uracil specific excision reagent user cloning dna fragments  (Thermo Fisher)

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Quantification of mRNA levels by RT-qPCR showing knockdown efficiency of target genes upon siRNA treatment. Expression was normalized to the non-targeting siRNA control (siCTR) set to 1 for each gene. n = 2 independent experiments. (b) Total receptor levels determined by GFP fluorescence intensity in DOR-RUSH HeLa cells treated with individual siRNAs as in (a). GFP intensity was measured by flow cytometry, and normalized to non-treated control cells (NT, set to 1). n = 3 independent experiments. Significance was determined by one-way Anova: *, P < 0.05; **, P < 0.01. (c) Flow cytometry histograms showing total receptor levels based on GFP fluorescence in DOR-RUSH cells lacking CNIH1. Left: Cells treated with control (siCTR) or CNIH1-targeting siRNA. Right: Parental and CNIH1 KO DOR-RUSH cells. (d) Quantification of total receptor levels based on mean GFP intensity in cells depleted of CNIH1 using siRNA or CRISPR/Cas9-mediated KO measured by flow cytometry. n = 3 independent experiments.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Quantitative RT-PCR, Knockdown, Expressing, Control, Fluorescence, Flow Cytometry, CRISPR

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Western blot analysis of CNIH1 protein levels in parental and CNIH1 KO DOR-RUSH cells, and in cells treated with control (siCTR) or CNIH1-targeting siRNA (siCNIH1). CNIH1 signals were normalized to tubulin. n = 3 independent experiments. Significance was determined by one-way ANOVA and Tukey’s multiple comparisons tests; ****, P < 0.0001. (b, c) DOR PM levels at various time points after biotin addition in DOR-RUSH HeLa cells depleted of CNIH1 by siRNA (b) or CRISPR/Cas KO (c) shown relative to their respective controls. Surface DOR was quantified by flow cytometry normalized to the signal of parental DOR-RUSH cells at 24 h. n = 3 independent experiments. Significance was determined by two-way ANOVA with Šídák’s multiple comparisons tests; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. ( d ) Representative confocal microscopy images of parental and CNIH1 KO DOR-RUSH HeLa cells fixed 0.5 h after biotin addition. DOR (GFP signal) and the Golgi compartment (anti-Giantin immunolabeling) are shown. Images are maximum intensity Z-projections of confocal stacks. Scale bar, 10 μm. ( e ) Quantification of DOR fluorescence in the Golgi region in parental and CNIH1 KO DOR-RUSH cells after biotin addition. Cells were fixed and immunolabeled with anti-Giantin as in (d). Data are mean ± SEM error bands from 3 independent experiments. ( f ) Western blot analysis of DOR glycosylation 0, 2, 6 and 24 hours after biotin addition in parental and CNIH1 KO DOR-RUSH HeLa cells. DOR was isolated from cell lysates via GFP-Trap beads and treated with EndoH. Left panel: representative blot (anti-GFP). Right panel: Quantification of mature vs. immature receptor signals. n = 3 independent experiments. Significance was determined by two-way ANOVA with Šídák’s multiple comparisons tests; *, P < 0.05; **, P < 0.01; ***, P < 0.001. ( g ) Split-NanoLuc complementation assay measuring mGi1 recruitment to DOR in parental and CNIH1 KO DOR-RUSH cells expressing mGi1-LgBit and lyn11-SmBit. Cells were pre-incubated with biotin for 1.5 h or 24 h, or left untreated, and then stimulated with peptide agonist DADLE (100 nM, added at 3 min during acquisition). Graphs show mGi1 recruitment signal over time. Data are mean ± SEM error bands from 3 independent experiments. ( h ) Quantification of DADLE-induced mGi recruitment to DOR based on area under the curve (AUC) analysis in parental and CNIH1 KO cells as in (g). n = 3 independent experiments. Significance was determined by two-way ANOVA with Šídák’s multiple comparisons; *, P < 0.05. ( i ) Left: Western blot showing total ERK and phosphorylated ERK (pERK) levels in parental and CNIH1 KO DOR-RUSH cells after 5 min stimulation with 10 μM DPDPE (+) or vehicle (-). Right: Quantification of pERK/ERK ratios normalized to unstimulated control cells. n = 3 independent experiments. Significance was determined by two-tailed paired t-test; **, P < 0.01.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Western Blot, Control, CRISPR, Flow Cytometry, Confocal Microscopy, Immunolabeling, Fluorescence, Glycoproteomics, Isolation, Expressing, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Confocal microscopy image of HeLa cells immunostained for endogenous CNIH1 (magenta) and the cis-medial Golgi marker Giantin (cyan). Images are single confocal slices. The merged image shows strong co-localization in the Golgi region. The lower panel shows a magnified view of the boxed area. Line-scan analysis (right) displays the pixel intensity profiles of CNIH1 and Giantin along the indicated line. Representative image from n = 3 independent experiments. Scale bar, 10 μm. (b) Confocal microscopy image of HeLa cells immunostained for endogenous CNIH1 (magenta) and expressing the ERES marker mCherry-Sec23A (cyan). Images are single confocal slices. The merged image shows co-localization in the Golgi area and in puncta in the cytosol. The lower panel shows a magnified view of the boxed area, with a line-scan analysis (right) depicting pixel intensity profiles of CNIH1 and Sec23A across puncta at indicated line. Scale bar, 10 μm. (c) Left: Pearson’s correlation coefficient analysis to determine the degree of linear relationship between fluorescence intensities of CNIH1 and Giantin, Sec23A, or EEA1 in immunostained HeLa cells. Right: Manders’ colocalization coefficients M1 and M2 indicating overlap of CNIH1-positive pixels with Giantin or Sec23A (M1) or overlap of Giantin or Sec23A-positive pixels with CNIH1 (M2). The majority of Giantin- and Sec23A-positive pixels overlap with CNIH1. Cells analyzed: Giantin, n = 27; Sec23A, n = 17; EEA1, n = 9. (d) AlphaFold-predicted structural model of human CNIH1 (144 amino acids). The model has an average per-residue local distance difference test (pLDDT) confidence score of 84.3 and predicts a four-transmembrane-domain topology with the N-terminus (blue) and C-terminus (red) oriented toward the organelle lumen. The dotted line depicts the approximate membrane boundaries. (e) Confocal images of HeLa cells expressing CNIH1 (top) or CNIH4 (bottom), N-terminally fused to pmApple. Images are single confocal slices. Scale bar, 10 μm. (f) Percent identity between CNIH1 and other human CNIH paralogs. CNIH2 and CNIH3 show the highest identity.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Confocal Microscopy, Marker, Expressing, Fluorescence, Residue, Membrane

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Confocal microscopy images of parental (top) or CNIH1 KO (bottom) DOR-RUSH HeLa cells (no biotin added) immunostained for endogenous CNIH1 (magenta) and the cis-medial Golgi marker Giantin (cyan). DOR is shown by GFP fluorescence, nuclei were stained with DAPI. Images are maximum intensity Z-projections of confocal stacks. Scale bar, 10 μm. (b) Quantification of CNIH1 antibody staining intensity in the Golgi area in parental and CNIH1 KO DOR-RUSH HeLa cells. Data are mean ± SD, n = 4 independent experiments, each comprising 7-9 fields of view. (c) Confocal microscopy images of HeLa cells immunostained for endogenous CNIH1 (magenta) and either expressing the early endosomal marker Venus-EEA1 (cyan, top) or labeled with LysoTracker prior to fixation to visualize lysosomes (bottom). Merged images reveal no detectable co-localization between CNIH1 and endosomal or lysosomal compartments. Images are single confocal slices. Scale bar, 10 μm. (d) Confocal microscopy images of HEK293 (top) and SH-SY5Y (bottom) cells immunostained for endogenous CNIH1 (magenta) and the cis-medial Golgi marker Giantin (cyan). Nuclei were stained with DAPI. Images are maximum-intensity Z-projections of confocal stacks. Scale bar, 10 μm.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Confocal Microscopy, Marker, Fluorescence, Staining, Expressing, Labeling

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Confocal microscopy images of HeLa cells expressing CNIH1 (top) or CNIH4 (bottom) fused N-terminally to an HA tag. Cells were fixed and immunostained with anti-HA (magenta) and anti-Giantin (cyan) antibodies. Merged images show localization of CNIH1 in the Golgi area, which is less pronounced for CNIH4. Images are confocal slices. Scale bar 10 μm. (b) Heatmap of CNIH1-4 expression across diverse human tissues based on bulk RNA-sequencing data from the GTex portal ( https://www.gtexportal.org/home/ ), using the Multi-Gene Query tool. Expression values are shown as Transcripts per Million (TPM). (c) Relative RNA expression levels of CNIH1-4 in a published dataset of 12 human nociceptor cell types ( https://sensoryomics.shinyapps.io/RNA-Data/ ), known for endogenous DOR expression. Expression values of CNIH1-4 shown in colors indicated in the figure, with 75th and 25th percentile of overall RNA expression levels within each cell type shown as lines across the graph.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Confocal Microscopy, Expressing, RNA Sequencing, RNA Expression

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Flow cytometry histograms showing total cargo expression based on GFP fluorescence in various GPCR-RUSH and GFP-GPI-RUSH cell lines. Cargo expression levels are comparable to those observed for DOR-RUSH cells. (b) Quantification of CNIH1 protein levels in HeLa cells treated with control siRNA (siCTR) or siRNAs targeting CNIH1 (siCNIH1) or CNIH4 (siCNIH4), based on western blot analysis. CNIH1 signals were normalized to tubulin. n = 3 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. *, P < 0.05.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Flow Cytometry, Expressing, Fluorescence, Control, Western Blot

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Kinetics of PM arrival of CCR3, P2YR1, GPR35, H1R, M5R and GFP-GPI in transgenic cargo-RUSH HeLa cells, measured by flow cytometry. The AF647 cargo surface signals at each time point were normalized to the condition with the highest surface levels (set to 100%) for each receptor. Data are mean ± SD from 3 independent experiments. (b, c) Effects of siRNA-mediated depletion of CNIH1 (b) and CNIH4 (c) on cell surface delivery of secretory cargos 1.5 hours after biotin addition. For each respective RUSH HeLa cell line, cells treated with non-targeting control siRNA (siCTR) served as a control (set to 100%). n ≥ 3 independent experiments. Significance was determined by one sample t-test. *, P < 0.05; **, P < 0.01. (d) Western blot analysis of CNIH1 (left) and CNIH4 (right) protein levels in HeLa cells treated with control siRNA (siCTR) or siRNAs targeting CNIH1 (siCNIH1) or CNIH4 (siCNIH4). Tubulin levels serve as a loading control. (e) DOR PM levels in DOR-RUSH CNIH1 KO cells further depleted of CNIH4 by siRNA after 1.5 h of biotin incubation. Signals were normalized to DOR-RUSH parental cells at 1.5 h biotin addition (set to 100%). Significance was determined by paired t-test. *, P < 0.05. (f) Western blot analysis of CNIH4 levels in DOR-RUSH CNIH1 KO treated with control siRNA (siCTR) or siRNA targeting CNIH4 (siCNIH4). Tubulin levels serve as a loading control.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Transgenic Assay, Flow Cytometry, Control, Western Blot, Incubation

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Co-immunoprecipitation of DOR and CNIH1 in DOR-RUSH HeLa cells at 0, 1, and 24 h after biotin addition using GFP-Trap beads. Interaction was detected in the presence of the DSP crosslinker, tubulin serves as a negative control. Quantification of CNIH1 co-precipitation signals at different time points is shown normalized to the 0 h time point. Significance was determined by one sample t-test. *, P < 0.05. (b) Co-isolation of MOR and CNIH1 upon co-expression of His-tagged MOR and Strep-tagged CNIH1 in Sf9 insect cells. Sequential purification steps included: 1) Ni-NTA affinity purification of His-MOR; 2) StrepTactin affinity purification of Strep-CNIH1; and 3) size-exclusion chromatography. Elution fractions from each step were analyzed by SDS-PAGE and Coomassie stain or by western blot using anti-Strep and anti-His antibodies. Expected molecular weights of CNIH1 (open arrowhead) and MOR (filled arrowhead) are indicated. (c) Predicted structure of the CNIH1-DOR complex generated with Alphafold3 (interface predicted template modelling score, ipTM = 0.73). The interaction is predominantly predicted along DOR’s TMD6 and CNIH1’s TMD1, with a polar interface at the cytoplasmic side involving arginine residues in DOR (light blue) and aspartic acid (D32), glutamic acid (E33), and tyrosine (Y36) in CNIH1. The putative COPII binding site in CNIH1 (aa 106-112) is highlighted in green, with the sequence indicated. (d) Rescue of DOR export 1.5 h after biotin addition in DOR-RUSH CNIH1 KO cells by expressing CNIH1 constructs or human CNIH paralogs. CNIH1 KO cells were transfected with pm-Apple fused CNIH1 wild-type, CNIH1 DOR-interface mutant (D32A/ E33A/Y36A), CNIH1 COPII-binding mutant (aa 106-112 replaced by alanines), or CNIH2, CNIH3, CNIH4. Control (CTR) cells were transfected with pmApple. Parental DOR-RUSH cells transfected with pmApple serve as a control for DOR export at 1.5 h. n = 3-7 independent experiments. Significance was determined by one sample t-test. *, P < 0.05, **, P < 0.01. (e) Confocal time-lapse images of DOR-RUSH CNIH1 KO HeLa cells expressing or lacking (arrowhead) pmApple-CNIH1, acquired at the indicated time points after biotin addition (80 μM). Images are single confocal slices. The right panel presents an enlarged view of the boxed region, highlighting co-localized puncta. Scale bar, 10 μm.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Immunoprecipitation, Negative Control, Isolation, Expressing, Purification, Affinity Purification, Size-exclusion Chromatography, SDS Page, Staining, Western Blot, Generated, Binding Assay, Sequencing, Construct, Transfection, Mutagenesis, Control

Journal: bioRxiv

Article Title: CRISPR screen identifies CNIH1 as a selective driver of GPCR export

doi: 10.1101/2025.10.27.684930

Figure Lengend Snippet: (a) Flow cytometry plots showing gating strategy for selecting pmApple-positive cells to assess effects on DOR export (related to ). Right: histograms of non-transfected and pmApple-N1-transfected cells used to define the pmApple-positive gate. (b) Representative flow cytometry histograms showing expression of pmApple-fused CNIH1 wild-type and DOR or COPII interface mutants in DOR-RUSH CNIH1 KO cells. Expression levels are comparable across constructs. (c) Confocal images of HeLa cells expressing CNIH1 wild-type or DOR and COPII interface mutants N-terminally fused to pmApple (magenta), fixed and immunostained for the cis-medial Golgi marker Giantin (cyan). Single confocal slices are shown. Scale bar, 10 µm. (d) Representative flow cytometry histograms showing expression levels of pmApple-fused CNIH1, CNIH2, CNIH3, or CNIH4 in DOR-RUSH CNIH1 KO cells. Expression levels are comparable across constructs.

Article Snippet: N-terminally tagged CNIH1 constructs, including CNIH1 wild-type, the DOR-interface mutant (D32A/E33A/Y36A), and the COPII-binding mutant (amino acids 106–112 replaced by alanine), were generated by cloning synthesized CNIH1 DNA fragments (Invitrogen GeneArt Synthesis) separated by a 10 amino-acid (2xGGGGS) flexible linker, with an N-terminal signal peptide and pmApple- or HA-tag, into pcDNA3.1.

Techniques: Flow Cytometry, Transfection, Expressing, Construct, Marker