Screening for functional transcriptional and splicing regulatory variants with GenIE

Abstract

Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.

Figure 1.

GenIE overview. (A) Proportion of GWAS lead SNPs in different genomic regions; 69% fall in transcribed regions (introns/exons/splice sites/UTRs/ncRNAs). (B) Deletion profiles from Cas9 editing at MUL1 intronic SNP rs6700034 in hiPSCs, assayed in genomic DNA (gDNA, left) and complementary DNA (cDNA) generated from RNA (right). (top row) count of sequencing reads having a deletion at each nucleotide position relative to the cut site; (bottom row) profile of each unique Cas9-induced deletion. (C) Schematic of GenIE assay. Edited pools of cells contain a mixture of WT, edited point mutation (SNP) and a variety of deletion alleles (indel 1, 2, etc.), expression from each of which can be quantified by amplicon sequencing of cDNA and gDNA extracted from the same population of cells.

Figure 2.

GenIE identifies effects of intronic enhancer elements. (A) Violin plots of eQTLs for MUL1 and ABHD4 in hiPSCs. (B) (left) Genomic position of targeted SNPs within MUL1 and ABHD4 enhancer elements; (right) pie chart showing the corresponding editing rates. (C) Barplots of GenIE-measured expression of alternative alleles (HDR-introduced allele or deletion alleles) in hiPSCs, relative to WT allele. (D) CLU gene region, showing ATAC-seq profiles from hiPSCs, hiPSC-derived neurons, and primary microglia, with positions of 11 targeted SNPs indicated. (E) GenIE-measured expression of HDR-introduced alleles or deletions, relative to the WT allele in hiPSCs. Effect sizes (fold change) of significant results are shown above each bar. (F) GenIE expression for CLU SNPs 6–8, relative to WT, with editing in an hiPSC line homozygous for the opposite haplotype; for each SNP, the edit was in the opposite direction to the edit in panel (c). All error bars represent 95% confidence intervals.

Figure 3.

GenIE identifies splicing regulatory elements. (A) Violin plots of splicing QTLs for TAF1C (usage of junction 1, chr16:84184989–84186901) and SDF4 (junction 1, chr1:1228946–1231892) in hiPSCs. (B) (left) Genomic region showing differential splicing at TAF1C and SDF4 loci; (right) fraction of reads for HDR, deletion (NHEJ) and wild-type (WT) alleles for TAF1C and SDF4. (C) GenIE-measured expression of targeted SNP alleles. (D) Deletion profiles of the top 16 alleles by read count from GenIE targeting of rs1171830. Shown on the right is the expression of each allele relative to the WT (reference) SNP allele, coloured by whether the canonical splice site motif is retained (blue) or disrupted (red). (E) GenIE-measured expression for dense mutagenesis near the CCDC6 exon1-intron1 splice site. All error bars represent 95% confidence intervals. (F) (top) Sequence logo showing that GenIE recapitulates the consensus splice site motif, with letter size proportional to the inverse of the GenIE effect size when mutated to that nucleotide (relative to the WT/consensus, set to 1); (bottom) Scatter plot showing correlation between GenIE-measured effect size and SpliceAI score for donor splice site loss.