Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [bisulphite-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. 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. For bisulphite sequencing of iPSCs and neurons, genomic DNA was extracted from cultured cells with the Qiagen DNEasy Blood and Tissue kit (Qiagen, 69504) or the Zymo QuickDNA Miniprep kit (Zymo D3024). gDNA was stored at -80C. 250ng of gDNA were input into the bisulphite conversion reaction. For bisulphite conversion and purification, we used the Zymo EZ DNA Methylation Gold kit as instructed (Zymo, D5005). 2uL of bisulphite-converted gDNA was used as input for PCR as described above, with an annealing temperature of 57C. PCR1 reactions were then cleaned with Ampure XP beads (Beckman, A63881) at a ratio of 1.8x. 1/10th of PCR1 was used as input for PCR2 with Q5 polymerase. PCR2 added i5 and i7 barcodes and P5 and P7 overhangs for dual-index Illumina short read sequencing. PCR2 products were purified with Ampure XP beads. Products were visualized via electrophoresis on an Agilent TapeStation 4200 and quantified with Qubit dsDNA HS assay kit (Invitrogen, Q32851) on a Qubit fluorometer (Invitrogen). Libraries were pooled and sequenced on an Illumina Miseq instrument with a Miseq v3 600 cycle kit (Illumina, MS-102-3003) with read lengths of 250x250. Raw reads were trimmed using Trimmomatic to remove low quality reads and adapters with the following settings: HEADCROP: 25; ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10; TRAILING: 20; SLIDINGWINDOW:4:15; MINLEN:40 Bismark was used to create a bisulphite-converted reference genome from the hg19 genome build. Trimmed, paired-end reads were aligned to the genome (hg19) and analysed with Bismark version 0.22.3. The output coverage files were used to determine percentage of methylated cytosine for each CpG site in the amplicon. Bismark coverage (cov) files were retrieved, and cov files were filtered for CpG sites located on chr15 within the target amplicon (24 CpG sites total) and compiled into a filtered methylation table for all samples. Methylation coverage and counts at CpG sites within the target amplicon were then compared between treatments. Processed output files: methylation coverage tables for all samples in each group (combined Bismark .cov files)