ice crispr analysis tool  (Synthego Inc)

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: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity

doi: 10.64898/2026.04.03.715992

Figure Lengend Snippet: a, Schematics of key differences in Tfh cell maturation between humans (left) and mice (right). b, Gene expression signatures of all CD4 + T cell clusters from tonsillar T cell scRNA-seq. c, Volcano plot showing differential gene expression of GC-Tfh cells (left) vs. CXCL13 + GC-Tfh cells (right). d, Select subset-specific marker gene expression in Tfh, GC-Tfh, and CXCL13 + GC-Tfh cells. e, Schematic overview for assessment of CXCL13 production by non-, pre-, and GC-Tfh cells. f, Exemplary gating for non-Tfh (PD-1 - CXCR5 - ), pre-Tfh (PD-1 lo CXCR5 lo ), and GC-Tfh cells (PD-1 hi CXCR5 hi ). g, CXCL13 secretion by different sorted T cell subtypes cultured with or without B cells (n = 4). h, Schematic representation for CRISPR/Cas9-based editing of tonsil samples. i, CXCL13 ELISA of tonsillar cell culture supernatants for indicated transcription factor knockouts vs. scramble control (n = 14; ≤ 30 yo). Data were analyzed using two-way ANOVA with Šídák’s multiple comparison test ( g ) and one-way ANOVA with Dunnett’s multiple comparisons test ( i ). * P < 0.05, ** P < 0.01.

Article Snippet: Editing efficiencies ( Table S3 ) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout.

Techniques: Gene Expression, Marker, Cell Culture, CRISPR, Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: bioRxiv

Article Title: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity

doi: 10.64898/2026.04.03.715992

Figure Lengend Snippet: a, Number of differentially expressed genes (DEGs, >50yo vs. <30yo) over pseudotime during Tfh cell development from naïve CD4 + T cells (left) to CXCL13 + GC-Tfh cells (right). False discovery rate (FDR) is set to < 0.05. b, Volcano plot showing differential gene expression of Tfh cells from older (left) vs. younger donors (right). c, Schematic representation for CRISPR/Cas9-based editing of naïve CD4 + T cells followed by T cell polarization and flow cytometric analysis. d, Expression of CXCR5, FOXP3, and PD-1 in naïve CD4 + T cells cultured under Tfh-polarizing conditions (dark grey) or non-polarizing conditions (light grey). e, Log2 fold change relative to matched scramble controls for Th1 (left) and Th17 (right) cell frequency in TF-knockouts under Th1- or Th17-polarizing conditions. f, Log2 fold change relative to matched scramble controls for Tfh cell frequency in TF-knockouts under Tfh-polarizing conditions (gated on CD4 + CD19 - FOXP3 - CXCR5 + PD-1 + cells). Data were analyzed using a mixed effects model and Dunnett’s multiple comparisons test ( f ), one sample t test ( e ). Error bars SEM ( e-f ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Editing efficiencies ( Table S3 ) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout.

Techniques: Gene Expression, CRISPR, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Virus-Like Particles: The Next Frontier in Livestock Gene Editing

doi: 10.64898/2026.03.30.715406

Figure Lengend Snippet: A : Bar graph showing ICE analysis results of targeting of SMAD4, B2M, KDM6A and TP53 in pKDNFs using Cas9 VLPs. Blue bars: INDEL scores, green bars: KO scores compared to WT control (n≥3; SD shown). B : Bar graph showing ICE analysis results of targeting of IL-10 in developed blastocysts (B1-B6) using multiplexed Cas9 VLPs. Blue bars: INDEL scores, green bars: KO scores compared to WT control. C : Detected INDELs of blastocyst 2 (B2) report that in 98% of the cases, the whole fragment was deleted.

Article Snippet: Sequencing results were analyzed for the percentage of INDELs using the online ICE analysis tool by Synthego ( https://ice.editco.bio ).

Techniques: Control

Journal: Nature Immunology

Article Title: Engineering NK and T cells with metabolite-sensing receptors to target solid tumors

doi: 10.1038/s41590-026-02473-y

Figure Lengend Snippet: a , Enzymatic oxysterol synthesis pathway. Sequential cholesterol degradation by CH25H and CYP7B1 enzymes generates the GPR183 ligand 7α,25-OHC, and sequential cholesterol degradation by CYP27A1 and CYP7B1 generates the GPR183 ligand 7α,27-OHC. b , Expression levels of oxysterol-synthesizing enzymes in MDA-MB-231 cells in monoculture with and without IFNγ and TNF treatment ( n = 3 biological replicates per group). Expression levels were measured via quantitative real-time PCR (qPCR; ) and normalized to GAPDH . Data are representative of two independent experiments, are presented as the mean ± s.e.m. and were analyzed by two-tailed unpaired Student’s t -test. c , CH25H - and CYP7B1 -deficient MDA-MB-231 cells were generated using CRISPR knockout. Knockout efficiency was validated using the Synthego ICE analysis tool (v3). Control cells (transduced with NTC sgRNA) are shown in gray. The dashed black bar indicates the sequence alignment window, the solid black bar indicates the ICE interference window, and the gray dotted line marks the start of the guide sequence; KO, knockout. d , e , Transwell assay with flow cytometry-based quantification of NK-92 cell migration to CM from isogenic MDA-MB-231 cells with and without knockout of oxysterol synthesis enzymes ( d ; n = 3 technical replicates per group) and to tumor and lung lysates ( e ; n = 9 technical replicates per group). f , GPR183 cell surface expression in GPR183 OE and control primary human NK (pNK) cells. g , h , Transwell assay with flow cytometry-based quantification of control and GPR183 OE primary NK cell migration to 7α,25-OHC ( g ; n = 3 technical replicates per group) and to tumor and lung lysates ( h ; n = 6 technical replicates per group). i , In vivo co-transfer experiment. NK-92 cells were transduced with either control–GFP or GPR183–mCherry. GFP + and mCherry + cells were mixed at a 1:1 ratio and intravenously transferred into MDA-MB-231-bearing NSG mice. Spleens, lungs and tumors were dissected, and tissue-infiltrating cells were assessed by flow cytometry. The GPR183:control ratio was calculated across tissues ( n = 7 mice). Experimental scheme (left), representative flow cytometry plots (middle) and a bar plot with each dot depicting the ratio per mouse (right) are shown. j , Proliferation of GPR183 A+ cells and control NK-92 cells quantified via decay of cell-trace dye. Top, histogram plots. Bottom, relative mean fluorescence intensity (MFI) quantification ( n = 3 technical replicates per group). Data are representative of two independent experiments. Two independent experiments were pooled for e , h and i . Data are representative of two independent experiments for d and g . Data are presented as mean values ± s.e.m. for d , e and g – j . Data analysis was performed using one-way analysis of variance (ANOVA) with a Dunnett’s multiple comparisons test for d , g and i and two-way ANOVA with a Tukey’s multiple comparisons test for e and h . Panel i scheme created in BioRender; Jerby Lab https://biorender.com/c1gnk9g (2026).

Article Snippet: Knockout efficiency was validated using the Synthego ICE analysis tool (v3).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Generated, CRISPR, Knock-Out, Control, Transduction, Sequencing, Transwell Assay, Flow Cytometry, Migration, In Vivo, Fluorescence