Fig. 5: Comparison of nuclear Cas13d with cytosolic RNA-guided RNA-targeting systems and Cas13d-based splicing suppression and enhancement.
From: Non-invasive and high-throughput interrogation of exon-specific isoform expression
a, Binding sites of RfxCas13d–NLS (cyan), PspCas13b–NES (orange) and amiRNA (dark red) on the mature MAPT 4R mRNA. b, Isoform specificity of cytosolic targeting of mature mRNA by Cas13b compared to amiRNA. The normalized FLuc, NLuc and NLuc/FLuc values after transfection of EXSISERSMAPT:10NLuc-11FLuc cells with different RNA systems and positions (shown in a) are displayed. c, G-rich/SR-rich domains were fused to a nuclease-inactivated version of RfxCas13d–NLS (dRfxCas13d–NLS) to create RNA-guided splice suppressors (G-rich) or enhancers (SR-rich). d, The binding site of the splice donor (SD)-targeting crRNA; the position of the crRNA 10 is shown in a. e, Different dRfxCas13d–NLS fusions were tested for their ability to modulate MAPT exon 10 splicing. The normalized FLuc, NLuc and NLuc/FLuc values after transfection of EXSISERSMAPT:10NLuc-11FLuc cells with different RNA systems and positions (shown in a, c and d) are displayed. d, dRfxCas13d–NLS; SR-rich, serine/arginine-rich domain of SC35; G-rich, glycine-rich domain from HNRNPA1 isoform A1-B; d–SR, fusion of dRfxCas13d–NLS to the SR-rich domain; d–G, fusion of dRfxCas13d–NLS to the G-rich domain. For b and e, data are mean ± s.d., n = 3 biological replicates. Selected results of Bonferroni MCT after one-way ANOVA are shown; **P < 0.01, ***P < 0.001, ****P < 0.0001 (full statistical results are provided in Supplementary Table 1). Source data are available online.
