Synthego (Redwood City, CA).

Techniques: CRISPR, Isolation, Amplification, Sequencing, Infection, Two Tailed Test, Generated, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production

doi: 10.1016/j.omtm.2025.101598

Figure Lengend Snippet: CRISPR-Cas9 RNA-guided editing in the IGH locus leads to VRC01 knockin (A) Schematic of the engineered VRC01 antibody and its VH4a promoter. (B) B cells were isolated from healthy donor (uninfected) RM. The cells were stimulated for 2 days in culture before electroporation in the presence of a CRISPR-Cas9 gRNA complex followed by transduction 30 min later with an scAAV-6 virus encoding the donor template. The genomic DNA was isolated 5 and 8 days post-editing, PCR-amplified, and sequenced. The Sanger sequencing was analyzed by ICE to quantify the indels. This figure is representative of three independent experiments using B cells from three different donors. Paired t test was performed using Prism with a resulting p value of 0.3514. ns, nonsignificant. (C) Schematic of the in-out PCR. Arrows: forward and reverse primers; UHA: upstream homology arm; DHA: downstream homology arm; VH4a: B-cell-specific promoter; VRC01: engineered anti-HIV bNAb; SS: splicing site. (D) VRC01 in-out PCR demonstrates VRC01 cassette knockin at the expected location in the IGH locus. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus.

Article Snippet: The analyses were performed using Inference of CRISPR Edits (ICE) software by Synthego (Redwood City, CA).

Techniques: CRISPR, Knock-In, Isolation, Electroporation, Transduction, Virus, Amplification, Sequencing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production

doi: 10.1016/j.omtm.2025.101598

Figure Lengend Snippet: B cell editing leads to VRC01 bNAb expression (A) Total RNA was extracted from edited B cells 5 days post-electroporation and -transduction. VRC01 mRNA amplification was analyzed by reverse transcription PCR. (B) VRC01 bNAb at the cell surface was also detected by flow cytometry at 8 days post-editing using an antibody directed against the Strep-tag II linker (Strep-Tactin PE). This experiment is representative of three independent experiments using three different donor animals. A summary of the three replicates is shown in (C). (D and E) The secreted VRC01 absorbances (D) and concentrations (E) were measured by ELISA using a secondary antibody directed against the Gly-Ser-StrepII linker (D) or anti-monkey IgG secondary antibody (E). B cell supernatants from 5 days (d5) and 8 days (d8) post-editing were either undiluted or diluted 1:2 or 1:4. The data are representative of a single experiment. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 virus transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus. Paired t test statistics were performed using GraphPad Prism. ns, nonsignificant; ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The analyses were performed using Inference of CRISPR Edits (ICE) software by Synthego (Redwood City, CA).

Techniques: Expressing, Electroporation, Transduction, Amplification, Reverse Transcription, Flow Cytometry, Strep-tag, Enzyme-linked Immunosorbent Assay, Virus, CRISPR