exon targeting guide rna  (Broad Clinical Labs)

Journal: Experimental & Molecular Medicine

Article Title: Reversibility and therapeutic feasibility of DNM1L -associated neurodevelopmental disorders

doi: 10.1038/s12276-026-01660-z

Figure Lengend Snippet: a Mutation information and representative MRI images of two patients. The T2 axial view of patient 1 was taken at the age of 3 years and 9 months. The FLAIR axial view of patient 2 was taken at the age of 1 year and 10 months, with increased signal intensity indicating atrophic change. b Cross-species conservation of protein sequences near the mutation residue. c The structure of the DRP1 tetramer (PDB, 4BEJ) shown as a ribbon diagram, with each protomer colored differently. Mutated residues are highlighted as sticks and labeled in a zoom-in box. L650 (UniProt ID, O00429-1) corresponds to L624 in DNM1L isoform 2 (UniProt ID, O00429-3) in the displayed structure. d Perturbation modeling of previously reported (G350R and G362S) and novel (L416P and L650R) DNM1L mutants using FoldX. A, B, C and D represent chain IDs in the tetramer structure, while AB, ABC and ABCD correspond to the dimer, trimer and tetramer, respectively. e Representative images of mitochondrial morphology in human NPCs transfected with DNM1L variants. Red, Mitotracker CMXRos; green, GFP; blue, DAPI. Scale bar, 10 μm. f Quantification of mitochondrial morphology in human NPCs transfected with DNM1L variants. n = 3 with at least 60 cells in each condition analyzed for an independent experiment. g Representative images of peroxisomal morphology in human NPCs transfected with DNM1L variants. Red, PMP70; green, GFP; blue, DAPI. Scale bar, 20 μm. h Quantification of peroxisomal morphology in human NPCs transfected with DNM1L variants. Control, n = 21; wild type, n = 34; G350R, n = 25; L416P, n = 31; L650R, n = 40. Bar graphs indicate mean ± s.e.m. Statistical significance is determined by two-way ANOVA with Tukey’s post hoc test for e and the Kruskal–Wallis test with Dunn’s post hoc test for h . ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Single-guide (sg)RNA targeting endogenous human DNM1L exon 1 was designed with Synthego online tool (Supplementary Table ).

Techniques: Mutagenesis, Residue, Labeling, Transfection, Control

Journal: Experimental & Molecular Medicine

Article Title: Reversibility and therapeutic feasibility of DNM1L -associated neurodevelopmental disorders

doi: 10.1038/s12276-026-01660-z

Figure Lengend Snippet: a , b , Representative images and quantification of GFP signal in postnatal brain slices electroporated with control vector or DNM1L G350R . IUE timelines are indicated above. Black, GFP. Scale bar, 100 μm. b Quantification of GFP + area ratio (G350R/control) in postnatal brain slices. n = 5. c , d Representative images and quantification of P7 mouse brain sections immunostained for CC3. Black arrows indicate positive cells. Black, CC3. Scale bar, 200 μm. n = 5. e IUE scheme for piggyBac system to label neurons and glia. f , g Representative images and quantification of P7 mouse brain sections electroporated with piggyBac system and immunostained with anti-GFP. Black, GFP. Scale bar, 200 μm. n = 4. Bar plot indicates mean ± s.e.m. Statistical significance is determined by the Kruskal–Wallis test with Dunn’s post hoc test for b , the Mann–Whitney test for d and one-way ANOVA with Dunnett’s post hoc test for g . ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant. Panel e created with BioRender.com .

Article Snippet: Single-guide (sg)RNA targeting endogenous human DNM1L exon 1 was designed with Synthego online tool (Supplementary Table ).

Techniques: Control, Plasmid Preparation, MANN-WHITNEY

Journal: Experimental & Molecular Medicine

Article Title: Reversibility and therapeutic feasibility of DNM1L -associated neurodevelopmental disorders

doi: 10.1038/s12276-026-01660-z

Figure Lengend Snippet: a Scheme of RNA sequencing sampling from DNM1L iKO NPCs and differentiated neurons. Cells were treated with DMSO (DNM1L ON ) or DOX (DNM1L OFF ) during culture. b , c Functional enrichment analysis of upregulated and downregulated DEGs between DNM1L OFF and DNM1L ON conditions in NPCs ( b ) and neurons ( c ). The bar color indicates the cluster categories of the GO term. d Representative images of neurospheres immunostained with CC3. Red, CC3; blue, DAPI. Scale bar, 50 μm. e Quantification of neurospheres immunostained with CC3. Control Dox−, n = 22; control Dox+, n = 31; DNM1L iKO Dox−, n = 26; DNM1L iKO Dox+, n = 31. f RRHO analysis. The overlap of gene expression alterations by DNM1 L dysfunction ( x axis, DNM1L OFF versus DNM1L ON ) and by neuronal differentiation ( y axis) was compared. Genes were ordered on the basis of their Wald test statistics (log 2 fold change divided by the standard error of the log 2 fold change). Each point on the plot indicates the significance level of the overlap between the two ranked gene lists. Bar graphs indicate mean ± s.e.m. Statistical significance is determined by Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test for e . **** P < 0.0001.

Article Snippet: Single-guide (sg)RNA targeting endogenous human DNM1L exon 1 was designed with Synthego online tool (Supplementary Table ).

Techniques: RNA Sequencing, Sampling, Functional Assay, Control, Gene Expression

Journal: Experimental & Molecular Medicine

Article Title: Reversibility and therapeutic feasibility of DNM1L -associated neurodevelopmental disorders

doi: 10.1038/s12276-026-01660-z

Figure Lengend Snippet: a Schematic diagram of rescue conditions. Red arrows indicate DOX treatment during culture. b Heat map of sample-to-sample distances based on variance-stabilized transformation of read count data for total gene expression. The clustering of RNA sequencing samples illustrates the genetic relationships, as indicated by the intensities of the square colors. c Overlapped DEGs between the indicated comparison groups are shown as a Venn diagram. DEGs of each group were identified by comparison with the DNM1L ON group. Restored and persistent DEGs in DNM1L OFF condition are filled with green and pink, respectively. d , e Heat maps (left) and functional enrichment analysis (right) of persistent ( d ) and restored ( e ) DEGs. The color gradient in the heat map represents the Z score. Functional enrichment of upregulated and downregulated genes was shown in separate bar plots. The bar color indicates the cluster categories of the GO term.

Article Snippet: Single-guide (sg)RNA targeting endogenous human DNM1L exon 1 was designed with Synthego online tool (Supplementary Table ).

Techniques: Transformation Assay, Gene Expression, RNA Sequencing, Comparison, Functional Assay

Journal: Experimental & Molecular Medicine

Article Title: Reversibility and therapeutic feasibility of DNM1L -associated neurodevelopmental disorders

doi: 10.1038/s12276-026-01660-z

Figure Lengend Snippet: a Scatter plot presenting reversibility of Reactome pathways, color-coded by keyword and sized by gene set size. The x axis is the percentage of reversible genes among the Reactome pathway gene sets, and the y axis is the difference in the extent of change caused by DNM1L dysfunction and under rescue conditions compared with control, expressed as a Z score. b Heat map of PPAR–PGC1α signaling pathway genes in different conditions. The color gradient represents Z scores. c Scheme of mimicking reversible effects by overexpressing the mitochondrial biogenesis master regulator PPARGC1A . d , e Representative images and quantification of P7 PPARGC1A -coexpressing mouse brain sections immunostained with anti-GFP. Black, GFP. Scale bar, 100 μm. n = 5. f , g Representative images and quantification of mouse primary cortical neurons electroporated with control or DNM1L G350R , treated with DMSO vehicle or bezafibrate. Green, GFP. Scale bar, 50 μm. The viability of mouse primary cortical neurons was compared with the starting point DIV 4. n = 6 from two independent experiments for all conditions. h Scheme of bezafibrate administration during the perinatal period. i , j Representative images of P7 mouse brain sections treated with DMSO vehicle or bezafibrate and immunostained with anti-GFP. Black, GFP. Scale bar, 100 μm. n = 5. Bar graphs indicate mean ± s.e.m. Statistical significance is determined by one-way ANOVA with Tukey’s post hoc test for e and j and two-way ANOVA with Bonferroni’s post hoc test for g . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Panels c and h created with BioRender.com .

Article Snippet: Single-guide (sg)RNA targeting endogenous human DNM1L exon 1 was designed with Synthego online tool (Supplementary Table ).

Techniques: Control

Journal: Journal of Neuroinflammation

Article Title: Rebamipide (Mucosta®), a clinically approved drug, alleviates neuroinflammation and dopaminergic neurodegeneration in a Parkinson’s disease model

doi: 10.1186/s12974-025-03461-z

Figure Lengend Snippet: Chemical structure of rebamipide. A Overview of the experimental design. B Protective effects of rebamipide on α-synuclein + MPP + -induced cytotoxicity in BV2 microglia cells. C BV2 microglia cells were treated with rebamipide for 1 h and then stimulated with α-synuclein + MPP + for an additional 11 h. Lactate dehydrogenase (LDH) ( C ), TNF-α ( D ), IL-6 ( E ), IL-1ß ( F , G , and I ), pro-IL-1ß ( H ), and IL-18 ( G ) levels were evaluated. Inhibitory effects of rebamipide on NLRP3 ( J ), ASC ( K ), pro-caspase-1 ( L ), and p20 ( M ) in BV2 microglia cells were measured using ELISA. Molecular docking illustrating the binding interactions of rebamipide with the NLRP3-NEK7 complex and NLRP3 alone ( N ). The left panels depict 3D docking models, whereas the right panels represent 2D interaction diagrams highlighting key binding residues. Hydrogen bonds are indicated by purple arrows, halogen bonds by yellow arrows, and salt bridges by red and blue lines. Rebamipide interacts with ASN978, TYR1009, and PRO1034 of NLRP3 and LYS163 of NEK7, suggesting a role in disrupting the NLRP3-NEK7 interaction and modulating inflammasome activation. Surface plasmon resonance (SPR) sensorgrams demonstrating the real-time binding kinetics of rebamipide to NLRP3-NEK7 complex ( O ) and NLRP3 alone ( P ). Rebamipide exhibited dose-dependent binding, with a higher binding affinity for NLRP3-NEK7 complex than NLRP3 alone. Overview of the experimental design ( Q ). Inhibitory effects of rebamipide on NLRP3-induced upregulation of IL-1ß ( R ) and IL-18 ( S ) in BV2 microglia cells. BV2 microglia cells were treated with rebamipide for 1 h and then stimulated with MSU, nigericine, ATP, and hemozoin for an additional 11 h. IL-1ß and IL-18 levels were evaluated ( T - X ). Data are presented as mean ± standard error of mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the α-synuclein + MPP + or NLRP3 inducers-treated groups

Article Snippet: A single-guide RNA (sgRNA) targeting exon 5 of the NLRP3 gene and Cas9 protein tagged with a nuclear localization signal were procured from Macrogen, Inc. to generate NLRP3 KO mice (Fig. A).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Activation Assay, SPR Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Rebamipide (Mucosta®), a clinically approved drug, alleviates neuroinflammation and dopaminergic neurodegeneration in a Parkinson’s disease model

doi: 10.1186/s12974-025-03461-z

Figure Lengend Snippet: NLRP3 inhibition neutralizes the protective effect of rebamipide following α-synuclein + MPP + intoxication. Overview of the experimental design. A BV2 microglia cells were treated with NLRP3 inflammasome inhibitor (MCC950) for 6 h, treated with rebamipide for 1 h, and stimulated with α-synuclein + MPP + for an additional 11 h. IL-1ß ( B ) and IL-18 ( C ) levels were evaluated using enzyme-linked immunosorbent assay kits (ELISA). Overview of the experimental design ( D ). Effects of NLRP3 on IL-1ß and IL-18 regulation ( E ) in NLRP3 siRNA-transfected BV2 microglia cells. NLRP3 inhibition neutralized the protective effect of rebamipide following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. An MCC950 or saline was injected once daily in the MPTP intoxication model before rebamipide treatment. Overview of the experimental design. F Microglial activations were visualized 3 d after the last MPTP treatment using Iba-1-specific immunostaining. Iba-1-immunopositive microglia in the substantia nigra pars compacta (SNpc) were counted 3 d after the last MPTP treatment (G and N). IL-1ß (H) and IL-18 ( I ) levels were evaluated using enzyme-linked immunosorbent assays (ELISA). Moreover, dopaminergic neurons were visualized 7 d after MPTP injection, using tyrosine hydroxylase (TH)-specific immunostaining. TH-immunopositive neurons in the SNpc ( J and O ) were counted, and the relative TH fluorescence intensity in the striatum (ST) ( K and O ) was measured. Dopamine levels in the ST were measured using HPLC ( L ). Latency time on the rotarod was recorded 7 d post-MPTP injection, with a 300 s cutoff limit ( M ). Data are expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the α-synuclein + MPP + or MPTP-treated group

Article Snippet: A single-guide RNA (sgRNA) targeting exon 5 of the NLRP3 gene and Cas9 protein tagged with a nuclear localization signal were procured from Macrogen, Inc. to generate NLRP3 KO mice (Fig. A).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Transfection, Saline, Injection, Immunostaining, Fluorescence, Control

Journal: Journal of Neuroinflammation

Article Title: Rebamipide (Mucosta®), a clinically approved drug, alleviates neuroinflammation and dopaminergic neurodegeneration in a Parkinson’s disease model

doi: 10.1186/s12974-025-03461-z

Figure Lengend Snippet: NLRP3 KO using CRISPR/Cas9 neutralizes the protective effect of rebamipide following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. Schematic of the NLRP3 KO using CRISPR/Cas9 ( A ) Sequences for CRISPR-Cas9 ( B ) and primer sequences for real-time RT-PCR ( C ) NLRP3 KO (hetero or homo) was confirmed using real-time PCR ( D ) Overview of the experimental design ( E ). Inhibitory effects of rebamipide on MPTP-induced levels of IL-1ß ( E ), IL-18 ( F ), and microglia activation ( G and H ) in the substantia nigra pars compacta (SNpc) 3 d after MPTP injection. Moreover, dopaminergic neurons were visualized using tyrosine hydroxylase (TH)-specific immunostaining 7 d after the last MPTP treatment. TH-immunopositive neurons in the SNpc were counted ( I ), and the relative TH fluorescence intensity in the striatum (ST) was measured ( J ). Dopamine levels in the ST were measured using HPLC ( K ). Representative photomicrographs of the SNpc and ST ( L ). Data are expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the MPTP-treated group

Article Snippet: A single-guide RNA (sgRNA) targeting exon 5 of the NLRP3 gene and Cas9 protein tagged with a nuclear localization signal were procured from Macrogen, Inc. to generate NLRP3 KO mice (Fig. A).

Techniques: CRISPR, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Activation Assay, Injection, Immunostaining, Fluorescence, Control

Journal: Journal of Neuroinflammation

Article Title: Rebamipide (Mucosta®), a clinically approved drug, alleviates neuroinflammation and dopaminergic neurodegeneration in a Parkinson’s disease model

doi: 10.1186/s12974-025-03461-z

Figure Lengend Snippet: NLRP3 KO neutralizes rebamipide’s protective effects on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor impairment in Cas9 Ctrl and NLRP3 KO mice. Representative images of mouse movement in the open field box, as captured by the video tracking system. The total distance covered by the mice was quantified in the open field box 7 d after MPTP injection ( A and E ), and the latency time on the rotarod was recorded, with a 300 s cutoff limit ( B ). Moreover, the time it took to turn completely downward ( C ) and the time to fall off the rod onto the floor ( D ) were recorded 7 d postinjection, with a 60 s cutoff limit. Data are presented as the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the MPTP-treated group

Article Snippet: A single-guide RNA (sgRNA) targeting exon 5 of the NLRP3 gene and Cas9 protein tagged with a nuclear localization signal were procured from Macrogen, Inc. to generate NLRP3 KO mice (Fig. A).

Techniques: Injection, Control

Journal: Journal of Neuroinflammation

Article Title: Rebamipide (Mucosta®), a clinically approved drug, alleviates neuroinflammation and dopaminergic neurodegeneration in a Parkinson’s disease model

doi: 10.1186/s12974-025-03461-z

Figure Lengend Snippet: Proposed mechanism through which rebamipide protects via NLRP3 inhibition against Parkinson’s disease pathogenesis. Rebamipide (Mucosta®), a clinically approved drug, alleviates neuroinflammation and dopaminergic neurodegeneration in a Parkinson’s disease model

Article Snippet: A single-guide RNA (sgRNA) targeting exon 5 of the NLRP3 gene and Cas9 protein tagged with a nuclear localization signal were procured from Macrogen, Inc. to generate NLRP3 KO mice (Fig. A).

Techniques: Inhibition