Fig. 6: Truncation of the top RBP hits identify splice-enhancing domains that can be repurposed for artificial splicing factors.

a–c, Domain structures of top hits used for truncation experiments; D-NTD and D-CTD represent N-terminal and C-terminal domains, respectively, containing MobiDB-lite consensus disorder prediction. All tested truncations are shown. Hits are separated into their position dependence from the initial screen: position-independent hits (a), hits that primarily activated the lucMAPT-30D reporter (b) and hits that primarily activated the lucMAPT-30U reporter (c). d–f, Bar graphs displaying reporter readout from both lucMAPT-30U and lucMAPT-30U of the full-length proteins next to their associated truncations (mean ± s.d., n = 3 replicate transfections). Graphs are separated by position dependence of full-length protein from the initial screen: position-independent hits (d), hits that primarily activated the lucMAPT-30D reporter (e) and hits that primarily activated the lucMAPT-30U reporter (f). g, Left, top, schematic of truncation–dCas13d fusion used as artificial splicing factors. Left, bottom, schematic of MS2-free lucMAPT reporter used for reporter-based assessment of artificial splicing factors. Right, bar graphs displaying reporter output from MS2-free lucMAPT reporter after co-transfection of reporter with truncation–dCas13d fusion and gRNA-containing plasmid (mean ± s.d., n = 3 replicate transfections). h, Left, top, schematic of truncation–dCas13d fusion used as artificial splicing factor. Left, bottom, schematic of HNRNPD exon 7 used for endogenous splicing modulation, with the position of the two sets of three gRNAs that are co-transfected with the artificial splicing factors as gRNA arrays. Middle, agarose gel showing splicing of HNRNPD exon 7 of a sample replicate for both artificial splicing factors in co-transfection with both gRNA arrays and a non-targeting gRNA (NT). Right, bar graphs displaying quantification of inclusion/exclusion ratio normalized to the non-targeting gRNA (NT) from gels in Extended Data Fig. 6c,d (mean ± s.d., n = 3 replicate transfections). bp, base pairs.