crispr guide rnas grnas  (Benchling Inc)

Journal: bioRxiv

Article Title: Plasmids weaponize conjugation to eliminate non-permissive recipients

doi: 10.64898/2026.02.10.705089

Figure Lengend Snippet: (A) Model of coercive assimilation. In non-permissive recipients (pink oval), intracellular defense systems (e.g., CRISPR-Cas9, teal) degrade the incoming plasmid, preventing TrbK expression. The unshielded recipient remains susceptible to repetitive, T4SS punctures and uncontrolled DNA delivery (lethal zygosis). (B) Restriction-Modification (defense). Left: Schematic of the RM competition assay. RM+ recipients (non-permissive, pink) and RM-recipients (permissive, yellow) compete in the presence of varying amounts of donor cells carrying RK2 plasmids (blue) that are either methylated (protected from digestion) or unmethylated (susceptible to digestion). Right: Competitive index of RM+ recipients relative to RM-recipients in the presence of donors with methylated (left facet) or unmethylated (right facet) RK2. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Different letters (a - b) indicate statistically significant differences within and between facets. When donors carried unmethylated RK2, RM+ recipients suffered significant fitness costs compared to the methylated plasmid treatment (p < 0.05) (C) CRISPR-Cas defense. Left: Schematic of the CRISPR competition assay. Recipients with Cas9 and an RK2-targeting gRNA (non-permissive, pink) or a non-targeting gRNA (permissive, yellow) were competed in the presence of varying amounts of RK2 donor. Right: Competitive index of targeting recipients relative to non-targeting recipients under uninduced or induced conditions. Different letters (a - b) indicate statistically significant differences (p < 0.05). Circles represent the competitive index of replicates, bars represent the mean (n = 3).

Article Snippet: CRISPR guide RNAs were designed to target RK2’s oriV using the Benchling guide design tool ( ).

Techniques: CRISPR, Plasmid Preparation, Expressing, Modification, Competitive Binding Assay, Methylation

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: ALS is associated to altered nucleoporin content in spinal cord and brain cortex . A) Left panel shows a representative western-blot image of FG-repeats containing NUPs (reactive to monoclonal antibody MAb414) of lumbar spinal cord homogenates from ALS patients and healthy, age and sex matched controls. Right panel shows the quantified immunoreactivity. B) The left panel shows representative confocal microscopy images from isolated nuclei from spinal cord samples, quantified in the violin plots from the right panel, demonstrating in situ decreased nucleoporin content.C) Quantitative analyses of NUP107, FG-repeats, and NUP93 in isolated nuclei by flow cytometry analyses, showing decreased content of NUP107 in spinal cord samples from ALS patients. D) RNA Seq data in heatmap showing that the mRNAs content of scaffold NUPs and NUP153 (an FG-repeat containing NUP) was decreased in spinal cord from ALS patients.Right panel indicates TPM (transcrits per kilobase million). E) Left panel shows confocal microscopy images with anti-tubulin β III immunoreactivity in nuclei isolated from brain cortex (area 8) from ALS patients and healthy individuals. Middle panel indicates tubulin β III content distribution, with the vertical line situating median values. Right panel illustrates differences in relationship between the content of FG-repeat NPC and tubulin β III in nuclei, with reference to ALS and tubulin β III content. F) Left panel shows representative confocal microscopy images from isolated nuclei from spinal cord samples, quantified plots from the right panel, demonstrating a decreased amount of nuclei from neuronal cells, based on NeuN content. Bars show mean values ± SEM or % in F. ∗,∗∗, and ∗∗∗∗ indicate p < 0.05, p < 0.01 and p < 0.0001 with reference to control values by Student's T test, Chi-Square (F) or Mann-Whitney U test. In E) For controls, linear relationship equation Y = −0.2721∗X + 0.01782 (p < 0.0004) and for ALS, the equation is Y = 0.3195∗X + 0.008008 (p < 0.0001). Scale white bar length in B, E and F are 50 μm long. Ns: non statistically significant differences.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Western Blot, Confocal Microscopy, Isolation, In Situ, Flow Cytometry, RNA Sequencing, Control, MANN-WHITNEY

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: NUPs are present in motor neurons and glial cells in human lumbar spinal cord, ventral horn. Representative immunohistochemical images of cellular distribution of the scaffolding NUP TPR (A–D) and the FG-repeat NUP107 (E,F). Samples from grey matter are shown in A,B,E and F, while as white matter cells (i.e. glial cells) are shown in C and D. Samples from healthy donors (A,C and E) and ALS patients (B,D and F) are presented. Arrows in A and B indicate non-nuclear staining of TPR, which were more frequent in ALS samples.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Immunohistochemical staining, Scaffolding, Staining

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: NUP107 silencing alters autophagy and induces TDP-43 pathology in human cells . A) Left panel indicates representative western-blot of different NUPs in CRISPR-mediated NUP-107 silencing of HEK293 cells. Right panel shows densitometry analyses of different experiments. B) Left panel shows representative western-blot of different autophagy and protein-turnover components in the same cells, with right panel indicating densitometric analyses. C) Confocal microscopy of TDP-43 immunoreactivity of HEK293 CRISPR-mediated NUP107 cells, showing altered TDP-43 immunoreactivity, quantified in the lower panel.D) Confocal microscopy of phospho-TDP-43 immunoreactivity of CRISPR-mediated NUP107-silenced HEK293 cells, showing altered TDP-43 immunoreactivity, quantified in lower panel microscopy. E) Western-blot analyses of TDP-43 and phospho-TDP-43 in CRISPR-mediated NUP107-silenced HEK293 cells, quantified in the right panel. F) Higher magnification of confocal microscopy images showing cytosolic aggregates of phospho-TDP-43 (yellow arrows) induced by CRISPR-mediated NUP107-silencing in HEK293 cells, quantified in the left panel (both size and number of phospho-TDP-43 aggregates). G)Left panel, representative western-blot analyses of NUPs and autophagy components in siRNA-mediated silencing of NUP107 in HeLa cells.Right panel shows the quantitative analyses. Bars show mean values ± SEM from N = 3–5 different experiments. ∗,∗∗, ∗∗∗ and ∗∗∗∗ indicate, respectively, p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 significant differences between silenced or non silenced cells by Student's T test or Mann Whitney U test. In B and D, white scale bar lenght is 50 μm long and 30 μm in F. ns: non statistically significant differences.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Western Blot, CRISPR, Confocal Microscopy, Microscopy, MANN-WHITNEY

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: Loss of TDP-43 induces alteration in nucleoporin homeostasis. A) Representative immunofluorescence analyses showing NUP107 and MAb414 in TDP-43 silenced cells for a HeLa inducible silencing clone. B) Violin plot showing the quantitative analyses of nuclear (i.e. colocalizing with DAPI) content of NUP107 and MAb414 of cells in A. C) Left panel, western-blot analyses of TPR-content in HeLa cells with inducible TDP-43 silencing, with right panel showing quantitative analyses. D) Western-blot analyses of selected NUPs and LC3B in HeLA cells with doxycycline-inducible TDP-43 silencing under autophagy induction by 24 h of nutrient deprivation (Depriv). E) Quantitative analyses of western-blots shown in D. Bars show mean values ± SEM from N = 3–5 different experiments. ∗,∗∗, and ∗∗∗∗ indicate, respectively, p < 0.05, p < 0.01, and p < 0.0001 significant differences between silenced or non silenced cells by Student's T test, Mann Whitney U test or post-hoc LSD after two-way ANOVA. In A white scale bar lenght is 50 μm long. ns: non statistically significant differences.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Immunofluorescence, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: The DND1–NANOS3 complex shapes the primordial germ cell transcriptome via a heptanucleotide sequence in mRNA 3′ UTRs

doi: 10.1101/2025.09.25.678639

Figure Lengend Snippet: ( a ) Schematic illustrations of human DND1 and NANOS3 protein domains. Protein constructs used in this study are indicated below. ( b ) Crystal structure of DND1 (purple) and NANOS3 (pink) reveals a continuous surface to recognize the 7-nt N3-DRE RNA. ( c-i ) Hydrogen bond, salt bridge, and stacking interactions formed between DND1–NANOS3 and N3-DRE nucleotides. Hydrogen bonds and salt bridges are indicated by dotted lines. Water molecules that mediate side chain- nucleotide interactions are represented by red oxygen spheres. Residues that stack with RNA bases are shown with transparent surfaces. ( j ) DND1 and NANOS3 recognize N3-DRE nt A5. Protein- protein and protein RNA interfaces near N3-DRE nts A4-A5 are highlighted. A primary DND1–NANOS3 interaction surface includes DND1 helix 1. ( k ) In vitro RNA-binding assays demonstrate that DND1 and NANOS3 ZF+C bind with high affinity to N3-DRE. RBP(s) included (left); K d values measured by MST are shown as a bar graph (right). See also Supplementary Table 2. K d values were determined from 3 independently pipetted measurements. ( l ) High-affinity RNA binding requires an intact N3-DRE sequence. Mutations of DND1 or NANOS3 binding sites (D1mut, N3mut, D1-N3mut) or separation of DND1 (A1-A4) and NANOS3 (A5-U7) interaction sequences by a spacer of three cytosines (spacer) abolishes high-affinity binding. ( m ) DND1–NANOS3 ZF+C selectively recognize G3. K d values measured by MST are shown for binding to CDK1 N3-DRE RNAs with G3 (wt) or substituted with the other nts. Fold differences (right) indicate the range of differences relative to G3 (wt).

Article Snippet: We purchased a cocktail of three small guide RNAs (sgRNAs) (Santa Cruz Biotechnology, Inc.; cat# sc-434074), cloned individual sgRNA plasmids, and used pairs of the sgRNAs for the targeted deletions of the zinc-finger region of the Nanos3 locus.

Techniques: Construct, In Vitro, RNA Binding Assay, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: The DND1–NANOS3 complex shapes the primordial germ cell transcriptome via a heptanucleotide sequence in mRNA 3′ UTRs

doi: 10.1101/2025.09.25.678639

Figure Lengend Snippet: (a) Amino acid residues of DND1 (purple) and NANOS3 (pink) interacting with the N3-DRE. Residues with filled shading contact RNA bases through their side chains and were substituted with alanine to assess their importance for DND1–NANOS3 function. (b) Schematic of DND1–NANOS3 co-expression construct with mutated amino acids indicated (top) or DND1 and NANOS3 truncations highlighted in blue (bottom). (c) Schematic of assays performed with cells expressing constructs from (b). (d) Results from cell-based assays for DND1–NANOS3 constructs expressing DND1 point mutants. Left panels show results from IncuCyte growth assays. The middle panels show the effect of expression of mutant constructs on N3-DRE-containing RNAs. n = number of genes in each group; ML = med.lfc. Right panels show protein expression levels of three N3-DRE containing target genes (CDK1, EHMT2, and CNOT6) determined by Western Blot after expression of mutant constructs. The y-axis in each graph represents mean confluence, cumulative fraction, and relative protein level, respectively. (e) Same as in (d) except for the expression of DND1-NANOS3 constructs with point mutations in NANOS3. (f) Same as in (d) except for expression of DND1- NANOS3 constructs with truncations in DND1 or NANOS3. All experiments were performed in three independent biological replicates. *p < 0.05, ** p < 0.01 .

Article Snippet: We purchased a cocktail of three small guide RNAs (sgRNAs) (Santa Cruz Biotechnology, Inc.; cat# sc-434074), cloned individual sgRNA plasmids, and used pairs of the sgRNAs for the targeted deletions of the zinc-finger region of the Nanos3 locus.

Techniques: Expressing, Construct, Mutagenesis, Western Blot