Rohm Dahlia, Black Joshua B, McCutcheon Sean R, Barrera Alejandro, Berry Shanté S, Morone Daniel J, Nuttle Xander, de Esch Celine E, Tai Derek J C, Talkowski Michael E, Iglesias Nahid, Gersbach Charles A
Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Center for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USA.
Center for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USA; Department of Biostatistics and Bioinformatics, Duke University, Durham, NC 27708, USA.
Cell Genom. 2025 Feb 12;5(2):100770. doi: 10.1016/j.xgen.2025.100770.
Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi syndrome (PWS) results from loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control the expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.
利用CRISPR-dCas9等靶向DNA的技术进行表观基因组编辑，可用于剖析基因调控机制，并有可能治疗相关疾病。例如，普拉德-威利综合征（PWS）是由15号染色体q11.2-q13.3区域父本表达的印记基因缺失所致，尽管母本等位基因完好无损，但在表观遗传上处于沉默状态。通过在人类诱导多能干细胞（iPSC）中进行CRISPR抑制和激活筛选，我们鉴定出了控制父本和母本染色体上PWS基因SNRPN表达的基因组元件。我们发现，靶向转录激活或DNA去甲基化均可激活沉默的母本SNRPN及下游PWS转录本。然而，这两种方法在不同区域发挥作用，优先激活不同的转录变体，并涉及不同的表观遗传重编程机制。值得注意的是，靶向脱甲基酶的瞬时表达可导致PWS iPSC中母本SNRPN的稳定、长期表达。这项工作揭示了靶向表观遗传操作可对疾病相关的印记位点进行重编程，并提出了可能的治疗干预措施。
