Fig. 3: High-fidelity RfxCas13d has improved specificity and can mediate targeting in cells derived from an ALS patient.
From: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD
a RfxCas13d domain organization with RfxCas13d-N2V7 and RfxCas13d-N2V8 mutations shown. Relative all-V and V3 mRNA in b HEK293T and c SH-SY5Y cells transfected with RfxCas13d, RfxCas13d-N2V7 and RfxCas13d-N2V8 with crRNA-13. All values normalized to untreated cells (n = 3). d Volcano plot of the RNA-seq analysis comparing HEK293T cells transfected with (left) RfxCas13d or (right) RfxCas13d-N2V8 with crRNA-13 to each variant with a NTG crRNA (n = 3). Lines denote a > 1.25-fold change (FC) and an FDR-adjusted P < 0.01. e Number of differentially expressed genes (DEGs) [>1.25-FC, FDR-adjusted P < 0.01] from (d). f Gene ontology (GO) and biological process (BP) term analysis for the DEGs in (d). Line denotes FDR-adjusted P < 0.05. g Venn diagram of overlapping DEGs. h Immunostaining of C9-ALS neurospheres. Scale bar, 75 µm. i Brightfield and fluorescent images of C9-ALS neurospheres 14 days after treatment with PHP.eB-EGFP-KASH. h, i Immunostaining and visualization were conducted once. Relative j V3 to all-V mRNA ratio and k CBLN1 mRNA in C9-ALS or wild-type neurospheres treated with PHP.eB-RfxCas13d-N2V8-crRNA (n ≥ 3). j Values normalized to NTG crRNA. k Values normalized to NTG in C9-ALS cells. l Number of DEGs [>1.25-FC from wild-type cells, FDR-adjusted P < 0.01] in C9-ALS cells treated with PHP.eB-RfxCas13d-N2V8-crRNA. Values indicate means and error bars indicate SD. b, c All-V and V3 for each RfxCas13d variant compared to untreated cells using a two-tailed unpaired t-test, with exact P values shown. d–f FDR-adjusted P values were determined by a global FDR correction across pairwise comparisons. j, k crRNA-13 compared to NTG using a one-tailed unpaired t-test, with exact P values shown. k NTG for C9-ALS iMNs compared to NTG for WT iMNs using a one-tailed unpaired t-test, with the exact P value shown. l FDR-adjusted P values were determined by a global FDR correction across pairwise comparisons. All data points are biologically independent samples. Source data are provided in the Source Data file.
