dcas9 vp64  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Efficient activation of hundreds of LTR12C elements reveals cis -regulatory function determined by distinct epigenetic mechanisms

doi: 10.1093/nar/gkae498

Figure Lengend Snippet: The dCas9-SunTag-based CRISPRa systems induce LTR12C expression from proximal promoter regions. ( A ) Percentage of upregulated LTR families in human cell lines following 5-aza-CdR treatment. The four cell lines were treated with PBS (control) or 300 nM 5-aza-CdR for 24 h and harvested at 5 days after the treatment. The upregulated LTR copies [fold change (FC) >2 by comparison with PBS-treated human cell lines] are categorized into each LTR family. The top 10 most highly abundant LTR families are highlighted with distinct colors. ( B ) Schematics of dCas9 constructs and fusion proteins. The constructs contain dCas9, SunTags separated by 22-amino-acid linkers (Multi SunTag), 2A self-cleaving peptide (2A), single-chain variable fragment (ScFv), green fluorescence protein (GFP), VP64/p300 and gRNA. The fusion proteins with single gRNA are inferred to transactivate multiple copies of LTR12C. ( C ) A sequence alignment of LTR12C elements deposited in the RepeatMasker database. Each row represents one LTR12C element. Heatmap indicates percent identity (0 to 90%); blank indicates gaps. Arrowhead indicates gRNA positions. Promoter and enhancer region is defined as 400 bp upstream region from TSS. ( D ) Relative expression of LTR12C based on reverse transcription qPCR (RT-qPCR) after transfection of the dCas9-SunTag-VP64 construct with represented gRNAs in HEK293T cells. Error bars represent standard error of the mean (SEM) from three independent biological replicates.

Article Snippet: The VP64 sequence was amplified from pcDNA-dCas9-VP64 plasmid (Addgene #47107) by polymerase chain reaction (PCR).

Techniques: Expressing, Control, Comparison, Construct, Fluorescence, Sequencing, Reverse Transcription, Quantitative RT-PCR, Transfection

Journal: Nucleic Acids Research

Article Title: Efficient activation of hundreds of LTR12C elements reveals cis -regulatory function determined by distinct epigenetic mechanisms

doi: 10.1093/nar/gkae498

Figure Lengend Snippet: The transactivation capacity of the dCas9-SunTag-VP64 system for LTR12C is higher than that of the dCas9-SunTag-p300 system. ( A ) Relative RNA expression of LTR12C and selected coding genes for RHOXF2B , IL1RN and OCT4 following represented CRISPRa as determined by RT-qPCR in HEK293T cells. Primers of LTR12C were designed at multiple loci. Error bars represent SEM from three independent biological replicates. P -values were calculated using the two-tailed Student’s t -test: *** P < 0.001. ( B ) The volcano plots show expression changes of LTR copies (not only LTR12C) after transfection of represented constructs with LTR12C-targeting gRNA in HEK293T cells. The log 2 FC values and −log 10 ( P -value) were calculated by comparison with expression of LTR copies in cells transfected with constructs expressing nontargeting gRNA. Dashed lines are the threshold of the two-tailed Wilcoxon signed-rank test P -value <0.05 (horizontal) or FC > 2 (vertical). The red (upper-right area) and blue (upper-left area) points represent upregulated and downregulated LTR copies with statistical significance, respectively. The pie charts show percentage of the upregulated LTR copies in each LTR family. The top five most highly abundant LTR families are highlighted with distinct colors. ( C ) The distribution of expression level (RPKM) of LTR12C in the represented groups in HEK293T cells. ‘All LTR12C’ refers to LTR12C copies that were deposited in RepeatMasker. Each box represents the data between the 25th and 75th quartiles. The whiskers are drawn down to the 10th percentile and up to the 90th percentile. White bars indicate median values. Adjusted P -values were calculated using Mann–Whitney U test and Holm’s method: *** P < 0.001. ( D ) LTR12C copies overlapping among upregulated LTR12C by dCas9-SunTag-VP64, dCas9-SunTag-p300 and 5-aza-CdR in HEK293T cells.

Article Snippet: The VP64 sequence was amplified from pcDNA-dCas9-VP64 plasmid (Addgene #47107) by polymerase chain reaction (PCR).

Techniques: RNA Expression, Quantitative RT-PCR, Two Tailed Test, Expressing, Transfection, Construct, Comparison, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Efficient activation of hundreds of LTR12C elements reveals cis -regulatory function determined by distinct epigenetic mechanisms

doi: 10.1093/nar/gkae498

Figure Lengend Snippet: The off-target events by single gRNA-based dCas9-SunTag-VP64 were limited. ( A ) Percentage of dCas9-HA binding LTR families represented among the LTRs upregulated after dCas9-SunTag-VP64-based CRISPRa in HEK293T cells, as detected in Figure , left panel. ( B ) Representative genomic regions showing LTR12C-specific transactivation in HEK293T cells. The track views represent RNA expression based on RNA-seq (upper) and dCas9-HA binding sites based on ChIP-seq (lower). Green (bottom) bars indicate genomic position of retrotransposons with their strand orientation. Arrowhead (blue) on the retrotransposons indicates gRNA targeting sites with one mismatch. Arrowhead (green) on the track view indicates poly(A) signal site. ( C ) Representative genomic region comprising LTR12C and other retrotransposons that gained robust expression in HEK293T cells.

Article Snippet: The VP64 sequence was amplified from pcDNA-dCas9-VP64 plasmid (Addgene #47107) by polymerase chain reaction (PCR).

Techniques: Binding Assay, RNA Expression, RNA Sequencing, ChIP-sequencing, Expressing