Discovery of target genes and pathways of blood trait loci using pooled CRISPR screens and single cell RNA sequencing

Abstract

The majority of variants associated with complex traits and common diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown regulatory effects in cis and trans. By leveraging biobank-scale GWAS data, massively parallel CRISPR screens and single cell transcriptome sequencing, we discovered target genes of noncoding variants for blood trait loci. The closest gene was often the target gene, but this was not always the case. We also identified trans-effects networks of noncoding variants when cis target genes encoded transcription factors, such as GFI1B and NFE2. We observed that GFI1B trans-target genes were enriched for GFI1B binding sites and fine-mapped GWAS variants, and expressed in human bone marrow progenitor cells, suggesting that GFI1B acts as a master regulator of blood traits. This platform will enable massively parallel assays to catalog the target genes of human noncoding variants in both cis and trans.

Conditional resampling improves calibration and sensitivity in single-cell CRISPR screen analysis

“…Finally, we implement the algorithm such that it can run in parallel on hundreds or thousands of processors on a computer cluster. (We used this approach in our independent study of noncoding blood trait GWAS loci 14 .) Overall, we estimate that SCEPTRE can analyze 2.5 million gene-gRNA pairs on a dataset of 200,000 cells in a single day using 500 processors.…”

“…As an example application of SCEPTRE, we highlight STING-seq , a platform that we developed in parallel to the current work in an independent study 14 . STING-seq leverages biobank-scale GWAS data and single-cell CRISPR screens to map noncoding, disease-associated variants at scale.…”

“…SCEPTRE demonstrated excellent calibration and sensitivity on the data and revealed hundreds of new regulatory relationships, validated using a variety of orthogonal functional assays. In the Discussion we describe an independent work conducted in parallel to the current study in which we leveraged biobank-scale GWAS data, single-cell CRISPR screens, and SCEPTRE to dissect the cis and trans effects of noncoding variants associated with blood diseases 14 . This work highlights what we see as a primary application of SCEPTRE: dis-secting regulatory mechanisms underlying GWAS associations.…”

A genome-wide CRISPR screen identifies regulators of beta cell function involved in type 2 diabetes risk

“…Perturbation studies can provide more compelling evidence of causation, but only if conducted in authentic models and using disease-relevant phenotypic readouts 6 . The strongest such evidence arises from disease-associated coding variants that provide a readout in free-living humans of the consequences of perturbations of gene and protein function, but the low frequency of most such variants limits this approach 2 .…”

“…The strongest such evidence arises from disease-associated coding variants that provide a readout in free-living humans of the consequences of perturbations of gene and protein function, but the low frequency of most such variants limits this approach 2 . The availability of human cellular models and CRISPR based technologies provides an increasingly attractive alternative approach for generating genome-wide profiles of the phenotypic consequences of gene perturbation and thereby inform understanding of disease biology 6 . Central to this aspiration is confidence in the disease relevance of a cell type.…”