Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [RNA-seq]

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) is caused by 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 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. Overall design: iPSCs were maintained in StemCell mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging. Stable polyclonal Tet1-dCas9 or VP64-dCas9-VP64 cell lines were established by transducing with lentivirus and selecting for transgene-expressing cassette with 1.5 ug/mL blasticidin for 5 days. Then, cells were transduced with gRNA lentivirus and selected for gRNA-expressing cassette with puromycin (1ug/mL) for 3 days. 14 days after gRNA virus transduction, cells were harvested with Accutase, pelleted, and total RNA was extracted using Norgen Total RNA Plus Micro kit. Total RNA was submitted to Genewiz (Azenta) for library prep and total RNA sequencing. RNA libraries were prepared by Genewiz/Azenta Life Sciences. Total RNA library was constructed with rRNA depletion method. Raw reads were trimmed using Trimmomatic toe remove adapters. Trimmed reads were aligned to hg19 genome using STAR aligner. Gene counts were obtained with featureCounts from the subread package (v1.4.6-p4) using the comprehensive gene annotation in Gencode v22. Differential expression analysis was determined with DESeq2 where gene counts are fitted into negative binomial generalized linear models (GLMs) and Wald statistics to determine significantly differentially expressed genes. Genome: hg19