Journal: medRxiv
Article Title: Gene-Gene Interactions Between A LMNA Variant and Common Polymorphisms Drive Early-Onset Atrial Fibrillation
doi: 10.1101/2025.05.05.25326834
Figure Lengend Snippet: a. Schematic showing the CRISPR activation design. A closed chromatin conformation at the SCN5A-SCN10A locus inhibits transcription of SCN5A. RNA-guided binding of dCas9-VPR at the SCN5A promoter and dCas9-p300/CBP histone acetyltransferase at the SCN10A enhancer region initiates transcription of SCN5A. b. Representative INa traces show restoration of sodium current after CRISPR activation. c. Current-voltage (I/V) curve showing reduced INa in LMNA-S143P iPSC-aCMs, rescued by transfection of CRISPR activation complexes. d. Current-voltage (I/V) curve quantified in a bar graph. (n = 4-6 cells per group; unpaired t-test with Welch’s correction p<0.05). e. CRISPR activation restores expression of SCN5A ; bar graph quantifying changes in expression by qPCR. n=4 replicates representative of three independent differentiations, ordinary one way ANOVA with multiple comparisons, p<0.05)
Article Snippet: Tracr RNAs targeting the SCN5A promoter and SCN10A intronic enhancer were cloned into backbones with sgRNA scaffold (addgene#47108). iPSC-aCMs were co-transfected with individual plasmids with sgRNAs, dCas9-VP64-p65-Rta (addgene#63798) or dCas9 CBP/ p300(addgene # 61357) and a plasmid with a GFP expression cassette (addgene#40973). dCas9-VP64-p65-Rta was used for activation of SCN5A promoter while dCas9 CBP/ p300 was used for SCN10A enhancer activation.
Techniques: CRISPR, Activation Assay, Binding Assay, Transfection, Expressing
Addgene inc is a verified supplier 
