Browsing by Subject "Massively parallel reporter assay"

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    Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing
    (bioRxiv, 2024-12-04) Zheng, Yanjiang; Mayourian, Joshua; King, Justin S.; Li, Yifei; Bezzerides, Vassilios J.; Pu, William T.; VanDusen, Nathan J.; Pediatrics, School of Medicine
    The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/Cas9 gRNAs and donor templates via adeno-associated virus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.
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    Dynamic changes in P300 enhancers and enhancer-promoter contacts control mouse cardiomyocyte maturation
    (Elsevier, 2023) Zhou, Pingzhu; VanDusen, Nathan J.; Zhang, Yanchun; Cao, Yangpo; Sethi, Isha; Hu, Rong; Zhang, Shuo; Wang, Guangyu; Ye, Lincai; Mazumdar, Neil; Chen, Jian; Zhang, Xiaoran; Guo, Yuxuan; Li, Bin; Ma, Qing; Lee, Julianna Y.; Gu, Weiliang; Gupta, Weiliang; Yuan, Guo-Cheng; Ren, Bing; Chen, Kaifu; Pu, William T.; Pediatrics, School of Medicine
    Cardiomyocyte differentiation continues throughout murine gestation and into the postnatal period, driven by temporally regulated expression changes in the transcriptome. The mechanisms that regulate these developmental changes remain incompletely defined. Here, we used cardiomyocyte-specific ChIP-seq of the activate enhancer marker P300 to identify 54,920 cardiomyocyte enhancers at seven stages of murine heart development. These data were matched to cardiomyocyte gene expression profiles at the same stages and to Hi-C and H3K27ac HiChIP chromatin conformation data at fetal, neonatal, and adult stages. Regions with dynamic P300 occupancy exhibited developmentally regulated enhancer activity, as measured by massively parallel reporter assays in cardiomyocytes in vivo, and identified key transcription factor-binding motifs. These dynamic enhancers interacted with temporal changes of the 3D genome architecture to specify developmentally regulated cardiomyocyte gene expressions. Our work provides a 3D genome-mediated enhancer activity landscape of murine cardiomyocyte development.