Fig. 4: Optimization of Cas13d for isoform-specific perturbation of MAPT.
From: Non-invasive and high-throughput interrogation of exon-specific isoform expression
a, Binding sites of RfxCas13d–NLS/NES with a standard (22 nucleotides; 1022) and an extended (30 nucleotides; 10) spacer targeting MAPT exon 10. 30 nucleotides was used as the default length for all of the subsequent experiments such that we omit the subscript in the notation. b, Optimization of spacer length for RfxCas13d measured through the depletion of induced tau, tracked using bioluminescence from NLuc (4R-tau) and FLuc (pan-tau) 72 h after transfection. RLU of the bioluminescence signals are given for FLuc and NLuc separately and as the NLuc/FLuc ratio calculated from each sample. Nuc, nuclear localization by NLS; Cyt, cytoplasmic localization by NES. c, Binding sites of the different RfxCas13d crRNAs on the mature MAPT 0N, 3R and 4R mRNAs. d, The normalized FLuc, NLuc and NLuc/FLuc values are shown after transfection of EXSISERSMAPT:10NLuc-11FLuc cells with different MAPT-targeting crRNAs to increase isoform specificity of RfxCas13d–NLS. For b and d, data are mean ± s.d., n = 3 biological replicates. Selected results of Bonferroni MCT after two-way ANOVA (b) and one-way ANOVA (d) analysis are shown; ***P < 0.001, ****P < 0.0001; NS, not significant (full statistical results are provided in Supplementary Table 1). Source data are available online.
