Fig. 3: Simultaneous activation and repression of two independent exons by RBFOX1N-dCasRx-C.

a Schematic of the CASFx-1, various gRNA architectures, as well as the RG6 and SMN2 minigenes. SMN2-DN gRNAs is a pool of three gRNAs (SMN2-gRNA-1 through 3), each expressed by a separate plasmid, targeting the corresponding numbered locations on the SMN2 minigene. RG6-SA targets splice acceptor of RG6 cassette exon (CX). DR-SMN2-2-DR is SMN2 target gRNA 2 flanked by two direct repeats (DR). DR-RG6-SA-DR contains spacer against RG6-CX splice acceptor flanked by two DRs. SMN2-DN-RG6-SA is a polycistronic pre-gRNA with spacers targeting three DN sites on SMN2 downstream intron and RG6-CX splice acceptors intervened by DRs. b Upper panel shows inclusion/exclusion (inc/exc) ratio fold-changes assayed by qRT-PCR on SMN2 minigene transcripts in cells co-transfected with the two minigene plasmids, CASFx-1 (RBFOX1N-dCasRx-C) and the indicated gRNAs. SMN2 and RG6 splicing changes are represented by gray and slash pattern filled bars, respectively. Fold-changes are relative to cells transfected with GFP control (set to 1). Data are represented as mean ± SD (n = 3). Lower panel shows a gel image of semi-quantitative splicing RT-PCR of RG6 and SMN2 minigene transcripts in cells co-transfected with the two minigene plasmids, CASFx-1 and the indicated gRNAs. Uncropped gel images and qRT-PCR values are included in the Source Data file.