Journal: The EMBO Journal

Article Title: CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of maternal-to-zygotic transition in teleosts

doi: 10.1038/s44318-025-00617-8

Figure Lengend Snippet: ( A ) Heatmap plot of relative translation data during early developmental stages (0, 2, 5, and 12 hpf) of the kinases and phosphatases selected for the maternal screening. Data from Bazzini et al, and Chan et al, . ( B ) Schematic illustration of the experimental setup used for the maternal screening. (1) RfxCas13d protein (Cas13d, 3 ng per embryo) was mixed with a pool of 3 gRNAs (1000 pg per embryo) and injected into one-cell-stage zebrafish embryos. (2) Knockdown conditions were evaluated and considered positive when epiboly defects were observed at 6 hpf. Control embryos injected only with RfxCas13d protein were at a normal gastrulation stage. (3) Transcriptomic analysis was performed at 4 hpf to evaluate mRNA depletion and the global analysis of pure zygotic genes (PZG). Knockdown conditions with an epiboly defect and global downregulation of PZG were considered ZGA candidates. ( C ) Stacked barplots showing the percentage of phenotypes observed at 6 hpf under maternal screening conditions. Control conditions, either uninjected embryos, injected with RfxCas13d protein alone or with RfxCas13d plus non-targeting gRNAs (designed for gfp or rfp mRNAs), are indicated with a gray background. The positive control, targeting nanog mRNA, is indicated with a red background. Positive candidates (purple background) were considered when more than 35% of the embryos showed a developmental delay (30–50% epiboly at 6 hpf where control embryos were at shield stage). Negative candidates are indicated with an orange background. mRNAs encoding for kinases are labeled in blue and mRNAs encoding for phosphatases are labeled in green. The results are shown as the averages ± standard error of the mean of each developmental stage from at least two independent experiments. Number of embryos evaluated ( n ) is shown for each condition. Representative pictures of different zebrafish epiboly-stages from bckdk mRNA knockdown are shown in the upper panel. 30% epiboly, 50% epiboly, germ ring, and shield stages correspond to 4.6, 5.3, 5.7, and 6 hpf in uninjected embryos growing in standard conditions, respectively (scale bar, 0.05 mm). ( D ) Boxplot with individual values showing the percentage of mRNA depletion using the RfxCas13d approach for seven positive candidates (purple dots) and seven negative candidates (orange dots) from maternal screening measured by Bulk-RNA-Seq at 4 hpf. The mean of mRNA depletion is represented together with the first and third quartile. Vertical lines indicate the variability outside the upper and lower quartiles. ( E ) Scatter plot of Normalized Enrichment Score and adjusted p value associated with gene set enrichment analysis of pure zygotic genes (PZG) defined by Lee et al, from positive (purple dots), negative candidates (orange dots) and negative controls (gray dots) (see Methods for details). Bulk-RNA-Seq data from panel ( D ). Vertical and horizontal dashed lines indicate −2 and 2 normalized enrichment score (NES) and adjusted p value = 0.01, respectively. NES and adjusted p value were calculated using gene set enrichment analysis (GSEA) (Subramanian et al, ; Mootha et al, ) (see Methods for details). ( F ) Scatter plots representing the log2 fold change in mRNA levels and p value from two biological RNA-seq replicates ( n = 20 embryos/biological replicate) at 4 hpf in zebrafish embryos injected with Cas13d protein and three gRNAs targeting bckdk (left), mknk2a (middle) or vrk3 (right) mRNA. p values were calculated using the Wald test. Pure zygotic genes mRNAs (PZG) defined by Lee et al, are indicated in blue. .

Article Snippet: All designed gRNAs for maternal screening were chemically synthesized by Synthego (Synthego Corp., CA, USA). gRNAs for phf10 mRNA knockdown were generated by fill-in-PCR with a universal primer and three specific primers (Dataset ) with the Q5 High-Fidelity DNA Polymerase.

Techniques: Injection, Knockdown, Control, Positive Control, Labeling, RNA Sequencing

Journal: The EMBO Journal

Article Title: CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of maternal-to-zygotic transition in teleosts

doi: 10.1038/s44318-025-00617-8

Figure Lengend Snippet: ( A ) Schematic representation of SLAM-Seq workflow. Zebrafish embryos were injected with 4-thiouridine (S 4 U, 25 pmol/embryo) in one-cell-stage together with RfxCas13d protein alone or with a mix of two gRNAs targeting bckdk mRNA. Total RNA was extracted at 4 hpf from 20 embryos. Total RNA was chemically modified before library preparation. After library preparation T > C ratio conversion was used to differentiate between unlabeled (preexisting RNA; Maternal) and labeled (newly synthesized RNA; Zygotic) reads (see Methods for further details). ( B ) Scatter plot showing newly transcribed RNAs (SLAM-seq data in Counts per million reads; CPM) of zebrafish embryos at 4 hpf from two and four biological replicates ( n = 25 embryos/biological replicate) from embryos injected with RfxCas13d protein alone (Cas13d protein) or with a mix of two gRNAs targeting bckdk mRNA (Bckdk KD), respectively. Dashed and dot-dash lines indicate a 2 and 5-fold difference between RNA levels, respectively. Up- and down-regulated genes are represented by yellow (between 2- and 5-fold change) and brown (>5-fold change) or pink (between 2- and 5-fold change) and purple (>5-fold change) dots, respectively. Pure zygotic genes (PZG) defined by Lee et al, are indicated by blue outline dots. The number of mRNAs in each category is shown ( n ). ( C ) Scatter plot of normalized enrichment score and adjusted p value associated of pure zygotic genes (PZG) defined by Lee et al, (PZG: Lee et al,) or Baia Amaral et al, (PZG: Baia Amaral et al,) from Bckdk KD data generated by RNA-Seq (gray dot) or by SLAM-Seq with labeled (orange dots) or unlabeled (brown dot) reads. NES and adjusted p value were calculated using gene set enrichment analysis (GSEA) (Subramanian et al, ; Mootha et al, ) (see Methods for details). ( D ) Plot profile (top) and heatmaps (bottom) representing normalized ATAC intensity signal for less accessible regions (red line; Down peaks) and more accessible regions (blue line; Up peaks) from the comparison between two biological replicates ( n = 80 embryos/biological replicate) from zebrafish embryos injected with RfxCas13d protein (Cas13d) alone or together with a mix of two gRNAs targeting bckdk mRNA (Bckdk KD). Number of less accessible (Down peaks) or more accessible (Up peaks) regions are shown ( n ). ( E ) Plot profile (top) and heatmaps (bottom) representing normalized H3K27ac intensity signal for all detected peaks ( n = 5.428) from CUT&RUN data at 4 hpf from two biological replicates per condition in zebrafish embryos ( n = 75 embryos/biological replicate) injected with RfxCas13d protein alone (Cas13d) or together with a mix of two gRNAs targeting bckdk mRNA (Bckdk KD). ( F ) Differential analyses of H3K27ac levels between zebrafish embryos injected with RfxCas13d protein alone or co-injected with 2 gRNAs targeting bckdk mRNA from CUT&RUN data described in ( E ). The Log2 fold change (FC) and p value associated are represented. Peaks with a significant decrease in H3K27ac are represented as purple dots (FC <−1.5 and p value <0.05). The number of downregulated peaks is indicated ( n ). p values were calculated using the Wald test. .

Article Snippet: All designed gRNAs for maternal screening were chemically synthesized by Synthego (Synthego Corp., CA, USA). gRNAs for phf10 mRNA knockdown were generated by fill-in-PCR with a universal primer and three specific primers (Dataset ) with the Q5 High-Fidelity DNA Polymerase.

Techniques: Injection, Modification, Labeling, Synthesized, Generated, RNA Sequencing, Comparison

Journal: The EMBO Journal

Article Title: CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of maternal-to-zygotic transition in teleosts

doi: 10.1038/s44318-025-00617-8

Figure Lengend Snippet: ( A ) Stacked barplots showing developmental phenotypes at 6 hpf in zebrafish embryos injected with RfxCas13d protein (3 ng/embryo) or RfxCas13d mRNA (150 pg/embryo) alone or together with different amounts of S 4 U (25, 50, or 75 mM). The phenotype selection criteria were the same as described in Fig. . The results are shown as the averages ± standard error of the mean of each developmental stage from at least two independent experiments. The number of embryos evaluated ( n ) is shown for each condition. ( B ) Volcano plot representing the log2 fold change in mRNA level from unlabeled reads (SLAM-Seq data) and the associated p value from two and four biological replicates ( n = 25 embryos/biological replicate) at 4 hpf from embryos injected with RfxCas13d protein alone or with a mix of two gRNAs targeting bckdk mRNA, respectively. p values were calculated using the Wald test. bckdk mRNA is represented in red. Pure zygotic genes mRNAs (PZG) determined by Lee et al, data were depicted in blue. Volcano plots representing the fold change in mRNA level from labeled reads and the associated p value from three biological SLAM-Seq replicates of zebrafish embryos at 4 hpf from two and four biological replicates ( n = 25 embryos/biological replicate) from embryos injected with RfxCas13d protein alone or with a mix of two gRNAs targeting bckdk mRNA, respectively. p values were calculated using the Wald test. bckdk mRNA is represented in red. Pure zygotic genes mRNAs (PZG) determined by Lee et al, data were depicted in blue in panel ( C ) and an updated list of pure zygotic genes mRNAs (PZG) determined by Baia Amaral et al, data were depicted in blue in panel ( D ). ( E ) Stacked barplot showing percentage of upregulated genes in SLAM-Seq data upon bckdk mRNA depletion that belong to different categories. Genes were classified according to Baia Amaral et al, . No MZT: Genes that are not present between 0 and 7 hpf; Maternal: Genes maternally provided as mRNA but not zygotically transcribed; Maternal-and-Zygotic: Genes maternally provided and zygotically transcribe between 4 and 7 hpf; or pure zygotic: genes not maternally provided as mRNA and zygotically transcribed between 4 and 7 hpf. ( F ) Stacked barplot showing percentage of upregulated MZT genes (maternal-and-zygotic or pure zygotic genes) in SLAM-Seq data upon bckdk mRNA depletion that belong to different categories. Genes were classified according to transcript levels in wild-type conditions at 4 hpf in labeled data. Higher transcribed: Genes with more than ten CPM (counts per million) in labeled data at 4 hpf in WT conditions; Earlier transcribed: Genes with less than ten CPM in labeled data at 4 hpf in WT conditions. ( G ) Gene Ontology enrichment analyses of biological processes for down-(Down) or up-regulated genes (Up) from the comparison of SLAM-Seq data between zebrafish embryos injected with RfxCas13d alone and together with two gRNAs targeting bckdk mRNA. Terms with a false discovery rate (FDR) lower than 0.05 and with more than 20 genes represented are shown and considered as enriched. ( H ) Differential analyses of chromatin accessibility between zebrafish embryos injected with RfxCas13d protein alone or co-injected with two gRNAs targeting bckdk mRNA from ATAC-seq data at 4 hpf from two biological replicates per condition ( n = 80 embryos/biological replicate). The log2 fold change (FC) and p value associated is represented. p values were calculated using the Wald test. Regions with a significant decrease in accessibility are represented as purple dots (log2FC <−1 and p value <0.05), and regions with a significant increase in accessibility are represented as orange dots (log2FC >1 and p value <0.05). Vertical and horizontal dashed lines indicate 1.5-fold and p value = 0.05, respectively. Motif enrichment analyses from the decreased ( I ) and increased ( J ) ATAC regions (down and up peaks, respectively) in bckdk mRNA knockdown condition. The top five motifs are represented with their motif logos, transcription factor name, percentage of peaks containing the motif and enrichment p value. ( K ) Violin plots showing the distribution of log2 fold change of RNA levels (SLAM-Seq) from all differential expression genes (DEGs) and those associated with less accessible (Down) or more accessible (Up) regions from ATAC-Seq data. Dash lines and dot lines inside the violin plots indicate the mean and quartiles, respectively. Gray dot line and dash line outside the violin plots indicate 0-fold and 1.5-fold in RNA levels, respectively. Exact p values are indicated above, Mann–Whitney U -test. The number of differentially expressed genes ( n ) for each category are shown. ( L ) Representative western blot images for H3K27ac and H3 of embryos injected with RfxCas13d protein alone (Cas13d) or together with two gRNAs targeting bckdk mRNA (Bckdk KD) (left). Barplots represent H3K27ac or H3 levels relative to total proteins as the averages ± standard error of the mean at least four biological replicates from two or three independent experiments. Zebrafish embryos were collected at 4 hpf ( n = 25 embryos/biological replicate). Exact p values are indicated above (Welch’s t -test) (Right). ( M ) Stain-free signal (Gürtler et al, ) of the gels employed as loading control for Western blot in panel ( L ). .

Article Snippet: All designed gRNAs for maternal screening were chemically synthesized by Synthego (Synthego Corp., CA, USA). gRNAs for phf10 mRNA knockdown were generated by fill-in-PCR with a universal primer and three specific primers (Dataset ) with the Q5 High-Fidelity DNA Polymerase.

Techniques: Injection, Selection, Labeling, Comparison, Knockdown, Quantitative Proteomics, MANN-WHITNEY, Western Blot, Staining, Control

Journal: The EMBO Journal

Article Title: CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of maternal-to-zygotic transition in teleosts

doi: 10.1038/s44318-025-00617-8

Figure Lengend Snippet: ( A ) Cumulative distribution of mRNA levels (TPM; Transcript per million reads) in control (Cas13d; purple line) and bckdk mRNA knockdown embryos (Bckdk KD; orange line) that are degraded by four different pathways determined by Vejnar et al, (maternal, zygotic, miR-430) and described by Medina-Muñoz et al, (miR-430 golden candidates were defined as downregulated mRNAs, fourfold, from the comparison between WT and maternal-zygotic dicer mutant embryos at 6 hpf). Number of mRNA controlled by each pathway is shown ( n ). Distance (D) and p value calculated by Kolmogorov–Smirnov test are indicated. ( B ) RT-qPCR analysis showing relative primary miR-430 (pri-miR-430) transcript levels at 4.3 hpf (Dome) and 6 hpf (Shield). Results are shown as the averages ± standard error of the mean from three experiments with two biological replicates per experiment ( n = 10 embryos/biological replicate) for RfxCas13d protein alone (Cas13d) and RfxCas13d plus two gRNAs targeting bckdk mRNA (Bckdk KD), respectively. taf15 mRNA was used as normalization control. Exact p values are indicated above, unpaired t -test. ( C ) RT-qPCR analysis showing levels of mature miR-430 isoforms (miR430-a, red; miR430-b, orange; and miR430c, yellow) at 4.3 hpf (Dome) and 6 hpf (Shield). Results are shown as individual values and the mean from three experiments with two biological replicates per experiment ( n = 10 embryos/biological replicate) for RfxCas13d protein alone (Cas13d) and RfxCas13d plus 2 gRNAs targeting bckdk mRNA (Bckdk KD). ncRNA u4atac was used as normalization control. Exact p values are indicated above, unpaired t -test). ( D ) Cumulative distribution of Log2 fold changes (Log2FC) of mRNAs degraded by miR-430 (miR-430 described by Vejnar et al, , miR-430 golden described by Medina-Muñoz et al, ), or not dependent on the activity of this microRNA, from the comparison between zebrafish embryos injected with RfxCas13d protein and two gRNAs targeting bckdk mRNA, either alone (Bckdk KD, orange line) or co-injected with a mix of mature miR-430 isoforms (miR-430 rescue, green line) at 6 hpf. The number of mRNAs is shown ( n ). Distance (D) and p value calculated by the Kolmogorov–Smirnov test are indicated. .

Article Snippet: All designed gRNAs for maternal screening were chemically synthesized by Synthego (Synthego Corp., CA, USA). gRNAs for phf10 mRNA knockdown were generated by fill-in-PCR with a universal primer and three specific primers (Dataset ) with the Q5 High-Fidelity DNA Polymerase.

Techniques: Control, Knockdown, Comparison, Mutagenesis, Quantitative RT-PCR, Activity Assay, Injection

Journal: bioRxiv

Article Title: Comprehensive profiling of transcription factors for reprogramming human astrocytes to neuronal cells through endogenous CRISPR-based gene activation

doi: 10.1101/2025.10.11.681828

Figure Lengend Snippet: A. Schematic of CRISPRa screening with TFome gRNA library. B. Summary of TFome gRNA library. C. Significance (P adj ) versus fold change in gRNA abundance between MAP2-high and MAP2-low populations in TFome CRISPRa screen. D. Selected enriched biological processes for positive and negative hits from the TFome screen. Statistical significance was determined using a two-tailed Fisher’s exact test followed by Benjamini–Hochberg correction. E. Expression level of hit TFs in astrocytes or neurons in the Human Protein Atlas Single Cell Type data. F. Validations of selected hit factors for MAP2 protein expression 10 days post-transduction. **p < 0.01, ***p < 0.001 by global one-way ANOVA with Dunnett’s post hoc test comparing all groups to non-targeting 1, gating set to 1% positive for non-targeting gRNAs; error bars represent SEM. G. Validations of selected hit factors for NeuN RNA levels 10 days post-transduction. *p < 0.05, ***p < 0.001 by global one-way ANOVA with Dunnett’s post hoc test comparing all groups to non-targeting 1. Error bars represent SEM. H. Validation of selected hit factors for NeuN, MAP2, and neuronal morphology by immunofluorescence staining.

Article Snippet: For each TF that had <6 gRNAs present in the referenced library, additional unique gRNAs were subset from an earlier published gRNA library , for a total library size of 10,233 gRNAs. gRNAs were then ordered as an oligo pool from Twist Biosciences and cloned into an AIO CRISPRa vector by Gibson assembly.

Techniques: Two Tailed Test, Expressing, Transduction, Biomarker Discovery, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Comprehensive profiling of transcription factors for reprogramming human astrocytes to neuronal cells through endogenous CRISPR-based gene activation

doi: 10.1101/2025.10.11.681828

Figure Lengend Snippet: A. Summary of TFome hit library. B. Schematic and resulting UMAP of Perturb-seq screen of TFome hits. C. Dot plot of expression of astrocyte and neuron marker genes in each UMAP cluster. D. Module scores of neural cell type gene signatures from scRNA-seq atlases overlaid on UMAP. E. Fold change in target gene expression for negative (n=35) and positive (n=70) hit gRNAs. F. Differentially expressed genes (DEGs, significant gRNA–gene links) were defined using a two-tailed MAST test with Bonferroni correction . Significance of differences in number of DEG numbers were determined by a global one-way ANOVA with Dunnett’s post hoc test comparing all groups to NT, *p<0.05, ***p < 0.001. G. Expression level of neuron marker genes and genes associated with mitotic DNA replication by gRNA.

Article Snippet: For each TF that had <6 gRNAs present in the referenced library, additional unique gRNAs were subset from an earlier published gRNA library , for a total library size of 10,233 gRNAs. gRNAs were then ordered as an oligo pool from Twist Biosciences and cloned into an AIO CRISPRa vector by Gibson assembly.

Techniques: Expressing, Marker, Targeted Gene Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Comprehensive profiling of transcription factors for reprogramming human astrocytes to neuronal cells through endogenous CRISPR-based gene activation

doi: 10.1101/2025.10.11.681828

Figure Lengend Snippet: A. Pearson’s correlation-based distance matrix comparing the scaled expression of variable genes. Categories represent transcriptomes pseudobulked by gRNA. All positive hit or non-targeting gRNAs included. B. Module scores of all neural cell type gene signatures from scRNA-seq atlases, ordered from high to low with top 3 scoring modules labeled. Perturbations are grouped on the X axis by similarity in . C. Left: Cells in the relevant UMAP cluster colored in blue. Middle and right: Expression heatmap of key neuronal subtype markers. D. Selected enriched biological processes for neuronal subtype clusters. Statistical significance was determined using a two-tailed Fisher’s exact test followed by Benjamini–Hochberg correction. E. Enrichment for gRNAs in UMAP clusters determined by Seurat’s FindMarkers function with the hurdle model implemented in MAST. Weighted FC = −log10(p adj ) * abs(fold change). F. Expression of INSM1, LHX6, or ZNF276 in each t-SNE cluster of Allen Brain Cell Atlas . G. Validation of CRISPR perturbations identified to lead to neuronal or oligodendrocyte cell states in . RNA levels of key glutamatergic (glut), GABAergic (GABA), or oligodendrocyte (oligo) marker genes shown. Genes expected to be upregulated by each gRNA highlighted in blue-grey. *p < 0.05, **p < 0.01, ***p < 0.001 by global one-way ANOVA with Dunnett’s post hoc test comparing all groups to non-targeting gRNA.

Article Snippet: For each TF that had <6 gRNAs present in the referenced library, additional unique gRNAs were subset from an earlier published gRNA library , for a total library size of 10,233 gRNAs. gRNAs were then ordered as an oligo pool from Twist Biosciences and cloned into an AIO CRISPRa vector by Gibson assembly.

Techniques: Expressing, Labeling, Two Tailed Test, Biomarker Discovery, CRISPR, Marker

Journal: Frontiers in Genome Editing

Article Title: In vivo and in vitro genome editing to explore GNE functions

doi: 10.3389/fgeed.2022.930110

Figure Lengend Snippet: Generation of Sol8 Gne KO cells. (A) , schematic representation of the 3 gRNAs location in Gne exon 3. (B) , sequences of clone 12 and clone 13 DNAs, showing the respective deletions generated by Crisp/Cas9. (C) , schematic representation of the location of the primers used to test the cDNA sequences of clones 12 and 13. (D) , sequences of the cDNAs from clones 12 and 13 aligned with the respective DNA. (E,F) , sequences at the junction regions between the different exons in clone 12 and clone 13 respectively.

Article Snippet: Three gRNAs targeting three different sites in exon 3 of Sol8 Gne were chosen and purchased as sense and antisense strands (Hylabs, Israel), at a concentration of 100 µM.

Techniques: Generated, Clone Assay

Journal: Frontiers in Genome Editing

Article Title: In vivo and in vitro genome editing to explore GNE functions

doi: 10.3389/fgeed.2022.930110

Figure Lengend Snippet: Validation of the GNE KO status in Sol8 manipulated cells. (A) , schematic representation of the location of the primers used to verify Gne KO homogeneity; (B) , agarose gels illustrating the PCR products of clones 12 and 13 (yellow arrows) compared to controls (blue arrows); (C) , immunostaining of Sol8 Gne KO cells with anti-GNE Ab. (D) , FACS analysis of PSA positive cells (% of positive cells) ( n = 4 replicates); (E) , Incucyte confluency analysis; (F) , cell size determination (n > 300 cells); (G) , FACS analysis of anti Ki67 Ab immunostained Sol8 cells (% of positive cells) (n = 3 replicates).

Article Snippet: Three gRNAs targeting three different sites in exon 3 of Sol8 Gne were chosen and purchased as sense and antisense strands (Hylabs, Israel), at a concentration of 100 µM.

Techniques: Clone Assay, Immunostaining

Journal: Frontiers in Genome Editing

Article Title: In vivo and in vitro genome editing to explore GNE functions

doi: 10.3389/fgeed.2022.930110

Figure Lengend Snippet: Cell cycle analysis of Sol8 Gne KO cells. (A) , determination of the cell cycle stages by FACS analysis of 7AAD stained cells at 24 h after plating (upper panel) and at 48 h after plating (lower panel) (% of positive cells) ( n = 6 replicates). (B) , FACS analysis of PI stained (dead) cells: upper panel, % of positive cells; lower panel, staining intensity (MFI units) ( n = 3 replicates). (C) Differentiated cells stained with phalloidin (F-actin).

Article Snippet: Three gRNAs targeting three different sites in exon 3 of Sol8 Gne were chosen and purchased as sense and antisense strands (Hylabs, Israel), at a concentration of 100 µM.

Techniques: Cell Cycle Assay, Staining

Journal: Frontiers in Genome Editing

Article Title: In vivo and in vitro genome editing to explore GNE functions

doi: 10.3389/fgeed.2022.930110

Figure Lengend Snippet: Sol8 Gne KO cells cultured in stress conditions. (A) , confluency curves determined by Incucyte (in duplicates; each point depicts the average of 9 fields). (B) . Morphology of the cells by phase contrast microscopy. (C) , determination of cell size at day 3 ( n > 300 cells). (D) , staining of the cells with anti alpha actinin Ab. (E) , confluency curves of Sol8 Gne KO cells after AAVGNE infection. (F) , staining of the infected cells with anti GNE Ab. (G) , Immunostaining of Sol8 Gne KO and WT cells for p53BP1; (H) , Image -Pro Plus 7 analysis of area and intensity of γ-H2AX staining (relative units). (I) , FACS analysis of γ-H2AX stained cells (MFI units) ( n = 3 replicates).

Article Snippet: Three gRNAs targeting three different sites in exon 3 of Sol8 Gne were chosen and purchased as sense and antisense strands (Hylabs, Israel), at a concentration of 100 µM.

Techniques: Cell Culture, Microscopy, Staining, Infection, Immunostaining

Journal: Frontiers in Genome Editing

Article Title: In vivo and in vitro genome editing to explore GNE functions

doi: 10.3389/fgeed.2022.930110

Figure Lengend Snippet: Genes and proteins differentially expressed in Sol8 differentiated and proliferative Gne KO versus WT cells. (A) , PCA plot for the RNA seq triplicate samples of: Sol8WT proliferative cells (WT_P1-WT_P3), Sol8 WT differentiated cells (WT_D1-WT_D3), Sol8 Gne KO proliferative cells (KO_P1-KO_P3), Sol8 Gne KO differentiated cells (KO_D1-KO_D3). (B) , Venny representation of Sol8 Gne KO differentiated and proliferative differentially expressed genes. (C) , Venny representation of Sol8 Gne KO differentiated and proliferative differentially expressed proteins. (D) , Venny representation of the differentially expressed genes and proteins in both differentiated and proliferative Sol8 Gne KO cell versus WT cells.

Article Snippet: Three gRNAs targeting three different sites in exon 3 of Sol8 Gne were chosen and purchased as sense and antisense strands (Hylabs, Israel), at a concentration of 100 µM.

Techniques: RNA Sequencing Assay

Journal: Frontiers in Genome Editing

Article Title: In vivo and in vitro genome editing to explore GNE functions

doi: 10.3389/fgeed.2022.930110

Figure Lengend Snippet: Enriched processes in Sol8 Gne KO versus Sol8 WT cells. Major enriched pathways in proliferative (Prol) and differentiated (Diff) Sol8 cells for RNA expression (A) , protein expression (B) , phosphorylated proteins (C) and ubiquitinated proteins (D) . Enrichment pathways represented were obtained with the GeneAnalytics software except for enrichment of proteins (B) and of ubiquitinated proteins (D) in the proliferative cells that were generated by the IPA software.

Article Snippet: Three gRNAs targeting three different sites in exon 3 of Sol8 Gne were chosen and purchased as sense and antisense strands (Hylabs, Israel), at a concentration of 100 µM.

Techniques: RNA Expression, Expressing, Software, Generated