crispr guide rna design tool  (Benchling Inc)

Journal: Journal of Human Immunity

Article Title: Ribosomal RNA processing impairments in a B cell immunodeficient patient with WDR75 variants

doi: 10.70962/jhi.20250061

Figure Lengend Snippet: U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site (guide sequence) of CRISPR/Cas9 modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: .

Article Snippet: KO score for each sequence was evaluated using Inference of CRISPR Edits (RRID:SCR_024508) software (Performance Analysis, 2019. v3.0; Synthego).

Techniques: Expressing, Sequencing, CRISPR, Modification, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Northern Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Dual inhibition of intercellular adhesion molecule-1 and nucleolin reduces RSV infection efficiency in human respiratory organoids

doi: 10.1016/j.omtn.2026.102932

Figure Lengend Snippet: ICAM-1- and EGFR-KO iPSCs were established using CRISPR-Cas9 system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.

Article Snippet: Analysis was done using inference of CRISPR edits (ICE) ( https://www.synthego.com/help/ice ) showed that the KO scores for ICAM-1-KO and EGFR-KO iPSCs were nearly 100 ( A and A).

Techniques: CRISPR, Knock-Out, Generated, Immunofluorescence, Expressing, Derivative Assay, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Deletion of mitochondrial calcium uniporter enhances calcium signals by slowing calcium clearance and triggers adaptive transcriptomic remodeling

doi: 10.1016/j.jbc.2026.111473

Figure Lengend Snippet: CRISPR/Cas9-mediated knockout of Mcu in RBL-2H3 cells. A , exon structure of rat Mcu indicating the target sites of guide RNA1 and guide RNA2. B , quantitative real-time PCR analysis of Mcu mRNA expression in wild-type (WT) RBL-2H3 cells and Mcu knockout (KO) clones A4 and D10 (n = 30 wells; 3 biological replicates). Medians ( bars ) with 95% confidence intervals (error bars ) are displayed on the scatter dot plots. C , immunoblot analysis of WT and Mcu KO clones A4 and D10 using MCU antibodies raised against either the C terminus or N terminus. Vinculin served as a loading control. Molecular weights are indicated in kilodaltons (kDa). MCU, mitochondrial Ca 2+ uniporter.

Article Snippet: Two different guide (g)RNAs targeting rat Mcu (Ensembl Gene ID: ENSRNOG00000045920 ; chromosomal location chr20 29,038,480—29,199,224 [-]; genome: Rnor_6.0 (rn6, Rattus norvegicus )) were designed using the Benchling CRISPR design webtool ( https://benchling.com/crispr ).

Techniques: CRISPR, Knock-Out, Real-time Polymerase Chain Reaction, Expressing, Clone Assay, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Deletion of mitochondrial calcium uniporter enhances calcium signals by slowing calcium clearance and triggers adaptive transcriptomic remodeling

doi: 10.1016/j.jbc.2026.111473

Figure Lengend Snippet: Mcu knockout by CRISPR/Cas9 causes widespread and divergent transcriptomic changes across clones generated using distinct guide RNAs. A and B , volcano plots showing differentially expressed genes (DEGs) in Mcu −/− clones A4 ( A ) and D10 ( B ) relative to WT cells. Red and blue dots represent significantly upregulated and downregulated genes, respectively. Mcu is shown as a green dot . Gray dots indicate unchanged genes, either with below absolute fold change 2.0 cutoff ( light gray ) or having false discovery rate(FDR) higher than 0.05 ( dark gray ). C , volcano plot comparing A4 and D10 clones directly. D and E , Venn diagrams of downregulated genes ( D ) and upregulated genes ( E ) in A4 versus WT and D10 versus WT, highlighting both overlapping and clone-specific expression changes. F , ingenuity pathway analysis (IPA) of the 213 DEGs common to both clones (64 downregulated and 149 upregulated) with a z-score cutoff of 1.0. G , bubble plot displays enriched canonical pathways predicted by IPA. The y -axis on the right shows enriched pathway names ordered according to hierarchical clustering of their z-scores across two conditions (A4 versus WT and D10 versus WT), with the corresponding dendrogram displayed on the left . The x -axis labels the two conditions. Each pathway is represented by two circles , one for each condition. Circle size corresponds to the gene ratio for that pathway in the given condition. The fill color of each circle reflects the z-score: red for positive z-scores (activation), blue for negative z-scores (inhibition), transparent for values near zero, and gray where the z-score is NA. Threshold of z-score was set to absolute z-score ≥ 1.5. Below cutoff p values ( i.e. , p value > 0.05) are indicated by small lines on the right of the bubble. MCU, mitochondrial Ca 2+ uniporter.

Article Snippet: Two different guide (g)RNAs targeting rat Mcu (Ensembl Gene ID: ENSRNOG00000045920 ; chromosomal location chr20 29,038,480—29,199,224 [-]; genome: Rnor_6.0 (rn6, Rattus norvegicus )) were designed using the Benchling CRISPR design webtool ( https://benchling.com/crispr ).

Techniques: Knock-Out, CRISPR, Clone Assay, Generated, Expressing, Activation Assay, Inhibition

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