p426 snr52p grna  (Addgene inc)

Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Optogenetic manipulation of proximity between repetitive genomic loci. (A) Scheme of OptoLoop consisting of a fusion between dCas9 and the optogenetic protein CRY2. OptoLoop is targeted to specific genomic loci by introducing specific sgRNAs. CRY2–CRY2 interactions activated by blue light bridge targeted loci to form a chromatin loop. (B) Left panel, region of chromosome 19 showing sgIDR3 and sgTCF3 target sites, representative Hi-C contact map (data from ) and BACs used in DNA-FISH to label the IDR3 (magenta) and TCF3 loci (green). Right panel, mCherry channel images of U2OS dCas9–3XmCherry–CRY2 cells transfected with sgIDR3 and sgTCF3, kept in dark or illuminated with blue light for 3 h (1 s pulses every 10 s), and fixed. Scale bars: 5 µm. (C) Left panel, representative image of DNA-FISH for IDR3 and TCF3 with specific BAC FISH probes in U2OS cells. Right panel represents a single cell highlighted in left panel (yellow box); the expansion shows a single allele in this cell. Dashed line denotes the distance between the two FISH signals. Scale bars: 20 µm (left panel), 5 µm (right panel), 1 µm (expansion). (D) IDR3–TCF3 distances, calculated for U2OS dCas9–mCherry–CRY2 polyclonal cells transfected with indicated combinations of sgIDR3 and sgTCF3, kept under dark or illuminated for 3 h (1 s pulses every 10 s). Violin plot corresponds to a representative experiment, with black lines representing median distances. Bar plot represents means of two independent experiments. Each dot represents the median of typically 5000–10,000 alleles analyzed per experiment. (E) Fraction of alleles with IDR3-TCF3 distance <0.27 µm measured from DNA-FISH images for U2OS dCas9–mCherry–CRY2 polyclonal cells and three clones of U2OS dCas9–3XmCherry–CRY2 cells, transfected with indicated combinations of sgIDR3 and sgTCF3, and kept in dark or illuminated for 3 h (1 s pulses every 10 s). Each dot represents the fraction of typically 5000–10,000 alleles analyzed per experiment. Bars represent means of two or three independent experiments. (F) Measurement of cell-to-cell heterogeneity in loop formation. Bars with green shades: observed fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm obtained from a representative experiment shown in E with 2500–5000 cells analyzed per sample. Bars with magenta shades: expected fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm assuming that alleles from a same cell are independent between each other (Eqn 2). * P <0.05; *** P <0.001; ns, not significant [two-way ANOVAs followed by post-hoc Tukey tests (D,E); paired two-tailed t -test (E); chi-squared test (F)].

Article Snippet: Synthetic single-guide RNAs (sgRNAs) were custom-ordered from Synthego (Reedwood City, CA, USA) and are listed in . sgRNAs were synthesized with a 2′-O-methyl modification at the three first and last bases, and 3′ phosphorothioate bonds between the third- and second-last bases for increased stability.

Techniques: Hi-C, Transfection, Single Cell, Clone Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Functional effects of genome organization manipulation on gene regulation. (A) Scheme of ‘telomere position effect over long distances’ model. Gene repression by long-range looping with telomere depends on telomere length. (B) Region of chromosome 5 showing sgSubtel (sgSubtel1+sgSubtel2) and sgTERT (sgTERT1+sgTERT2) target sites, and BACs used in DNA-FISH to label the 5p subtelomeric region (Subtel, green) and the TERT locus (magenta). (C) Left panel, representative image of DNA-FISH with Subtel and TERT BAC probes in HeLa cells. Scale bar: 5 µm. Right panel, fraction of alleles with Subtel- TERT distance <0.27 µm measured from DNA-FISH images of HeLa cells stably expressing dCas9-3XGFP-CRY2, transfected with none or sgSubtel and sgTERT sgRNAs, and kept under dark or illuminated with blue light for 3 h (1 s pulses every 10 s). Each dot represents the fraction of typically 6000–20,000 alleles analyzed per experiment. Bars represent the means of two experiments. Values are represented as relative to control (no sgRNAs, no light). (D) Representative image of RNA/DNA-FISH with RNA probes against TERT pre-mRNA and Subtel and TERT BAC probes in HeLa. Expansion shows a single allele (highlighted with a yellow square). Scale bars: 5 µm (left image), 1 µm (expansion). (E) Number of active TERT transcription sites (TSs) per cell (left panel) and total TERT pre-mRNA intensity (right panel) measured from RNA/DNA-FISH images from HeLa cells stably expressing dCas9–3XGFP–CRY2, transfected with none or sgSubtel and sgTERT sgRNAs, and illuminated with blue light for 4 h (1 s pulses every 10 s). Cells were binned according to their fraction of alleles with Subtel- TERT distance <0.27 µm. Average measurements from the whole population of cells (i.e. not binned) are also shown. Each dot represents the mean of typically 150–1500 cells analyzed per bin, per experiment. Bars represent the means of two experiments. RNA total intensity was normalized to that of control (bin ‘0’, no sgRNAs). (F) Fraction of alleles with Subtel- TERT distance <0.27 µm for alleles classified as active or inactive regarding TERT transcription. Allele transcription status was determined according to the presence or absence of an RNA-FISH signal at a distance <2.5 µm. Data obtained from RNA/DNA-FISH images of HeLa cells stably expressing dCas9–3XGFP–CRY2, transfected with none or sgSubtel and sgTERT sgRNAs, and illuminated with blue light for 4 h (1 s pulses every 10 s). Each dot represents the mean of typically 400–2000 alleles analyzed per condition, per experiment. Bars represent the means of two independent experiments. (G) Fraction of alleles with Subtel– TERT distance <0.27 µm (left panel) and average number of active TERT transcription sites per cell (right panel) measured from a representative RNA/DNA-FISH experiment shown in (E). Cells were binned according to their mean GFP nuclear intensity. Average measurements from whole population of cells (i.e. not binned) are also shown. Each dot represents the fraction of 300–1800 alleles analyzed per bin (left panel) or the mean of 100–600 cells analyzed per bin (right panel). * P <0.05; ** P <0.01; *** P <0.001 [two-way ANOVAs followed by post-hoc Tukey tests (C,E,F and G, right panel); Marascuilo's procedure (G, left panel)].

Article Snippet: Synthetic single-guide RNAs (sgRNAs) were custom-ordered from Synthego (Reedwood City, CA, USA) and are listed in . sgRNAs were synthesized with a 2′-O-methyl modification at the three first and last bases, and 3′ phosphorothioate bonds between the third- and second-last bases for increased stability.

Techniques: Functional Assay, Stable Transfection, Expressing, Transfection, Control

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs dampens 2′3′-cGAMP-mediated STING signaling. (A) Graph represents mean (±SEM, n = 3 independent experiment) CXCL10 and CCL5 levels as measured in supernatant of T98G cells treated or not with 2 µM NU7441 for 1 h prior to transfection or not of 10 µg/ml 2′3′-cGAMP for 6 h. Statistical significance was calculated by two-tailed Student's t test. (B) T98G cells were pretreated with the NU7441 (2 µM), NU7026 (10 µM), and AZD7648 (5 µM) DNA-PKcs inhibitors for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (C) T98G cells were pretreated with the NU7441 (2 µM) for 1 h prior to transfection with dsDNA (2 µg) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (D) T98G cells were transfected for DNA-PKcs– or KU70-targeting siRNAs or a control nontargeting siRNA for 48 h prior to analysis of knockdown efficiency by WB using the indicated antibodies. Representative WB; n = 3 independent experiments. (E) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB; n = 3 independent experiments. (F) THP-1 CTRL, THP1 cGAS−/− , and THP1 STING−/− were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. WB analysis was performed using the indicated antibodies. Representative WB of three to five independent experiments. (G) Densitometric quantification of band intensities of the p-IRF3/IRF3 ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (H) Densitometric quantification of band intensities of the pSTING/STING ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (I) Graph represents mean (±SEM, n = 3 independent experiment) IFNβ levels as measured in supernatant of THP1 CTRL , THP1 cGAS−/− , and THP1 STING−/− pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB of three independent experiments. (K) T98G cells engineered to express control nontargeting or STING-targeting gRNAs were treated or not with 2 µM of NU7441 prior to transfection or not of 10 µg/ml 2′3′-cGAMP and gene expression analysis. Graphs present the mean (±SEM, n = 5 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (L) As in K, except that WB analysis was performed using the indicated antibodies. Representative WB of three independent experiments. (M) As in L, except that T98G cells expressing an IFNAR-targeting gRNA were used. Representative WB of three independent experiments. (N) THP-1 cells were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection with dsDNA (2 µg) for up to 24 h. Gene expression analysis was conducted at 3, 6, 16, and 24 h. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (O) Cell culture supernatants were collected at 24 h in experiment performed as in N, and cytokine/chemokine levels were analyzed using a proteome profiler array. Heatmap representation of relative spot intensities is shown (mean of three independent experiments). (P) STING-deficient THP-1 cells engineered to express human STING haplotypes (STING-H232, STING-AQ, and STING-HAQ) were pretreated or not with the NU7441 (2 µM) for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (Q) As in P, except that IFNβ and CXCL10 levels were quantified by ELISA in supernatants. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to . Source data are available for this figure: .

Article Snippet: To generate the T98G IFNAR−/− knockout and control cell lines, lentiviral particles were produced by co-transfection of 2 × 10 6 293T cells with 5 μg of LentiCRISPRv2GFP plasmid (#82416; Addgene) expressing the gRNA targeting the gene of interest or nontargeting control gRNA, 5 μg of psPAX2, and 1 μg of pMD2.G, using the standard calcium phosphate transfection protocol.

Techniques: Transfection, Two Tailed Test, Gene Expression, Control, Knockdown, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs decreases the ability of STING agonists to trigger an antiviral response. (A) T98G cells were treated with the 10 µM diABZI, 1 µM E7766, or 10 mg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP for 16 h prior to DAPI nuclear staining and image acquisition. Images are representative of three independent experiments. (B) As in A, except that cells were infected with the MPXV clade 2b strain S2626 for 48 h. Images are representative of three independent experiments. (C) T98G cells engineered to express control nontargeting or DNA-PKcs–targeting gRNA were treated with 10 µg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP at MOI 0.3 for 16 h, prior to DAPI nuclear staining and image acquisition. Graph shows the mean (±SEM, n = 3 independent experiments) percentage of infected (GFP + ) cells as measured by fluorescent microscopy. Statistical significance was assessed using two-tailed Student's t test. (D) Gating strategy for macrophages used in . (E) Histograms show the percentage of infected (GFP + ) cells as measured by flow cytometry, following treatment with STING agonists, in the presence or absence of NU7441, at two different MOIs. (F) Primary macrophages from healthy donors 1, 2, and 3 were pretreated with NU7441 prior to STING agonist treatment. Cells treated with a STING agonist, and they were set as 100% infection, and the effect of adding a NU7441 was assessed relative to this condition. Scale bars, 500 μm. **: P < 0.01; *: P < 0.05. Data are from at least three independent experiments. Related to .

Article Snippet: To generate the T98G IFNAR−/− knockout and control cell lines, lentiviral particles were produced by co-transfection of 2 × 10 6 293T cells with 5 μg of LentiCRISPRv2GFP plasmid (#82416; Addgene) expressing the gRNA targeting the gene of interest or nontargeting control gRNA, 5 μg of psPAX2, and 1 μg of pMD2.G, using the standard calcium phosphate transfection protocol.

Techniques: Infection, Staining, Control, Microscopy, Two Tailed Test, Flow Cytometry