Functional interrogation of autoimmune disease genetics using CRISPR/Cas9 technologies and massively parallel reporter assays

Figures

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  Fig. 1. CRISPR/Cas9-based methods. When targeted to a region of interest (red box) by a single gRNA (blue ribbon), Cas9 (orange) predominately generates small deletions of a few base pairs (a); in coding regions, this is likely to knock out a gene. By fusing chromatin modifiers (red and green circles) to a catalytically inactivated Cas9 protein (dCas9), it is possible to activate or repress regions of interest (b), potentially switching on or off regulatory elements and downstream genes of interest (GOI). In much the same way as for MPRAs, it is possible to tile gRNAs and use lentiviral methods to scale up both of these approaches (c). Screens can be analysed using single-cell RNA sequencing (scRNA-seq), or more classically by measuring the abundance of gRNAs following selection (e.g. drug resistance or cell sorting based on a GOI)

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  Fig. 2. Experimental overview of massively parallel reporter assays (MPRAs). MPRAs can be targeted to GWAS loci (a) comprised of an index variant (red) and all credible co-inherited variants (grey). Oligos are synthesised across the entire region of interest (b) and cloned into reporter plasmids containing a minimal promoter, reporter gene and unique barcode (c). Plasmids are delivered to a disease-relevant cell type (d) and the effect of the synthesised oligos on reporter gene transcription determined. Generally reporter transcripts, quantified by RNA sequencing, are assigned to individual barcodes/oligos and the prevalence of these barcodes/oligos in a DNA-based library is used for normalisation (e). In addition to identifying potential enhancer elements (a–e), MPRAs can be designed to include oligos with both risk and protective alleles for variants of interest, thereby characterising allele-specific enhancer activity (f)