Fig. 1: RfxCas13d can be programmed to target C9ORF72.
From: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD
a Schematic of (top) the C9ORF72 gene and (bottom) the three main transcript variants expressed from it. V1 produces the short protein isoform (C9-S), while V2 and V3 produce the long protein isoform (C9-L). The inset shows the locations of the crRNA binding sites for RfxCas13d in exon 1a and intron 1a of the C9ORF72 transcript. b, c Schematic of the dual-reporter system used to evaluate crRNAs. The platform consists of a b Renilla luciferase-encoding plasmid, pSV40-RLuc, whose 3’ untranslated region (UTR) carries a fragment of C9ORF72 with 20 copies of the G4C2 repeat and 250- and 98-base pairs (bps) of the flanking upstream and downstream sequences, respectively, and c a firefly luciferase-encoding plasmid, pHSV-TK-FLuc, that was used as a proxy for collateral cleavage. d Relative Renilla and firefly luciferase luminescence in HEK293T cells co-transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d and one of the 15 candidate crRNAs. All values normalized to cells transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d with a non-targeted (NTG) crRNA (n = 3). Relative all-V and V3 mRNA in e HEK293T and f SH-SY5Y cells transfected with RfxCas13d and crRNAs 13, 7, and 1 or a NTG crRNA or one of two ASOs (n = 3). All-V and V3 for each crRNA and ASO normalized to untreated cells. Values indicate means and error bars indicate SD. d Renilla and firefly luciferase luminesence for each crRNA compared to NTG using a one-tailed unpaired t-test, with exact P values shown. e, f All-V and V3 for each cRNA and ASO compared to NTG using a two-tailed unpaired t-test, with exact P values shown. All data points are biologically independent samples. Source data are provided in the Source Data file.
