Using Single Cell, Noncoding CRISPR/Cas9-Based Regulatory Element Screening to Dissect Regulatory Mechanisms in Complex Genetic Loci

The Major Histocompatibility (MHC) Locus is the most SNP-dense region in the human genome and has been linked with >100 polygenic disorders. However, it remains unknown which, if any, of those variants disrupt regulatory interactions and drive specific phenotypes. To dissect regulatory mechanisms within this locus, we coupled CRISPR/Cas9-based regulatory element screening with single cell RNA-sequencing to identify putative regulatory element (pRE)-gene pairs in three diverse cell types: human induced pluripotent stem cells (hiPSCs), hiPSC-derived neural progenitor cells (hNPCs), and the immortalized myelogenous leukemia K562 cell line.

We designed a library (n=12,723 sgRNAs) targeting a union set of DNase hypersensitivity sites (DHSs) and previously identified POU5F1 enhancers (n = 581 elements) with 150 TSS-targeting controls and 1,273 nontargeting controls. We transduced hiPSCs, hNPCs, and K562s, expressing dCas9-KRAB, and profiled 250,000, 340,000, and 120,000 single cell transcriptomes, respectively, seven to nine days post-transduction. We then performed differential expression testing to identify sgRNA-gene pairs. Using a threshold based on significance and effect size, we recover >170 DHS-gene pairs in each cell type and find the same pREs can regulate different genes in different cell types, and certain genes are only regulated by the targeted elements in one cell type. In all cell types, we show that pREs regulate target genes nearby (<10kb) and further away (>0.5 Mb) and are located in promoters, introns, 3’ UTRs, and distal intergenic regions. We show that distal and promoter pRE regions have distinct characteristics. In hiPSCs and hNPCs, promoter pREs are enriched for active and repressive histone marks, suggesting that poised promoters may have additional regulatory function. We also demonstrate that targeted enrichment of the gene expression libraries enables detection of regulatory elements for lowly expressed genes.