Journal: Cellular and Molecular Life Sciences
Article Title: Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium
doi: 10.1007/s00018-023-05101-2
Figure Lengend Snippet: DNA methylation analysis of promoters. a Schematic of methylation-specific PCR (MSP) assay. Genomic DNA is subjected to bisulfite conversion. Converted DNA is used as input for PCR with primers specific for either methylated or unmethylated cytosines for the same site, and subsequently assessed for the presence or absence of product indicating the methylation status of the target region. Locations of MSP primer target sites measured for the CAG promoter are indicated. b WTC11 hiPSC lines, with genes driven by CAG that had been targeted to either the AAVS1 , hROSA26 , or CLYBL genomic loci, were subjected to bisulfite conversion, followed by PCR using primers targeting methylated (M) or unmethylated (U) DNA. Representative gel images are shown for analysis of the hypermethylated region of the CAG promoter. c MSP analysis of differentially methylated region of CAG promoter, using primers targeting methylated DNA, in CLYBL -targeted cell lines. d Genomic DNA from hiPSCs and hiPSC-CMs (Day 14) in CLYBL -targeted cell lines were subjected to methylation sensitive HpaII digestion. Quantitative PCR measures fraction of methylated DNA in the intronic region of the promoter. n = 1–4 biological replicates. 2-way ANOVA, followed by post-hoc Tukey-Kramer test, (Primer Set 1: CLYBL -CAG-mCherry vs. CLYBL -CAG-CRISPRa-mCherry, p = 0.0413; cell state : hiPSC vs. hiPSC-CM, p = 0.304; cell line:cell state : p = 0.772; Primer Set 2: CLYBL -CAG-mCherry vs. CLYBL -CAG-CRISPRa-mCherry, p = 0.037; cell state : hiPSC vs. hiPSC-CM, p = 0.650; cell line:cell state : p = 0.996). e Bisulfite PCR was performed to amplify the CAG intronic sequence. Sanger sequencing was used to assess and quantify percent methylation at individual CG sites within amplicon, spanning 33 CpGs. Plot shows percent methylation of CpGs in sampled PCR products from CLYBL cell lines. x-axis indicates CpG position within the PCR amplicon. f hiPSCs were treated with indicated concentrations of 5-azacytidine (5aza) for 24 h and harvested for mRNA expression analysis of CAG or dCas9-VPR by quantitative rt-PCR. Expression is quantified with respect to HPRT. One-way ANOVA, followed by post-hoc Tukey-Kramer test, was performed to calculate statistical significance. n = 2–7 biological replicates . g Day 16 hiPSC-CMs were treated with 5µM 5aza for 96 h and harvested for CAG transcript expression analysis by quantitative rt-PCR. Expression is quantified with respect to HPRT. A t-test was performed to determine statistical significance. All error bars represent SEM
Article Snippet: To generate the donor CRISPR-mCherry plasmids driven by muscle regulatory cassettes, the promoters were assembled with dCas9-VPR-P2A (Addgene #99373) and mCherry (a gift from Jacob Corn, Addgene #102245) and cloned into the pAAV-Neo-CAG donor backbone, replacing the CAG fragment (pAAV-[CK8e or TNT455]-dCas9-VPR-P2A-mCherry).
Techniques: DNA Methylation Assay, Methylation, MSP Assay, Real-time Polymerase Chain Reaction, Sequencing, Amplification, Expressing, Quantitative RT-PCR
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