Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR–Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR–Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR–Cas9 screens in defining mechanisms of neurodegenerative diseases.

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We thank E. Crane and B. Li for help with RNA-seq and helpful discussions; K. Han for helpful discussions and image analysis; and the Stanford Neuroscience Microscopy Service, supported by NIH NS069375. This work was supported by NIH grants R35NS097263 (A.D.G.), DP2HD084069 (M.C.B.), and R01NS097850 (J.K.I.), a National Science Foundation Graduate Research Fellowship (N.J.K.), a National Human Genome Research Institute Training Grant (M.S.H.), the Robert Packard Center for ALS Research at Johns Hopkins (A.D.G.), Target ALS (M.C.B. and A.D.G.), the Stanford Brain Rejuvenation Project of the Stanford Neurosciences Institute (M.C.B. and A.D.G.), the Muscular Dystrophy Association (J.K.I.), and Department of Defense grant W81XWH-15-1-0187 (J.K.I.). J.K.I. is supported as a New York Stem Cell Foundation–Robertson Investigator.

Author information

Author notes

    • Ana Jovičić

    Present address: Department of Molecular Biology, Genentech, South San Francisco, CA, USA

  1. These authors contributed equally: Nicholas J. Kramer and Michael S. Haney


  1. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    • Nicholas J. Kramer
    • , Michael S. Haney
    • , David W. Morgens
    • , Ana Jovičić
    • , Julien Couthouis
    • , Amy Li
    • , James Ousey
    • , Rosanna Ma
    • , Gregor Bieri
    • , C. Kimberly Tsui
    • , Michael C. Bassik
    •  & Aaron D. Gitler
  2. Neurosciences Graduate Program, Stanford University School of Medicine, Stanford, CA, USA

    • Nicholas J. Kramer
    •  & Gregor Bieri
  3. Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, CA, USA

    • Yingxiao Shi
    •  & Justin K. Ichida
  4. Department of Biology, Stanford University, Stanford, CA, USA

    • Nicholas T. Hertz
    •  & Marc Tessier-Lavigne


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N.J.K. and M.S.H. designed experiments, collected data, and wrote the manuscript. D.W.M. analyzed and performed statistics on screen data. A.J. helped design experiments for primary-neuron screens. A.L. and J.O. assisted with cloning. R.M. and G.B. contributed to primary-neuron experiments. J.C. contributed to RNA-sequencing analyses. C.K.T. contributed to the RAB7A-knockdown cell experiments. N.T.H. and M.T.-L. contributed to the dorsal root ganglia experiments. Y.S. and J.K.I. contributed to the ALS iMN experiments. M.C.B. and A.D.G. supervised the study and wrote the manuscript.

Competing interests

J.K.I. is a cofounder of Acurastem, Inc. All other authors declare no competing interests.

Corresponding authors

Correspondence to Michael C. Bassik or Aaron D. Gitler.

Integrated supplementary information

  1. Supplementary Figure 1 Localization of FLAG-tagged DPRs applied exogenously to K562 cells.

    (a) PR20 and (b) GR20 (8 µM added to culture media, overnight incubation) were observed to accumulate in nuclear puncta, as well as on the cell periphery, of K562 cells as visualized by immunocytochemistry (blue = DAPI, red = anti-FLAG).

  2. Supplementary Figure 2 Example genetic modifiers and specificity of the DPR screens compared with similar screens for ricin toxicity.

    (a) Distribution of sequencing from 10 KCNK5 sgRNAs (blue) compared to distribution of sequencing counts of all negative control sgRNA (grey) from PR20 screen replicate 1 (this is an example of a protective genetic modifier). (b) Distribution of sequencing from 10 RHOA sgRNAs (red) compared to distribution of sequencing counts of all negative control sgRNA (grey) from PR20 screen replicate 1 (this is an example of a sensitizing genetic modifier). Using CasTLE these sgRNA count distributions were used to generate effect scores, confidence scores and p-values for each gene (see Supplementary Table 1). (c) Volcano plot of effect vs. confidence scores for all human genes in the PR20 screen. Colored in blue are all the genes conferring resistance to PR20 when knocked out (10%FDR) and colored in red are all the genes conferring sensitivity to PR20 when knocked out (10% FDR). (d) Correlation of casTLE scores for all genes in the library in our screen for modifiers of PR20 toxicity compared to an independent screen for modifiers of another toxic protein, ricin performed in near identical conditions (same lab, same cell line, same sgRNA library). There was zero (R2 = 0) overall correlation between the hits for each screen suggesting a high degree of specificity in the modifiers identified in each screen. Note that TMX2 was only identified as a hit in the PR screen and not the ricin screen.

  3. Supplementary Figure 3 PR50 localization in primary mouse neurons and the effect of decreased Rab7 on PR20 localization.

    Lentiviral expression of GFP or PR50-FLAG using a synapsin promoter in primary mouse cortical neurons. Immunocytochemistry was used to visualize PR50 localization (blue = DAPI, red = anti-FLAG, green = anti-MAP2). (e) Immunocytochemistry to detect the subcellular localization of exogenously applied synthetic PR20-FLAG (green = anti-FLAG) in HeLa cells with reduced levels of Rab7 (Rab7-i.1) using CRISPR-i (blue = DAPI, magenta = LAMP1 (lysosomal marker). 1 µM PR20 was added to cell culture media for 1hr. before cells were fixed and subjected to immunocytochemistry.

  4. Supplementary Figure 4 Custom mouse sgRNA-library infection and gene knockdown by CRISPR in primary cortical neurons.

    (a) Visualization of the 3,000 sgRNA lentiviral library expressing mCherry in infected primary mouse neurons (grey = phase contrast, red = mCherry; live cells). (b, c) Validation of target protein reduction in Cas9+ primary neurons using sgRNAs targeting Xpo5 and Tmx2. Reduced abundance of target protein in primary neurons as measured by western blot was observed after more than 10 days of sgRNA expression (sgRNA transduction performed at DIV1). (d) Forest plots of all genes considered hits from each neuron screen with a non-zero effect estimate (95% C.I.) with estimated effect in center and error bars representing 95% credible interval of the effect estimate. Effect estimate is colored in blue if the gene was protective when knocked out and colored in red if it was sensitizing when knocked out.

  5. Supplementary Figure 5 Quality-control metrics of RNA-seq data from WT neurons expressing GFP and PR50.

    (a) Sample distance matrix using hierarchical clustering with Euclidian distance metric of total transcriptome of each sample. (b) Principal component analysis of total transcriptome of each sample. (c) Clustered heat map showing normalized expression levels of top 40 genes sorted by adjusted p-value. All analyses were performed using DEseq2 in R (Supplementary Table 3).

  6. Supplementary Figure 6 Quality-control metrics and summary of RNA-seq data from K562 cells and primary neurons treated with synthetic PR20.

    (a-c) K562 cells treated with 10 µM PR20 subjected to RNA-seq. (a) Sample distance matrix using hierarchical clustering with Euclidian distance metric of total transcriptome of each sample. (b) Principal component analysis of total transcriptome of each sample. (c) Clustered heat map showing normalized expression levels of top 40 genes sorted by adjusted p-value. All analyses were performed using DEseq2 in R (Supplementary Table 3). (d) Fold change of select ER-stress related, differentially expressed genes determined by DEseq2 (adjusted p-value < 0.001) from RNA-seq data from primary mouse neurons treated overnight with 1.5 µM PR20 (Supplementary Table 3).

  7. Supplementary Figure 7 Deceased TMX2 is protective against PR in K562 cells, and quality-control metrics of RNA-seq data from Tmx2-knockdown neurons expressing PR50 or GFP.

    (a) TMX2 protein reduction measured by immunoblot in K562 cells expressing Cas9 and sgRNAs targeting TMX2 or control sgRNAs (GAPDH levels were measured as loading control). (b) Enrichment of Tmx2 KO cells compared to WT in a K562 competitive growth assay after synthetic PR20 treatment. Cells measured 48 h after 10 µM PR20 treatment by flow cytometry. (c) Sample distance matrix using hierarchical clustering with Euclidian distance metric of total transcriptome of each sample. (d) Principal component analysis of total transcriptome of each sample. (e) Differential gene expression was determined using DEseq2, and the top differentially expressed genes as ranked by adjusted p-value were clustered using the R package pheatmap. Color indicates relative log2 fold change in normalized read counts from DEseq2. MA plot showing abundance of differentially expressed genes in red (adjusted p-value < 0.05) for pairwise comparisons of (f) control sgRNA neurons expressing PR50 vs GFP, (g) Tmx2 KO neurons expressing PR50 vs GFP, and (h) Tmx2 KO neurons expressing PR50 vs control sgRNA neurons expressing PR50 (Supplementary Table 3).

  8. Supplementary Figure 8 Survival of control iPSC-derived iMNs determined with the seven factor (7F) differentiation system.

    Quantification of the effect of TMX2 reduction by shRNA lentiviral transduction in two independent control iPSC lines, as performed in Fig. 7. Induced motor neurons were generated from iPSCs using a seven factor (7F) differentiation system. The survival of HB9-RFP+/shRNA-GFP+ iMNs was tracked by imaging after the addition of 10 µM glutamate to the cultures. All iMN survival experiments were analyzed by log-rank test, and statistical significance was calculated using the entire survival time course. *p < 0.05, ** p < 0.01 (survival assays were performed in triplicate with the indicated number of iMNs analyzed for each group, Supplementary Table 4).

  9. Supplementary Figure 9 iMN survival assays using an independent differentiation system (Dox-NIL).

    As an alternative to the 7F iMN differentiation procedure used in Fig. 7 and S8, iMNs were differentiated from C9-ALS and control iPSC lines using a Dox-NIL system. (a, b) Survival of Dox-NIL iMNs with or without TMX2 reduction by shRNA transduction. Results from two control (a) or two C9-ALS (b) lines were averaged to create the survival curves shown. (c, d) The same iMN data depicted in (b) but separated by individual C9-ALS cell line to show the variability in responses. (e) Representative images of GFP+ (shRNA expressing) C9-ALS iMNs taken during the survival experiments. (f) RNA was harvested from iMN survival experiments at the endpoint and TMX2 mRNA levels were measured by qRT-PCR (normalized to GAPDH levels). For information on the patient lines used and numbers of iMNs analyzed for survival analysis, see Supplementary Table 4.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–9

  2. Life Sciences Reporting Summary

  3. Supplementary Data 1

    Uncropped Western Blots

  4. Supplementary Table 1

    Genome-wide K562 DPR CRISPR Screens

  5. Supplementary Table 2

    Primary Neuron Targeted DPR Screen

  6. Supplementary Table 3

    RNA-seq DiffExp – All Datasets

  7. Supplementary Table 4

    iPSC motor neuron differentiation information

  8. Supplementary Table 5

    Primer Sequences

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