Journal: Nucleic Acids Research

Article Title: Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system

doi: 10.1093/nar/gkz134

Figure Lengend Snippet: Combining CRISPR system with peroxidase APEX2 to identify local chromatin interactions. ( A ) Schematic of CAPLOCUS system. ( B ) The three components of the CAPLOCUS system: TO-NLS_dCas9_NLS, the RNA binding partner fused with peroxidase APEX2_NLS, and target-specific sgRNA.

Article Snippet: To create the MS2-APEX2_NLS fusion protein expression vector, APEX2 was amplified by polymerase chain reaction (PCR) from pcDNA3 Connexin43-GFP-APEX2 (Addgene plasmid: 49385) and cloned into the pHAGE-EFS-MCP-3XBFPnls vector (Addgene plasmid: 75384) with BamHI and XhoI.

Techniques: CRISPR, RNA Binding Assay

Journal: bioRxiv

Article Title: PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

doi: 10.1101/2021.10.03.462944

Figure Lengend Snippet: a , Volcano plot showing log2-fold change (PAX3-FOXO1-APEX2/Parental) vs. -log10 of the p-value generated from the mass spectrometry results of PAX3-FOXO1-APEX2-mediated biotinylation (n=3, one-tail unpaired T-test). Blue dots, enriched in parental samples; red dots, enriched in PAX3-FOXO1-APEX2 samples. b , Heatmaps of selected PAX3-FOXO1-APEX2 biotinylated proteins from the APEX2-mass spectrometry analysis. Total spectral count is shown within each box. c , Heatmaps of CUT&RUN analysis of representative factors from the analysis in b plotted around all PAX3-FOXO1 binding sites. d , Histograms of the CUT&RUN signal of the indicated factors around all PAX3-FOXO1 peaks and the down-regulated eRNA peaks. Graphs of PAX3-FOXO1, H3K27ac, and BRD4 from are shown for comparison.

Article Snippet: The APEX2 sequence (Addgene #97421) or a 3xFLAG tag was cloned into the HDR donor plasmids to create the FOXO1-APEX2 and FOXO1-3xFLAG plasmids respectively.

Techniques: Generated, Mass Spectrometry, Binding Assay, Comparison