Fig. 1: Cellular efficacy assays of target transcript knockdown using ASOs or siRNAs.

A Structure of a typical human protein coding gene with 5’ and 3’ untranslated regions (UTRs), introns and exons. Splicing of the pre-mRNA (dashed lines) generates a mature mRNA. B Transcript knockdown by a gapmer ASO. The recognition of a target mature spliced mRNA (left) or pre-mRNA (right) occurs according to the rules of Watson-Crick hybridization. RNase H recognizes the DNA:RNA heteroduplex and cleaves the target RNA. In turn, the level of any encoded protein is reduced. C Transcript knockdown by an siRNA. A double-stranded siRNA is processed by the RNA-induced silencing complex (RISC), and the passenger (or sense) strand is released. The guide (antisense) strand guides RISC to the target RNA, which is cleaved by an Argonaute (Ago) protein. In turn, the levels of any encoded protein are reduced. D, E Knockdown of an endogenous transcript in patient cells or an immortalized cell line. D An ASO library is designed to span every nucleotide of splice variant 1 mature mRNA, although each ASO will also target the shared exons in splice variant 2. E To target splice variant 2 selectively, ASOs are designed against a single, unique exon. F–H Selective knockdown of a pathogenic variant allele. F ASOs spanning the mutated nucleotide position are screened in patient cells carrying the variant. G Mini-gene plasmids are introduced into immortalized cells; the co-expression of mutant and wild-type cDNAs that are differentially tagged allows for the relative quantification and thus determination of ASO allele selectivity. H In a reporter assay, the mutated target and a reporter (e.g., luciferase) gene are expressed in tandem. ASO activity on the target is quantified by monitoring the reporter’s activity (e.g., luciferase activity). A wild-type reporter construct can be assayed independently to generate comparative data on allele-selectivity. Some images created in BioRender; https://BioRender.com/lcouff4.