Journal: Nature Communications

Article Title: Specific multivalent molecules boost CRISPR-mediated transcriptional activation

doi: 10.1038/s41467-024-51694-y

Figure Lengend Snippet: a – d Relative mRNA expression of NTF3 , ASCL1 , MYOD1 , and IL1RN in HEK293T cells transfected with dCas9-VP64 or dCas9-VP64-FUS and a single guide RNA (gRNA) targeting each gene promoter as well as a scrambled gRNA (gScr) control. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. e , f Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells transfected with dCas9-VPR or dCas9-VPR-FUS and a gRNA targeting each gene promoter. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. g , h Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells transfected with dCpf1-VP64 or dCpf1-VP64-FUS and a gRNA targeting each gene promoter. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. Source data are provided as a Source data file.

Article Snippet: HEK293T cells transfected with Flag-tagged dCas9 activators along with gRNAs targeting NTF3 or IL1RN promoter were harvested and homogenized in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail (TargetMol, C0001) with 2 h of rotation.

Techniques: Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Specific multivalent molecules boost CRISPR-mediated transcriptional activation

doi: 10.1038/s41467-024-51694-y

Figure Lengend Snippet: a Boxplot illustrating the GFP intensity in HEK293R cells expressing different dCas9 activators together with gTetO. The results are presented as the median GFP intensity, along with the 25th and 75th quartiles, as well as the 5th and 95th percentiles, and are representative of three independent experiments. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Cell numbers from left to right ( n = 17198, 17379, 9902). b , c Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells expressing different dCas9 activators and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA versus the dCas9-VP64-FUS group. d , e Fluorescence recovery after photobleaching (FRAP) analysis of HEK293R cells expressing BFP-tagged dCas9-VP64-FUS or dCas9-VP64-TDP-43 with gTetO. Up, representative timelapse images after photobleaching. Yellow arrowheads indicate bleached condensates. Scale bar, 10 μm. Down: FRAP curves showing mean ± SD fluorescence recovery of condensates ( n = 5 puncta per group). f Boxplot illustrating the GFP intensity in HEK293R cells expressing different dCas9 activators together with gTetO. The results are presented as the median GFP intensity, along with the 25th and 75th quartiles, as well as the 5th and 95th percentiles, and are representative of three independent experiments. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Cell numbers from left to right ( n = 19,096, 16,734, 13,743, 14,080, 12,071, 17,691). g , h Relative mRNA expression of NTF3 and ASCL1 in HEK293T cells expressing different dCas9 activators and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA versus the dCas9-VP64-FUS group. i Co-immunoprecipitation (co-IP) of Flag-tagged dCas9 activators and BRG1, MED1 or RPB1. Three independent experiments were performed and similar results were obtained. j , k Enrichment of Flag-tagged dCas9 activators, BRG1, RPB1, and MED1 at the NTF3 or IL1RN promoter. Data are presented as mean values ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. Source data are provided as a Source data file.

Article Snippet: HEK293T cells transfected with Flag-tagged dCas9 activators along with gRNAs targeting NTF3 or IL1RN promoter were harvested and homogenized in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail (TargetMol, C0001) with 2 h of rotation.

Techniques: Expressing, Fluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Specific multivalent molecules boost CRISPR-mediated transcriptional activation

doi: 10.1038/s41467-024-51694-y

Figure Lengend Snippet: a , b Relative mRNA expression of ASCL1 and MYOD1 in HEK293T cells transfected with a single gRNA targeting the indicated genes along with the labeled dCas9-activators. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA versus the dCas9-VP64 or the dCas9-VP64-FUS group. c , d Relative mRNA expression of ASCL1 and MYOD1 in HEK293T cells transfected with dCas9-VP64-FUS-Shank3, dCas9-VP64-FUS-Shank3MA, or dCas9-VP64-FUS-Shank3 ME and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA test versus the dCas9-VP64-FUS-Shank3 group. e , f Relative mRNA expression of ASCL1 and MYOD1 in HEK293T cells transfected with dCas9-VP64-FUS-HOTag activators and a single gRNA targeting the indicated genes. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA test versus the dCas9-VP64-FUS group. g Relative mRNA expression of NTF3 , ASCL1 , MYOD1, and IL1RN in HEK293T cells transfected with a single gRNA targeting the indicated genes along with the dCas9-VP64-FUS-HOTag3 or dCas9-VP64-HOTag3-FUS. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical significance was determined by two-sided Welch’s t-test. h Boxplot showing relative GFP intensity in 1xTetO-GFP, 7xTetO-GFP or 14xTetO-GFP reporter cells expressing dCas9, dCas9-VP64, dCas9-VP64-FUS, dCas9-VP64-FUS-Shank3 or dCas9-VP64-FUS-HOTag3 together with gTetO. The results are presented as the relative median GFP intensity, along with the 25th and 75th quartiles, as well as the 5th and 95th percentiles, and are representative of three independent experiments. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Cell numbers from left to right ( n = 24,461, 17,298, 21,807, 17,611, 11,280, 22,233, 14,347, 16,136, 15,605, 13,372, 23,028, 11,938, 15,760, 15,641, 12,923). Source data are provided as a Source data file.

Article Snippet: HEK293T cells transfected with Flag-tagged dCas9 activators along with gRNAs targeting NTF3 or IL1RN promoter were harvested and homogenized in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail (TargetMol, C0001) with 2 h of rotation.

Techniques: Expressing, Transfection, Labeling