gpp sgrna designer  (Broad Institute Inc)

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Scheme of genome editing strategies used in this study. Cytidine deaminase Anc-APOBEC linked to dCas9 with CLCN4 -targeting sgRNAs was applied to hESCs (HUES6, 46XX). A target nucleotide sequence is highlighted in yellow. PAM sequence for each three sgRNA is marked in color-matched rectangles. (b) Allelic modification in CLCN4 verified by Sanger sequencing. Each sgRNA results in specific changes: sgRNA_A, CCG to TTG (A555L) in one allele; sgRNA_B, GCT to CTT (A556V) in one allele; and sgRNA_C, GTG to ATA (V557I) in both alleles. The hESC clones generated by each sgRNA were named Variants A, B, and C. (c) Representative whole-cell current traces from HEK293T cells expressing WT (black), reported mutant A555V (green), variant A (A555L, red), variant B (A556V, blue), and variant C (V557I, brown) hCLCN4 channels. Current traces are shown in 20-mV voltage increments for clarity. (d) Current-voltage (I-V) relationships of WT (black), A555V (green), A555L (red), A556V (blue), and V557I (brown) hCLCN4 channels. Current amplitudes were normalized to current densities. Each symbol represents mean ± SEM (WT, n=17; A555V, n=8; A555L, n=10; A556V, n=8; V557I, n=11). (e) Current density at voltage 150 mV. (One-way ANOVA, *** p =0.0003019 for A555V, **** p =0.00008188 for A555L, p =0.16391 for A5556, and p =0.1923 for A557I) (f) Representative images of day 50 forebrain organoids. SOX2 (magenta) and TUJ1 (yellow) were detected as makers for NPCs and neurons, respectively. Blue represents DAPI. Images are from a single plane of confocal images. Scale bar = 20 μm. (g) scRNA-seq results of day 50 forebrain organoids. The cell clusters are presented based on the gene expression pattern (left) and the differential abundance of cell population (right).

Article Snippet: Three sgRNAs targeting CLCN4 were designed using the Broad Institute GPP sgRNA designer.

Techniques: Sequencing, Modification, Clone Assay, Generated, Expressing, Mutagenesis, Variant Assay, Gene Expression

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Relative expression levels of CLCN4 mRNA during neurogenesis and in non-neuronal cells, normalized to ACTB expression. (n=3 or 4, mean ± SEM, one-way ANOVA, * p =0.0172) (b) NPCs (top) and day 4 neurons (bottom) derived from WT and CLCN4 -variant hESCs. NPCs were stained with SOX2 (magenta) and NESTIN (green), neurons with TUJ1 (green). DAPI is shown in blue. Scale bar = 20 μm. (c) Representative images of developing neurons used for neurite length analysis. Scale bar = 50 μm. (d) Quantification of average (left), total (middle), and maximum (right) neurite length. Average neurite length was calculated as total neurite length divided by the number of branches. The following numbers of neurons from three or more biological replicates were analyzed: day 2 (WT = 35; variant A = 38), day 4 (WT = 26; variant A = 25), and day 6 (WT = 28; variant A = 25). (mean ± SEM, two-way ANOVA with Sidak’s multiple comparisons test; * p =0.0288 at average; * p =0.0228 at total; ** p =0.0021 at maximum) (e) Schematic timeline for quantifying changes in neuronal cell numbers. (f) Quantification of relative neuronal cell numbers during neurogenesis. (n = 3 or 4, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test; Variant A, **p=0.0076 ; Variant B, *p=0.0259 ; Variant C, **p=0.0095 ). The magenta dashed line indicates the WT value. Each dot represents the relative ratio between the variant and WT from one biological replicate. (g) Relative TUNEL intensity in day 6 neurons. (n=3, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test; **** p <0.0001 for variant A, ** p =0.0054 for variant B, and ** p =0.0048 for variant C)

Article Snippet: Three sgRNAs targeting CLCN4 were designed using the Broad Institute GPP sgRNA designer.

Techniques: Expressing, Derivative Assay, Variant Assay, Staining, TUNEL Assay

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Representative images of day 6 neurons after Lysotracker treatment. Lysotracker is shown in magenta, and DAPI in blue. Scale bar = 20 μm. (b) Quantification of Lysotracker signal in day 6 neurons. The relative Lysotracker intensity of CLCN4 -variant neurons compared to the WT was presented. Each dot represents one biological replicate. (n=3-4, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test * p =0.039 for variant A; *** p =0.0007 for variant B; ** p =0.0092 for variant C) The magenta dashed line indicates the value for WT. (c) Relative acidity in acidic subcellular organelles. Each dot represents one biological replicate. (n=3, mean ± SEM, unpaired Student’s t-test; p =0.2894) (d-g) Dynamics of endo-lysosomal vesicles. RAB5, RAB7, RAB11 and LAMP1 indicates early endosome, late endosome, recycling endosome and lysosome markers, respectively. (RAB5, n=6; RAB7, n=3; RAB11, n=4; and LAMP1, n=3 or 4; mean ± SEM, unpaired Student’s t-test) (d) Representative images of a neuron with GFP-labeled vesicular organelles. TUJ1 and DAPI are shown in red and blue, respectively. Scale bar = 10 μm. (e) Relative GFP intensity expressed in vesicular organelles (* p =0.0265 for RAB5, * p =0.0158 for RAB7, * p =0.0475 for RAB11, and ** p =0.0067 for LAMP1) (f) Number of GFP-expressing vesicles in neurons. (* p =0.0186 for RAB5, * *p =0.0066 for RAB7, and p =0.9031 for LAMP1) (g) Average distance of GFP-expressing vesicles from the center of nucleus, representing the mean linear distance of puncta from the center of the nucleus in each cell. (** p =0.0011 for RAB5, * p =0.0463 for RAB7, and * p =0.0103 for LAMP1) (h) Scheme of autophagic flux analysis using tandem fluorescent protein fused to LC3. (i) Representative images of day 6 neurons expressing tandem fluorescent-tagged LC3. Red: RFP, green: GFP, white: TUJ1. Scale bar = 5 μm. (j) Quantification of the GFP-/RFP+ puncta. (n=5, mean ± SEM, unpaired Student’s t-test; * p =0.0164)

Article Snippet: Three sgRNAs targeting CLCN4 were designed using the Broad Institute GPP sgRNA designer.

Techniques: Variant Assay, Labeling, Expressing

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Volcano plots in day 2 and day 6 neurons total RNA seq. Horizontal dashed lines: threshold of adjust p-value, 0.05; dashed lines: threshold of |log2FoldChange|, 1. Blue and red dots indicate significantly down- and up-regulated genes, respectively. (b) TPM of CLCN4 in total RNA seq. (c) The confirmed expression of CLCN4 in CLCN4 -variant day 2 (left) and day 6 (right) neurons by real-time PCR. (n=5, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test, D2 neuron; ** p =0.0033 for variant A, ** p =0.0031 for variant B, and * p =0.0424 for variant C, D6 neuron; ** p =0.0047 for variant A, * p =0.0301 for variant B, and * p =0.0462 for variant C) (d) Top 5 significant gene list from total RNA seq. (e) The confirmed expression of MEG3 in CLCN4 -variant day 2 (left) and day 6 (right) neurons by real-time PCR. (n=3, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test, **** p < 0.0001) (f) Changes in expression of MEG3 during neurogenesis. (n=3, mean ± SEM, two-way ANOVA with Dunnett’s multiple comparisons test; *** p =0.0003 at D3, **** p < 0.0001 at D6, * p =0.0114 at D13, * p =0.0169 at D20, and *** p =0.0008 at D27)

Article Snippet: Three sgRNAs targeting CLCN4 were designed using the Broad Institute GPP sgRNA designer.

Techniques: RNA Sequencing, Expressing, Variant Assay, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Diagram of CRISPR/Cas9-based MEG3 inhibition system applied to WT NPC and neuron. (b) Relative expression level of MEG3 in WT NPCs after MEG3 inhibition. (n=3, mean ± SEM, unpaired Student’s t-test, **** p <0.0001) (c) Quantification of cell number ratio in WT neurons during neurodevelopment after MEG3 inhibition. (n=4, mean ± SEM, unpaired Student’s t-test; ** p =0.0083) (d) Diagram of CRISPR/Cas9-based MEG3 activation system applied in CLCN4 -variant A cells. (e) Relative expression of MEG3 in CLCN4 -variant NPCs after MEG3 activation. (n=3, mean ± SEM, unpaired Student’s t-test, ** p =0.0058) (f) Changes in cell number was compared in CLCN4 -variant neurons after MEG3 activation. (n=4, mean ± SEM, unpaired Student’s t-test, * p =0.0475) (g) Quantification of average (left), total (middle), and maximum (right) neurite length in CLCN4 -variant neurons. The following numbers of neurons were analyzed: day 2 (sgSCR = 34; sgMEG3-a = 36), day 4 (sgSCR = 11; sgMEG3-a = 10), and day 6 (sgSCR = 32; sgMEG3-a = 37). (n=3, mean ± SEM, two-way ANOVA with Sidak’s multiple comparisons test; * p =0.0372 at total; ** p =0.0028 at maximum) (h) Representative images of LAMP1 in day 6 neuron. LAMP1 (green), TUJ1 (red), and DAPI (blue) were shown. Scale bar = 5 μm. (i) Relative GFP intensity expressed in LAMP1-positive vesicles. (n=3, mean ± SEM, unpaired Student’s t-test, ** p =0.0078) (j) Representative images of day 6 neurons expressing tandem fluorescent-tagged LC3. Red: RFP, green: GFP, white: TUJ1. Scale bar = 5 μm. (k) Quantification of the GFP-/RFP+ puncta in CLCN4-variant neurons after MEG3 activation. (n=3, mean ± SEM, unpaired Student’s t-test, * p =0.0327)

Article Snippet: Three sgRNAs targeting CLCN4 were designed using the Broad Institute GPP sgRNA designer.

Techniques: CRISPR, Inhibition, Expressing, Activation Assay, Variant Assay