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CRISPR–Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity

Abstract

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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Fig. 1: Genome-wide CRISPR–Cas9 KO screens in human cells identify modifiers of C9ORF72 DPR toxicity.
Fig. 2: Summary of modifiers identified from genome-wide KO screens in K562 cells.
Fig. 3: CRISPR–Cas9 KO screens in primary mouse neurons.
Fig. 4: Validation of Rab7 and Tmx2 as modifiers of PR toxicity in an independent neuronal culture system.
Fig. 5: Transcriptional analysis of PR-treated primary neurons and K562 cells.
Fig. 6: Decreased Tmx2 protects primary neurons against PR-mediated toxicity.
Fig. 7: Decreased TMX2 improves the survival of C9-ALS iPSC-derived induced motor neurons.

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References

  1. Taylor, J. P., Brown, R. H. Jr. & Cleveland, D. W. Decoding ALS: from genes to mechanism. Nature 539, 197–206 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Ng, A. S., Rademakers, R. & Miller, B. L. Frontotemporal dementia: a bridge between dementia and neuromuscular disease. Ann. NY Acad. Sci. 1338, 71–93 (2015).

    CAS  PubMed  Google Scholar 

  3. Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133 (2006).

    CAS  PubMed  Google Scholar 

  4. Ling, S. C., Polymenidou, M. & Cleveland, D. W. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79, 416–438 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Renton, A. E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Mori, K. et al. The C9orf72GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338 (2013).

    CAS  PubMed  Google Scholar 

  8. Ash, P. E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zu, T. et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl. Acad. Sci. USA 110, E4968–E4977 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Gao, F. B., Richter, J. D. & Cleveland, D. W. Rethinking unconventional translation in neurodegeneration. Cell 171, 994–1000 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345, 1192–1194 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Peters, O. M. et al. Human C9ORF72 hexanucleotide expansion reproduces RNA foci and dipeptide repeat proteins but not neurodegeneration in BAC transgenic mice. Neuron 88, 902–909 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Wen, X. et al. Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron 84, 1213–1225 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kwon, I. et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345, 1139–1145 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Jovičić, A. et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat. Neurosci. 18, 1226–1229 (2015).

    PubMed  PubMed Central  Google Scholar 

  16. Boeynaems, S. et al. Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD. Sci. Rep. 6, 20877 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Lee, K. H. et al. C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167, 774–788.e17 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lin, Y. et al. Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target LC domain polymers. Cell 167, 789–802.e12 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Boeynaems, S. et al. Phase separation of C9orf72 dipeptide repeats perturbs stress granule dynamics. Mol. Cell 65, 1044–1055.e5 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kanekura, K. et al. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum. Mol. Genet. 25, 1803–1813 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Lopez-Gonzalez, R. et al. Poly(GR) in C9ORF72-related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons. Neuron 92, 383–391 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Shi, K. Y. et al. Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export. Proc. Natl. Acad. Sci. USA 114, E1111–E1117 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Gitler, A. D. & Lehmann, R. Modeling human disease. Science 337, 269 (2012).

    PubMed  Google Scholar 

  24. Gitler, A. D. et al. α-Synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity. Nat. Genet. 41, 308–315 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Yeger-Lotem, E. et al. Bridging high-throughput genetic and transcriptional data reveals cellular responses to α-synuclein toxicity. Nat. Genet. 41, 316–323 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Elden, A. C. et al. Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature 466, 1069–1075 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Treusch, S. et al. Functional links between Aβ toxicity, endocytic trafficking, and Alzheimer’s disease risk factors in yeast. Science 334, 1241–1245 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Sun, Z. et al. Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS. PLoS Biol. 9, e1000614 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Ju, S. et al. A yeast model of FUS/TLS-dependent cytotoxicity. PLoS Biol. 9, e1001052 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Armakola, M. et al. Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models. Nat. Genet. 44, 1302–1309 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Kim, H. J. et al. Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models. Nat. Genet. 46, 152–160 (2014).

    CAS  PubMed  Google Scholar 

  32. Liachko, N. F. et al. The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43. PLoS. Genet. 10, e1004803 (2014).

    PubMed  PubMed Central  Google Scholar 

  33. Jablonski, A. M. et al. Loss of RAD-23 protects against models of motor neuron disease by enhancing mutant protein clearance. J. Neurosci. 35, 14286–14306 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Berson, A. et al. TDP-43 promotes neurodegeneration by impairing chromatin remodeling. Curr. Biol. 27, 3579–3590.e6 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Couthouis, J. et al. A yeast functional screen predicts new candidate ALS disease genes. Proc. Natl. Acad. Sci. USA 108, 20881–20890 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Kim, H. J. et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 495, 467–473 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Becker, L. A. et al. Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice. Nature 544, 367–371 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).

    CAS  PubMed  Google Scholar 

  39. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    CAS  PubMed  Google Scholar 

  40. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    CAS  PubMed  Google Scholar 

  41. Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera, MdelC. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).

    CAS  PubMed  Google Scholar 

  42. Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Deans, R. M. et al. Parallel shRNA and CRISPR–Cas9 screens enable antiviral drug target identification. Nat. Chem. Biol. 12, 361–366 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Morgens, D. W., Deans, R. M., Li, A. & Bassik, M. C. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat. Biotechnol. 34, 634–636 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Zhang, K. et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56–61 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Freibaum, B. D. et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525, 129–133 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Prudencio, M. et al. Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS. Nat. Neurosci. 18, 1175–1182 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Christianson, J. C. et al. Defining human ERAD networks through an integrative mapping strategy. Nat. Cell. Biol. 14, 93–105 (2011).

    PubMed  PubMed Central  Google Scholar 

  50. Jonikas, M. C. et al. Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323, 1693–1697 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Wideman, J. G. The ubiquitous and ancient ER membrane protein complex (EMC): tether or not? F1000Res. 4, 624 (2015).

    PubMed  PubMed Central  Google Scholar 

  52. Shevtsova, Z., Malik, J. M., Michel, U., Bähr, M. & Kügler, S. Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp. Physiol. 90, 53–59 (2005).

    CAS  PubMed  Google Scholar 

  53. Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Westergard, T. et al. Cell-to-cell transmission of dipeptide repeat proteins linked to C9orf72-ALS/FTD. Cell Rep. 17, 645–652 (2016).

    CAS  PubMed  Google Scholar 

  55. Zhou, Q. et al. Antibodies inhibit transmission and aggregation of C9orf72 poly-GA dipeptide repeat proteins. EMBO Mol. Med. 9, 687–702 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Huang, L. et al. Missense mutations in ITPR1 cause autosomal dominant congenital nonprogressive spinocerebellar ataxia. Orphanet. J. Rare. Dis. 7, 67 (2012).

    PubMed  PubMed Central  Google Scholar 

  57. Ron, D. & Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell. Biol. 8, 519–529 (2007).

    CAS  PubMed  Google Scholar 

  58. Sidrauski, C. et al. Pharmacological brake-release of mRNA translation enhances cognitive memory. eLife 2, e00498 (2013).

    PubMed  PubMed Central  Google Scholar 

  59. Sidrauski, C., McGeachy, A. M., Ingolia, N. T. & Walter, P. The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly. eLife 4, e05033 (2015).

    PubMed Central  Google Scholar 

  60. Zhang, Y. J. et al. Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol. 128, 505–524 (2014).

    Google Scholar 

  61. Dafinca, R. et al. C9orf72 hexanucleotide expansions are associated with altered endoplasmic reticulum calcium homeostasis and stress granule formation in induced pluripotent stem cell-derived neurons from patients with amyotrophic lateral sclerosis and frontotemporal dementia. Stem Cells 34, 2063–2078 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Lynes, E. M. et al. Palmitoylated TMX and calnexin target to the mitochondria-associated membrane. EMBO. J. 31, 457–470 (2012).

    CAS  PubMed  Google Scholar 

  63. Ellgaard, L. & Ruddock, L. W. The human protein disulphide isomerase family: substrate interactions and functional properties. EMBO. Rep. 6, 28–32 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Seijffers, R. et al. ATF3 expression improves motor function in the ALS mouse model by promoting motor neuron survival and retaining muscle innervation. Proc. Natl. Acad. Sci. USA 111, 1622–1627 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Zhang, S. J. et al. A signaling cascade of nuclear calcium-CREB-ATF3 activated by synaptic NMDA receptors defines a gene repression module that protects against extrasynaptic NMDA receptor-induced neuronal cell death and ischemic brain damage. J. Neurosci. 31, 4978–4990 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Son, E. Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Davidson, Y. S. et al. Brain distribution of dipeptide repeat proteins in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathol. Commun. 2, 70 (2014).

    PubMed  PubMed Central  Google Scholar 

  68. Mackenzie, I. R., Frick, P. & Neumann, M. The neuropathology associated with repeat expansions in the C9ORF72 gene. Acta. Neuropathol. 127, 347–357 (2014).

    CAS  PubMed  Google Scholar 

  69. Schludi, M. H. et al. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing. Acta. Neuropathol. 130, 537–555 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Mackenzie, I. R. et al. Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers. Acta. Neuropathol. 130, 845–861 (2015).

    CAS  PubMed  Google Scholar 

  71. Gendron, T. F. et al. Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta. Neuropathol. 130, 559–573 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Gitler, A. D. & Tsuiji, H. There has been an awakening: emerging mechanisms of C9orf72 mutations in FTD/ALS. Brain. Res. 1647, 19–29 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Cheng, W. et al. C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2α phosphorylation. Nat. Commun. 9, 51 (2018).

    PubMed  PubMed Central  Google Scholar 

  74. Green, K. M. et al. RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response. Nat. Commun. 8, 2005 (2017).

    PubMed  PubMed Central  Google Scholar 

  75. Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell. Biol. 17, 5–15 (2016).

    CAS  PubMed  Google Scholar 

  76. Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5, e19760 (2016).

    PubMed  PubMed Central  Google Scholar 

  77. Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Joung, J. et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature 548, 343–346 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

    CAS  PubMed  Google Scholar 

  80. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome. Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

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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.

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

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

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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).

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.

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.

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.

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).

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).

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).

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).

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.

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Supplementary Text and Figures

Supplementary Figures 1–9

Life Sciences Reporting Summary

Supplementary Data 1

Uncropped Western Blots

Supplementary Table 1

Genome-wide K562 DPR CRISPR Screens

Supplementary Table 2

Primary Neuron Targeted DPR Screen

Supplementary Table 3

RNA-seq DiffExp – All Datasets

Supplementary Table 4

iPSC motor neuron differentiation information

Supplementary Table 5

Primer Sequences

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Kramer, N.J., Haney, M.S., Morgens, D.W. et al. CRISPR–Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nat Genet 50, 603–612 (2018). https://doi.org/10.1038/s41588-018-0070-7

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