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

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 or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging Each screen was performed in triplicate with independent transductions. For each replicate, 24 x 106 SNRPN-2A-GFP VP64dCas9VP64 (maternal) or dCas9KRAB (paternal) iPSCs were dissociated using Accutase (Stemcell Tech, 7920) and transduced in suspension across five matrigel-coated 15-cm dishes in mTesR (Stemcell Tech 85850) supplemented with 10 µM Rock Inhibitor (Y-27632, Stemcell Tech, 72304). Cells were transduced at a MOI of 0.2 to obtain one gRNA per cell and ~430-fold coverage of the gRNA library. The medium was changed to fresh mTesR without Rock Inhibitor 18-20 h after transduction. Antibiotic selection was started 30 h after transduction by adding 1 µg/mL puromycin (Sigma, P8833) directly to the plates without changing the medium. The cells were fed daily and passaged as necessary maintaining library coverage until harvest. Cells were harvested for sorting 9 d after transduction of the gRNA library for all screens. Cells were washed once with 1x PBS, dissociated using Accutase, filtered through a 30 µm CellTrics filter (Sysmex, 04-004-2326) and resuspended in FACS Buffer (0.5% BSA (Sigma, A7906), 2 mM EDTA (Sigma, E7889) in PBS). Before sorting, an aliquot of 4.8 x 106 cells were taken to represent a bulk unsorted population. The highest and lowest 10% of cells were sorted based on GFP expression and 4.8 x 106 cells were sorted into each bin. Sorting was done with a SH800 FACS Cell Sorter (Sony Biotechnology). FASTQ files were aligned to custom indexes (generated from the bowtie2-build function) using Bowtie 2. Counts for each gRNA were extracted and used for further analysis. All enrichment analysis was done with R. Individual gRNA enrichment was determined using the DESeq2 package to compare between high and low conditions for each screen. Only forward read (read 1) of paired-end sequenced sequencing files was used for data processing and enrichment analysis. Differentially-enriched gRNAs were gRNAs were identified via DESeq2 using an adjusted p-value cutoff of 0.05. processed output file: DESeq2 results table and sequencing counts for each condition