Journal: STAR Protocols

Article Title: Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells

doi: 10.1016/j.xpro.2024.103221

Figure Lengend Snippet:

Article Snippet: phH1 (Addgene #53186) , gRNA-FOXA2-Rank3-sense , 5′-TCCC A , GTGCCGAGCTGCCCCGAGG , .

Techniques: Virus, Recombinant, Knock-Out, Membrane, Gentle, Infection, Gel Extraction, Plasmid Preparation, TaqMan Copy Number Assay, Lysis, cDNA Synthesis, SYBR Green Assay, Software

Journal: STAR Protocols

Article Title: Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells

doi: 10.1016/j.xpro.2024.103221

Figure Lengend Snippet:

Article Snippet: phH1 (Addgene #53186) , gRNA-FOXA2-Rank3-sense , 5′-TCCC A , GTGCCGAGCTGCCCCGAGG , .

Techniques: Plasmid Preparation

Journal: STAR Protocols

Article Title: Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells

doi: 10.1016/j.xpro.2024.103221

Figure Lengend Snippet:

Article Snippet: phH1 (Addgene #53186) , gRNA-FOXA2-Rank3-sense , 5′-TCCC A , GTGCCGAGCTGCCCCGAGG , .

Techniques:

Journal: Cell reports

Article Title: Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

doi: 10.1016/j.celrep.2022.111817

Figure Lengend Snippet: (A) Pictorial representation of the γ-irradiated Mycobacterium tuberculosis (γ Mtb ) exposure assay. Partially sonicated clumps of γ Mtb are overlaid on THP-1 monolayers to model a primarily extracellular route of exposure that might be seen during longer courses of infection, such as in the granuloma. (B) qRT-PCR analysis of γ Mtb -exposed THP-1 macrophages at 8 h post exposure. VEGFA transcripts are upregulated in response to γ Mtb /DMSO-treated cells, but this upregulation is suppressed by treatment with 40 μM INCA-6, a chemical inhibitor that interferes with calcineurin binding to NFAT. Each data point represents the mean relative quantity from three technical replicates for each biological replicate within the single experiment; representative of three independent experimental replicates. Additional replicates are provided in and . Statistics from ANOVA with Tukey post-hoc honest significant differences test. (C) Supernatant ELISA of THP-1 macrophages at 24 h post exposure reveals potent induction of VEGFA only in the γ Mtb /DMSO-treated group. Representative of three independent experiments. Statistics from ANOVA with Tukey post-hoc honest significant differences test. (D) Immunofluorescence imaging reveals upregulation of VEGFA in γ Mtb -exposed THP-1 cells and substantial NFATC2 upregulation and nuclear translocation after treatment. Effects are both inhibited by treatment with INCA-6. DAPI channel labels nuclei; NFAT is detected by staining with anti-NFATC2 antibody; VEGFA detected by staining with anti-VEGFA antibody. Representative of three independent biological replicates. (E) Quantitation of the percentage of VEGFA + cells within entire images for (D). The first five images from each experimental group were selected and computationally blinded. Total cell number was counted in addition to the number of VEGFA + cells in each field. Each data point represents the percentage of VEGFA + cells in the field; 15 (5 of each of 3 total replicates) total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,264. Statistics from ANOVA with Tukey honest significant differences test. (F) Magnified immunofluorescence images demonstrating VEGFA inhibition by addition of INCA-6 with γ Mtb -exposed THP-1 cells. Note the white arrows showing nuclear localization of NFATC2 corresponding with VEGFA signal in the DMSO-treated condition while NFATC2 nuclear localization is disrupted in the INCA-6 condition and little VEGFA can be observed. (G) Quantitation of the percentage of cells with nuclear NFAT residence from the same images as (F). Each data point represents the percentage of NFAT nuclear-resident cells in the field; 15 total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,264. Statistics from ANOVA with Tukey honest significant differences test. (H) Quantitation from (F and G) of the percentage of total cells with nuclear NFAT residence and also expressing VEGFA out of the total number of cells. Each point represents one of 15 total fields counted for each group (5 from each of 3 independent experiments). Total number of cells counted = 5,264. Statistics from ANOVA with Tukey honest significant differences test.

Article Snippet: phH1 NFATC2 , This work, Addgene , Cat# 188711.

Techniques: Irradiation, Sonication, Infection, Quantitative RT-PCR, Binding Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Imaging, Translocation Assay, Staining, Quantitation Assay, Inhibition, Expressing

Journal: Cell reports

Article Title: Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

doi: 10.1016/j.celrep.2022.111817

Figure Lengend Snippet: (A) Immunofluorescence staining of THP-1 macrophages in the presence or absence of γ Mtb to identify potential NFAT isoforms of interest in human cells. NFATC2 is most robustly expressed and inducibly nuclear of these isoforms in macrophages. NFATC1 and NFATC3 are comparatively less expressed and less extensively translocated by 8 h post exposure. NFATC4 is very lowly expressed initially but, like all the isoforms, appears to be upregulated at the protein level after γ Mtb exposure. Initial staining from a single experiment, with NFATC2 validation from three biological replicates in (D–G). (B) Magnified images showing robust VEGFA expression in cells with nuclear NFATC2 compared with cells with nuclear NFATC1, NFATC3, and NFATC4, which lack the strong correspondence between NFAT nuclear localization and VEGFA induction, which is only seen with NFATC2 staining. (C) Blinded quantitation of the relationship between each NFAT isoform and the induction of VEGFA. We calculated the subset of cells expressing both VEGFA and demonstrating obvious NFAT nuclear localization and normalized to the total number of cells expressing VEGFA in that field. Initial staining from a single experiment, with validation in (D–G). Total number of cells counted = 5,859. Statistics from ANOVA with Tukey honest significant differences test. (D) THP-1 cells transduced with Cas9-expressing lentiviruses targeting either NFATC2 ( and ) or safe-targeting loci were selected with puromycin and then treated with γ Mtb or vehicle. Safe-targeting-transduced THP-1 cells robustly responded to γ Mtb with VEGFA production at 8 h post exposure, but fewer NFATC2-transduced cells produce VEGFA after stimulation and at lower staining intensity. Representative of three biological replicates. (E) Magnified images showing high VEGFA induction in γ Mtb -treated NFATC2-targeted THP-1 cells compared with γ Mtb -treated safe targeting (ST) THP-1 cells. The ST cells show robust VEGFA induction and NFAT nuclear translocation while NFATC2 cells show diminished VEGFA induction and disordered nuclear translocation and occasional lack of NFATC2 staining entirely; the antibody used to detect NFATC2 is N-terminal to the sgRNA sites, so residual expression is likely captured by the antibody. Representative of three biological replicates. (F) Blinded quantitation of the percentage of VEGFA + cells within entire images for (D and E). Each data point represents the percentage of VEGFA + cells in the field; 15 total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test. (G) Blinded quantitation of the percentage of VEGFA + and nuclear-localized NFAT out of the total number of cells with NFAT nuclear localization within entire images for (D) and (E). Fifteen total fields were counted for each groups (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test. (H) Blinded quantitation of the percentage of VEGFA + and nuclear-localized NFAT of the total number of cells in the images for (D) and (E). Fifteen total fields were counted for each group (5 from each of 3 independent experiments). Total number of cells counted = 5,029. Statistics from ANOVA with Tukey honest significant differences test.

Article Snippet: phH1 NFATC2 , This work, Addgene , Cat# 188711.

Techniques: Immunofluorescence, Staining, Biomarker Discovery, Expressing, Quantitation Assay, Transduction, Translocation Assay

Journal: Cell reports

Article Title: Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection

doi: 10.1016/j.celrep.2022.111817

Figure Lengend Snippet:

Article Snippet: phH1 NFATC2 , This work, Addgene , Cat# 188711.

Techniques: Control, Virus, Recombinant, Cell Culture, Adjuvant, Gas Phase Electrophoretic Molecular Mobility Analysis, Enzyme-linked Immunosorbent Assay, Derivative Assay, Software, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites

doi: 10.1016/j.omtn.2020.07.016

Figure Lengend Snippet: Parameters Used to Identify Novel HIV-1 LTR gRNAs (A) Local troughs of nucleotide diversity appeared in the core and enhancer regions in the HIV-1 LTR. The upper panel showed the 20-bp diversity plotted by leftmost position in any given 20-bp window. Details of the diversity calculation have been described in the method section. The middle panel showed the coordinates of the U3, R, and U5 regions of the LTR. The core and enhancer regions are at the 3′ end of U3 region (position 350–454). The sequence motifs that regulate HIV-1 transcription were shown on the lower panel. Shaded areas were the regions with locally low diversities. (B) Most potential gRNAs possessed a low number of sequences that were perfect 20-bp matches across LANL. The x axis represented the number of HIV-1 sequence variants that possessed identical 20-bp sequences to its paired gRNA along with 3-bp NGG PAM. y axis shows the number of gRNAs from the list of 75,885 possible gRNAs. High frequency 20-bp sequences next to NGG across LANL were more likely to make the gRNA to have broad coverage on HIV-1 variants. A list of 751 high frequency 20-bp sequences were selected as potential gRNA sequences for subsequent analysis. (C and D) A CFD score cutoff with high predicted accuracy was identified. (C) The receiver operating characteristic (ROC) curve using the CRISPR dataset described in results and two published scoring matrices for the prediction of CRISPR-mediated cleavage event by gRNA:target pair. CFD showed a better area under the curve (AUC) than MIT. (D) The precision rate of increasing cutoff of CFD score using CIRCLE-seq and CRISPOR datasets. A precision rate over 95% occurred at a CFD score of 0.569. The gRNA:target pair with a CFD score more than 0.569 may have more than a 95% chance to be cleaved by CRISPR-Cas9, whereas the pair with less than 0.569 would not be cleaved. (E) Scatter plot of HXB2- versus LANL-based gRNA CFD scores at same position. (F) Bar plot indicating the average CFD score for HXB2 and LANL-derived gRNAs. Error bars represent ± S.E.M. p value calculated by two-tailed t test. (G) Scatter plot of HXB2- versus LANL-based gRNA off-target hots from same position. (H) Bar plot indicating the average number of off target sites per HXB2 or LANL-derived gRNAs. Error bars represent ± SD. p value calculated by two-tailed t test.

Article Snippet: The gRNA expression vector phH1-gRNA (Addgene plasmid #53186) and Cas9 expression vector (catalog number # 41815) were obtained from Addgene. gRNA cloning was performed as previously described with some modifications.

Techniques: Sequencing, CRISPR, Derivative Assay, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites

doi: 10.1016/j.omtn.2020.07.016

Figure Lengend Snippet: The HIV-1 Reactivation Activity Was Significantly Reduced in the HIV-1 Latent Cell Line J-Lat 10.6 Treated with gNFKB1 J-Lat 10.6 cells were gated from a major cell population while excluding debris. Cells were then gated on live cells, then gated on RFP expression, and finally a histogram of GFP expression was used. Cells are J-Lat 10.6 cells without any treatment and the DMSO/PBS group are J-Lat 10.6 cells treated with DMSO or PBS. EV and NF-κB1 were transduced with LV encoding gRNA and Cas9 and later stimulated with PMA+Ionomycin or TNF-α. (A) Number of GFP + cells treated with gNFKB1 with PMA/I stimulation (50 ng/mL and 1 μM) after CRISPR treatment among all successfully transduced (RFP + ) cells. (B) Number of GFP + cells treated with gNFKB1 with TNF-α (20 ng/mL) stimulation after CRISPR treatment. The upper panel shows the raw data and distribution of fluorescein isothiocyanate A (FITC-A) intensity in treatments. The lower panel shows the relative percentage of GFP cells compared to the EV control (n = 3). p values were calculated using a one-tailed t test.

Article Snippet: The gRNA expression vector phH1-gRNA (Addgene plasmid #53186) and Cas9 expression vector (catalog number # 41815) were obtained from Addgene. gRNA cloning was performed as previously described with some modifications.

Techniques: Activity Assay, Expressing, Transduction, CRISPR, Control, One-tailed Test