A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

Abstract

An abnormal expansion of a GGGGCC (G₄C₂) hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we develop here a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G₄C₂ repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

Fig. 1. RfxCas13d can be programmed to target C9ORF72.

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.

Fig. 2. RfxCas13d can target the G₄C₂ repeat-containing RNA and reduce RNA foci in C9-BACexp mice.

a Cartoon of the injection scheme. Created in BioRender. Gaj, T. (2024) https://BioRender.com/a93i238. b The experimental plan to analyze C9ORF72 mRNA in EGFP-KASH⁺ nuclei isolated by fluorescence-activated cell sorting (FACS). Created in BioRender. Gaj, T. (2024) https://BioRender.com/w54s355. c Representative immunofluorescent staining of the hippocampus (HPC) and motor cortex (MC) in C9-BACexp mice two-months after injection with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-crRNA and PHP.eB-EGFP-KASH. Scale bar, 20 µm. Relative all-V and V3 mRNA in d the HPC and e MC of EGFP-KASH⁺ nuclei from C9-BACexp mice injected with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-crRNA and PHP.eB-EGFP-KASH. All-V and V3 for each crRNA normalized to the non-targeted (NTG) control (n ≥ 7). f Representative RNA FISH for the G₄C₂ repeat RNA (magenta) from the HPC of C9-BACexp mice injected with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-crRNA and PHP.eB-EGFP-KASH. Scale bar, 5 µm. Quantification of g the number of RNA foci per EGFP-KASH⁺ cell in the HPC and h the percentage of EGFP-KASH⁺ cells with 0, 1–2, 3–4, or >5 foci (n ≥ 4). g, h The total number of cells counted per biological replicate is described in Supplementary Table 1. RNA foci were counted by a blinded investigator. Values indicate means and error bars indicate SD. d, e All-V and V3 for each crRNA compared to NTG using a one-tailed unpaired t-test, with exact P values shown. g crRNAs 13 and 7 compared to NTG using a one-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.

Fig. 3. High-fidelity RfxCas13d has improved specificity and can mediate targeting in cells derived from an ALS patient.

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.

Fig. 4. High-fidelity RfxCas13d can target the G₄C₂ repeat-containing RNA and improve deficits in C9-BACexp mice.

a Cartoon of the injection scheme. Created in BioRender. Gaj, T. (2024) https://BioRender.com/a93i238. b Representative immunofluorescent staining of the hippocampus (HPC) and motor cortex (MC) in C9-BACexp mice two-months after injection with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-N2V8-crRNA and PHP.eB-EGFP-KASH. Scale bar, 30 µm. Relative all-V and V3 mRNA in EGFP-KASH⁺ nuclei from c the HPC and d MC of C9-BACexp mice injected with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-N2V8-crRNA with PHP.eB-EGFP-KASH. All-V and V3 for crRNA-13 normalized to the non-targeted (NTG) control (n ≥ 6). e Representative FISH for the G₄C₂ repeat RNA (magenta) in EGFP-KASH⁺ cells from the HPC of C9-BACexp mice injected with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-N2V8-crRNA and PHP.eB-EGFP-KASH. Scale bar, 5 µm. f Quantification of the number of RNA foci per EGFP-KASH⁺ cell in the (left) HPC and (right) MC of injected C9-BACexp mice (n ≥ 6). The total number of cells counted per biological replicate is described in Supplementary Table 3. g Soluble poly(GP) in the HPC of injected C9-BACexp mice (n ≥ 6). Volcano plot of the RNA-seq analysis from C9-BACexp mice injected with h PHP.eB-RfxCas13d-N2V8-NTG or i PHP.eB-RfxCas13d-N2V8-crRNA-13 and compared to wild-type littermates (n = 3–7). Lines denote a > 1.2-fold change (FC) and an FDR-adjusted P < 0.05. FC of the (j) down-regulated  or (k) up-regulated  DEGs from the analysis in (h, i). Values indicate means and error bars indicate SD. c, d All-V and V3 for crRNA-13 compared to NTG using a one-tailed unpaired t-test, with exact P values shown. f, g crRNA−13 compared to NTG using a one-tailed unpaired t-test, with exact P values shown. h, i FDR-adjusted P values were determined by a global FDR correction across pairwise comparisons. j, k crRNA-13 compared to NTG using a two-tailed unpaired t-test. All data points are biologically independent samples. Source data are provided in the Source Data file.