crispr guide rna design tool  (Benchling 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: Cryptic variation alters gene dosage sensitivity to shape inflorescence architecture in tomato

doi: 10.64898/2026.05.07.722400

Figure Lengend Snippet: a , b , Schematic representation of J2 ( a ) and EJ2 ( b ) with location of the CRISPR-Cas9 target sites and mutant alleles in domestic ( S. lycopersicum acc. S100) and wild ( S. pimpinellifolium acc. LA1589) tomato. c , d , Quantification of inflorescence branching in j2 null ej2 null segregating populations in domestic ( c ) and wild ( d ) tomato. e , f , Representative images of domestic ( e ) and wild ( f ) inflorescences with different strengths of branching from genotypes segregating j2 null ej2 null alleles. g , h , Quantification of inflorescence branching in j2 null ej2 null/hypo segregating populations in domestic ( g ) and wild ( h ) tomato. i , j , Representative images of domestic ( i ) and wild ( j ) inflorescences with different strengths of branching from genotypes segregating j2 null ej2 null/hypo alleles. Gene models in a and b : exons, untranslated regions, and Cas9 cleavage sites for guide RNAs are indicated by light gray boxes, dark gray boxes, and gray arrowheads, respectively. Per genotype, the number of individual plants for which 5 inflorescences were counted in c , d , g , and h is indicated by n . Scalebars and arrowheads in e , f , i , and j represent 1 cm and indicate inflorescence branching events, respectively. J2 , JOINTLESS2 ; EJ2 , ENHANCER OF J2 ; WT, wild-type.

Article Snippet: The PCR amplicons were either subjected to amplicon deep sequencing, or they were purified using ExoSAP-IT (Thermo Fisher Scientific) and analyzed by Sanger sequencing of the purified PCR amplicons, followed by decomposition of quantitative sequence trace data using Inference of CRISPR Editing (ICE) CRISPR Analysis Tool ( https://ice.synthego.com/ #/).

Techniques: CRISPR, Mutagenesis

Journal: bioRxiv

Article Title: Cryptic variation alters gene dosage sensitivity to shape inflorescence architecture in tomato

doi: 10.64898/2026.05.07.722400

Figure Lengend Snippet: a , Quantitative trait locus (QTL) sequencing using bulked segregants of plants with branched and suppressed inflorescences from a s2 x LA1589 F2 population showed two suppressor of branching ( sb ) loci from wild tomato (acc. LA1589) on chromosomes 1 and 2. The sb1 QTL contains a copy number variant of the MADS-box gene SISTER OF TM3 ( STM3 ). b , Physical positions of the QTL regions shown in ( a ) in wild (LA1589) and domestic (SL4.0) tomato. c , The sb2 locus was fine-mapped to an interval of ∼160 Kb between markers 43.46 Mb and 43.62 Mb (SL4.0 coordinates) on chromosome 2 in subsequent generations of sb2 segregating populations. Blue boxes indicate the fine-mapped sb2 locus in each population. d , The sb2 locus was fine-mapped to an interval of ∼83 Kb between markers 43.51 Mb and 43.59 Mb (SL4.0 coordinates) on chromosome 2. Each distinctive genotype is represented by a horizontal bar. Green and pink shading indicates homozygosity for s2 and LA1589, respectively. Per genotype, the number of individual plants for which 5 inflorescences were counted is indicated by n . e , Gene models and coordinates of the 15 genes in the sb2 locus including ANANTHA and 3βHSD2 . f , Representative images of strongly branched and suppressed inflorescences from plants with genotype 3 and 4 from ( d ), respectively. g , Schematic representation of ANANTHA with location of the CRISPR-Cas9 target sites and mutant alleles in domestic ( S. lycopersicum acc. S100) tomato. Gene model: exon, region encoding F-box, and Cas9 cleavage sites for guide RNAs are indicated by a light gray box, a lavender box, and gray arrowheads, respectively. g , h , Representative images of cauliflower inflorescences from anantha mutants in domestic (acc. S100) ( h ) and wild (acc. LA1589) ( i ) tomato. Scalebars and arrowheads in f , h , and i represent 1 cm and indicate inflorescence branching events, respectively. WT, wild-type; AN , ANANTHA ; 3βHSD2 , 3β-hydroxysteroid dehydrogenase/C4-decarboxylase 2 .

Article Snippet: The PCR amplicons were either subjected to amplicon deep sequencing, or they were purified using ExoSAP-IT (Thermo Fisher Scientific) and analyzed by Sanger sequencing of the purified PCR amplicons, followed by decomposition of quantitative sequence trace data using Inference of CRISPR Editing (ICE) CRISPR Analysis Tool ( https://ice.synthego.com/ #/).

Techniques: Sequencing, Variant Assay, CRISPR, Mutagenesis

Journal: bioRxiv

Article Title: Cryptic variation alters gene dosage sensitivity to shape inflorescence architecture in tomato

doi: 10.64898/2026.05.07.722400

Figure Lengend Snippet: a , Sequence encoding the F-box of the ANANTHA protein with location of the CRISPR-Cas9 target site (arrowhead) and mutant alleles in wild ( S. pimpinellifolium acc. LA1589) tomato. b , A 6.2 Kb and 929 bp region upstream and downstream, respectively, of the ANANTHA coding region showing open chromatin, conserved non-coding sequences (CNSs), predicted transcription factor binding sites (TFBSs), and genetic variants between the natural j2 TE ej2 W mutant ( s2 ) in domestic tomato ( S. lycopersicum ) and wild tomato ( S. pimpinellifolium acc. LA1589). A region 486–369 bp upstream of ANANTHA harbors a predicted AP2/ERF binding site (green) in s2 that is disrupted by an 8-bp deletion (bold) in wild tomato acc. LA1589. c , CRISPR genome editing with a PAM-less Cas9 variant (SpRY) to target the predicted AP2/ERF binding site (green) in a recombinant inbred line (RIL) homozygous for the domestic s2 mutant at the SB2 locus. d , Sequence logo for the predicted AP2/ERF binding site upstream of ANANTHA in the domestic s2 mutant. e , Representative images of inflorescences of the SB2 RIL and an reg lines. Scalebar represents 1 cm. f , Semi-quantification of inflorescence branching in an reg lines. Per genotype, the number of individual plants for which the level of inflorescence branching was quantified is indicated by n . Sequences targeted with CRISPR-Cas in a and c : Cas9 cleavage sites for guide RNAs, PAMs, and CRISPR-Cas edits are indicated by gray arrowheads, underlining, and pink-marked text, respectively.

Article Snippet: The PCR amplicons were either subjected to amplicon deep sequencing, or they were purified using ExoSAP-IT (Thermo Fisher Scientific) and analyzed by Sanger sequencing of the purified PCR amplicons, followed by decomposition of quantitative sequence trace data using Inference of CRISPR Editing (ICE) CRISPR Analysis Tool ( https://ice.synthego.com/ #/).

Techniques: Sequencing, CRISPR, Mutagenesis, Binding Assay, Variant Assay, Recombinant

Journal: bioRxiv

Article Title: Cryptic variation alters gene dosage sensitivity to shape inflorescence architecture in tomato

doi: 10.64898/2026.05.07.722400

Figure Lengend Snippet: a , Number of differentially expressed genes (DEGs; log₂ fold change |≥| 0.585 and FDR ≤ 0.05) between plants harboring the domestic SB2 and wild sb2 haplotype, based on RNA sequencing of dissected meristems at the transition and floral stage. b , Heatmap depicting z-score normalized expression of DEGs (n=488) between SB2 and sb2 plants. c , Z-score normalized expression profiles of DEG clusters with similar expression patterns identified by hierarchical clustering in ( b ). d , The 10 most enriched gene ontology (GO) categories for DEGs in clusters 1 and 4. No GO enrichment was detected for clusters 2 and 3. P values were obtained using the Benjamini-Hochberg (BH) method in clusterProfiler. e , f Volcano plots displaying downregulation of sterol-related genes in transition ( e ) and floral ( f ) meristems of sb2 compared with SB2 plants. g , h , Volcano plots displaying expression patterns for genes at the sb2 locus between SB2 and sb2 plants in transition ( g ) and floral ( h ) meristems. i , Schematic representation of 3βHSD2 with location of the CRISPR-Cas9 target sites and mutant alleles in domestic ( S. lycopersicum acc. S100) tomato. Gene model: exons, untranslated regions, and Cas9 cleavage sites for guide RNAs are indicated by light gray boxes, dark gray boxes, and gray arrowheads, respectively. j , Representative images of inflorescences from 3bhsd2 null1 , j2 null2 ej2 hypo1 /+, and j2 null2 ej2 hypo1 /+ 3bhsd2 null1 plants. Scalebars and arrowheads represent 1 cm and indicate inflorescence branching events, respectively. k , Quantification of inflorescence branching for 3bhsd2 null1 , j2 null2 ej2 hypo1 /+, and j2 null2 ej2 hypo1 /+ 3bhsd2 null1 plants. Dotted lines represent mean number of branching events for each genotype. Error bars denote standard deviation ( n =24–35). Circle areas represent the number of inflorescences per genotype. Statistical significance was determined by ANOVA followed by Tukey’s post-hoc analysis ( P < 0.05; indicated by different letters). suppressor of branching 2, sb2; DWF , DWARF ; SSR2 , STEROL SIDE CHAIN REDUCTASE 2 ; ROT3 , ROTUNDIFOLIA 3 ; GAME4 , GLYCOALKALOID METABOLISM 4 ; SMO , C-4 STEROL METHYL OXIDASE ; C5-SD2 , STEROL C-5 DESATURASE 2 ; MVK , MEVALONATE KINASE ; 8,7-SI , STEROL 8,7 ISOMERASE ; 3βHSD2 , 3β-hydroxysteroid dehydrogenase/C4-decarboxylase 2 ; AN , ANANTHA; J2 , JOINTLESS2 ; EJ2 , ENHANCER OF J2 ; WT, wild-type.

Article Snippet: The PCR amplicons were either subjected to amplicon deep sequencing, or they were purified using ExoSAP-IT (Thermo Fisher Scientific) and analyzed by Sanger sequencing of the purified PCR amplicons, followed by decomposition of quantitative sequence trace data using Inference of CRISPR Editing (ICE) CRISPR Analysis Tool ( https://ice.synthego.com/ #/).

Techniques: RNA Sequencing, Expressing, CRISPR, Mutagenesis, Standard Deviation

Journal: bioRxiv

Article Title: Cryptic variation alters gene dosage sensitivity to shape inflorescence architecture in tomato

doi: 10.64898/2026.05.07.722400

Figure Lengend Snippet: a , Sequence of 3βHSD2 targeted by genome editing with location of the CRISPR-Cas9 target sites (arrowheads) and mutant alleles in domestic ( S. lycopersicum acc. S100) tomato. b , Representative images of inflorescences from 3bhsd2 null2 , j2 null2 ej2 hypo1 /+ 3bhsd2 null /+, and j2 null2 ej2 hypo1 3bhsd2 null plants. Scalebars represent 1 cm.

Article Snippet: The PCR amplicons were either subjected to amplicon deep sequencing, or they were purified using ExoSAP-IT (Thermo Fisher Scientific) and analyzed by Sanger sequencing of the purified PCR amplicons, followed by decomposition of quantitative sequence trace data using Inference of CRISPR Editing (ICE) CRISPR Analysis Tool ( https://ice.synthego.com/ #/).

Techniques: Sequencing, CRISPR, Mutagenesis

Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following CRISPR/Cas9 editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.

Article Snippet: Sanger sequence traces were analyzed using the ICE (Inference of CRISPR Editing) deconvolution tool from Synthego. ( https://ice.synthego.com ; Synthego Performance Analysis, ICE Analysis.

Techniques: Sequencing, CRISPR, Expressing, Plasmid Preparation, Biomarker Discovery, Cell Culture, Imaging