Fig. 3

MPRA designs for studying gene regulation. MPRA modifies the design of canonical cis-regulatory MPRA (described in Fig. 2B) that contains a cis-regulatory element (CRE), a promoter, a reporter gene, and a unique barcode (BC). Elements of this construct can be replaced or rearranged to test different types of CREs. The red vertical line indicates where a variant can be located. A Promoter MPRA contains a promoter harboring a variant, a reporter gene (e.g., GFP), and a unique BC. Image adapted from [40]. B Enhancer MPRA contains a regulatory element harboring a variant, a (minimal) promoter, a reporter gene, and a unique BC. C Transcription factor binding MPRA (TransMPRA) can be broken down into two components: (1) a promoter with a guide RNA (gRNA) that targets a transcription factor (TF) of interest and (2) a promoter, a test enhancer sequence harboring a variant, and a unique BC. The gRNA brings catalytically dead Cas9 protein with an attached Krüppel-associated box (dCas9-KRAB) which silences the expression of the TF gene. If the silenced TF interacts with the test enhancer, the downstream barcode expression is decreased. Image adapted from preprint [41]. D Silencer MPRA (in a STARR-seq style) contains a (strong) promoter and a test silencer harboring a variant. The silencer sequence can prevent self-transcription by silencing the promoter. Image adapted from [42]. E Splicing MPRA has minigene constructs that are inserted between a split-GFP reporter (GFP-N terminus and GFP-C terminus) and a peptide 2A (P2A) upstream of an mCherry reporter. Variants can be located in the variable intron sections on either side of the exon or within the exon. Inclusion of the middle exon disrupts GFP fluorescence, and cells can be FACS sorted into bins based on GFP:mCherry ratios. The GFP with or without the exon are quantified for exon inclusion or skipping via DNA-seq of the plasmid in each sorted bin. Image adapted from [43]. F RNA modification MPRA contains a promoter, an arbitrary coding sequence (CDS), a putative pseudouridine (Ψ) sequence as 3′ untranslated region (UTR), and a unique barcode. Once the library is introduced, cells are treated with N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide (CMC) which binds to Ψ and prevents reverse transcription (RT). High-throughput sequencing of cDNA allows prediction of the exact base pair location of the Ψ RNA modification. Variants can be inserted anywhere in the CDS. Image adapted from [44]. G 3′ UTR MPRA consists of a promoter, a reporter gene, a 3′ UTR harboring a variant, and a BC. BC RNA counts reflect transcriptional stability modulated by 3′ UTRs. H RNA localization MPRA consists of a promoter, a mutated Sox2 gene that localizes in the cytoplasm (fsSox2), a lncRNA harboring a variant, and a unique barcode. Barcode expression from subcellular fractions is used to interrogate subcellular localization of lncRNA. Image adapted from [45]