DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells transfected with a methylated construct containing the FXS-related mutation upstream to an EGFP reporter gene

The primary mechanism causing Fragile X syndrome (FXS) is the expansion and silencing of a repetitive CGG sequence in the 5’-UTR of the FMR1 gene. To identify novel epigenetic pathways involved in FXS pathogenesis, we have established a model system for FMR1 silencing using a construct containing the FXS-related FMR1 CGG expansion upstream to an EGFP reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells. Library cells were then sorted to GFP-positive and GFP-negative populations. The analysis of the changes in abundance of mutants between the GFP-positive and GFP-negative populations has yielded a list of candidate genes whose functional loss reversed the methylation-induced silencing of the expanded FMR1 5'-UTR sequence. For the genome-wide screen, we used a CRISPR/Cas9 based genome-wide loss-of-function library of haploid hESCs, which contains 178,896 different gRNA constructs, targeting 18,166 genes. Library cells were transfected with the in-vitro methylated pFMR1-CGG(240)-EGFP constuct. 48 hours following transfection with methylated pFMR1-(240)CGG-EGFP construct, library cells were harvested and sorted to GFP-positive and GFP-negative populations. The abundance of different gRNAs represented in both populations was assessed by the amplification of the sgRNA-containing genomic DNA segment and high throughput sequencing. Following the mapping of the reads to the sgRNA sequences, an enrichment score was assigned to each gene by calculating the log2 fold change of its sgRNA counts between GFP-positive populations (n=4) and the GFP-negative population (n=3) using the EdgeR software.