Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [CUT&Run]

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. iPSCs were maintained in StemCell mTeSR or 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. 2 weeks after gRNA virus transduction, cells were harvested with Accutase, and cell concentration and cell number were assessed with a Countess II cell counter (Invitrogen) and Trypan Blue (Invitrogen). Library preparation was completed using the CUT&RUN Library Prep Kit with primer set 1 (Epicypher 14-1001). Libraries were sequenced on an Illumina Nextseq 2000 with a coverage of at least 5 million reads per sample. Sequencing data were processed using CUT&RUNTools 2.0, and peaks for H3K4me3 signal were annotated using MACS2 narrow peak calling. Data were visualised in a genome viewer as cpm-normalized bigwig files. Genome: hg19 processed file: cpm-normalized bigwig