CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9orf72 dipeptide repeat protein toxicity

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). 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 in 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 C9orf72 ALS patients. Together, this work demonstrates the promise of CRISPR-Cas9 screens to define mechanisms of neurodegenerative diseases.

genome using high complexity single-guide RNA (sgRNA) libraries (18)(19)(20)(21). Here, we used the CRISPR-Cas9 system to perform comprehensive genome-wide knockout screens in human cells and mouse primary neurons to identify genetic modifiers of C9orf72 DPR toxicity.
We engineered the human immortalized myelogenous leukemia cell line K562 to stably express Cas9 (22). Similar to previous reports in other human cell lines (9), synthetic polymers of PR and GR (PR 20 and GR 20 ) added to cell culture media were rapidly taken up by K562 cells, trafficked to the nucleus, and killed cells in a dose-dependent manner ( Fig. 1A-D, Fig. S1). To identify genetic modifiers of both PR 20 and GR 20 mediated-toxicity, we conducted genome-wide CRISPR knockout screens. We used a lentiviral sgRNA library comprised of ten sgRNAs per gene, targeting ~20,500 human genes, along with ~10,000 negative control sgRNAs (23) (Fig.   1E). We used deep sequencing to track the effect (protective, sensitizing, neutral) of each sgRNA in a pooled population of knockout cells. That is, sgRNAs that protected cells from DPR toxicity were enriched and those that sensitized cells were depleted from the pool. In order to identify both protective and sensitizing gene knockouts in the same screen, we treated the pooled population of cells with a dose of DPRs sufficient to kill 50% of cells and repeated this treatment four times over the course of the screen to amplify the selection. We performed separate screens for modifiers of PR 20 and GR 20 toxicity, each in duplicate, and repeated each screen two independent times.
We then used the casTLE algorithm as previously described (23) to detect statistically significant suppressors and enhancers of DPR toxicity. Using a false discovery rate (FDR) cutoff of 10%, the screens identified 215 genetic modifiers of PR 20 toxicity and 387 genetic modifiers of GR 20 toxicity (Fig. 1F, S2A-C, Supplemental Table 1). Results from the PR 20 and the GR 20 screen were well correlated (R 2 =0.61), suggesting similar mechanisms of toxicity (Fig.   1G). We validated individual knockout K562 lines by performing competitive growth assays with PR 20 treatment (Fig. 1H). Among the hits from these knockout screens were genes encoding some of the same nuclear import and export factors discovered in DPR modifier screens in model organisms (10,11,24,25), such as XPO6 and IPO11 (Fig. 1I), suggesting the same mechanisms are important in mammalian cells. In addition, we identified many novel genetic modifiers of PR and GR toxicity, including genes encoding RNA-binding proteins that have been shown to physically interact with PR and GR (12), such as NONO and HNRNPF. We also identified genes involved in RNA-splicing such as SPEN and PQBP1, a process previously observed to be dysregulated in C9FTD/ALS patient brains (26) and dramatically disrupted in human cell lines treated with DPRs (9). In addition to these previously implicated genes, some of the strongest hits in the screen were genes encoding ER-resident proteins, such as TMX2, CANX, almost all members of the endoplasmic reticulum membrane protein complex (EMC), a complex that is upregulated by ER stress and is thought to be involved in ER-associated degradation (ERAD) (27)(28)(29), and genes encoding proteasome subunits. Chromatin modifiers and transcriptional regulators, including genes involved in histone lysine methylation (KDM6A, KMT2D, KMT2A, and SETD1B) were also significant genetic modifiers of PR and GR toxicity.
To evaluate the role of the genetic modifiers identified in our human genome-wide screens in a more disease relevant context, we next designed a strategy to conduct pooled CRISPR-Cas9 screens in mouse primary neurons. Similar to human cell lines, synthetic PR 20 dipeptides were toxic to mouse primary neurons in a dose-dependent manner ( Fig. 2A).
However, to better model the endogenous production of DPRs, as in patient brains, we also utilized a lentiviral construct expressing codon optimized PR 50 driven by the neural-specific synapsin promoter (30). When expressed in primary neurons, PR 50 trafficked to the nucleus and elicited neurotoxicity in a time-dependent manner ( Fig. 2 B, C, S3). We constructed a custom lentiviral sgRNA library to test the top ~200 genes (5% FDR) found to be genetic modifiers of PR 20 toxicity in the genome-wide CRISPR screens. The custom library contained ten sgRNAs targeting each mouse gene and ~1,000 negative control sgRNAs. Using primary neuron cultures isolated from mice constitutively expressing Cas9 (31), we used immunoblotting to confirm efficient protein reduction of target genes after transduction with sgRNAs from this library following at least 10 days in culture ( Fig. S4 A-C).
We performed the primary neuron CRISPR screens two ways --either expressing PR 50 using lentivirus or treating with exogenous synthetic PR 20 . In this way, we could simultaneously validate hits from the genome-wide screens and disentangle the genes responsible for the uptake and spread of the DPRs throughout the cell from those responsible for mediating the toxic effects once inside the cell. We performed these targeted CRISPR KO screens such that there was a ~90% reduction in viability after one round of selection; only neurons with the strongest genetic modifiers would become enriched in the selection. We harvested the surviving neurons and assessed sgRNA enrichment compared to the untreated controls (Fig. 2C). We identified 13 modifier genes in the synthetic PR 20 screen and 17 genes in the endogenous PR 50 expression screen (determined by having a non-zero estimated effect with a 95% credible interval) (Fig. 2 Table 2). Strikingly, the genes that validated in this neuronal culture system showed strong specificity to the method of PR delivery to the neurons.

D-G, Supplementary
A top hit in the screen for modifiers of synthetic PR 20 induced toxicity was the endosomal trafficking gene Rab7a (Fig. 2D). We hypothesize that the endosomal trafficking pathway is necessary for the cellular uptake of the exogenously applied PR 20 . In contrast, none of the trafficking genes present in the mini-library were hits in the screen in which neuronal toxicity was induced by genetically encoded PR 50 expression. In this screen, the strongest genetic modifiers were genes that encode proteins predominantly localized to the nucleus and ER; a top protective knockout was a poorly characterized ER-resident thioredoxin protein Tmx2, which was also a protective genetic modifier in the primary neuron exogenous PR 20 screen (Fig. 2F).
The abundance of strong genetic modifiers involved in ER function and ER stress from our screens suggested that DPR accumulation might induce an ER stress response. To test this hypothesis, we performed RNA sequencing (RNA-seq) on primary neuron cultures transduced with a lentivirus expressing either PR 50 or GFP (Fig. 3A). 126 genes were significantly upregulated and 133 genes significantly downregulated following four days of PR 50 expression (adjusted p-value < 0.05; Fig. 3B, S5, Supplemental Table 3). The most significantly enriched Gene Ontology (GO) category among upregulated genes was 'apoptotic signaling pathway in response to endoplasmic reticulum stress' (Fig. 3C). These included genes encoding Atf4, Bbc3 (Puma), Chac1, Bax, and the ER Ca 2+ channel Itpr1, in which loss of function variants have been shown to cause spinocerebellar ataxia 29 (32) (Fig. 3D). We confirmed a time dependent induction of these ER stress genes by PR 50 expression using quantitative reverse transcription PCR (qRT-PCR) (Fig. 3E). To confirm that these stress responses are conserved in other models of DPR toxicity we performed RNA-seq on primary mouse neurons as well as human K562 cells, each treated with synthetic PR 20 . ATF4 and related ER stress response genes were induced in each of these models (Fig. 3F, S6, Supplemental Table 4). Finally, in addition to observing genetic signatures of ER stress, we also used a pharmacological approach to test the importance of this pathway in response to PR 20 . The small molecule ISRIB is a potent inhibitor of the cellular response to ER stress (33) preventing ATF4 induction by inhibiting eIF2α phosphorylation in human cells under ER-stress (34). Pre-treatment of K562 cells with 15nM ISRIB mitigated PR 20 toxicity (Fig. 3G), providing further evidence for a direct role of ER stress in DPR-mediated toxicity.
Several lines of evidence have suggested a potential role for ER stress in C9orf72 pathogenesis. Transcriptomic analyses of C9orf72 mutant ALS brain tissue reveals an upregulation in genes involved in the unfolded protein response (UPR) and ER stress (26). A specific PERK-ATF4 mediated ER stress response was reported in a separate study of c9ALS/FTD patient brain tissue when compared to sporadic ALS patient brain tissue (35).
Additionally, ER stress and disruptions to ER Ca 2+ homeostasis were observed in c9ALS/FTD patient derived iPSC motor neurons (36). Because C9orf72 mutations could cause disease pathology by several different mechanisms, including loss of function, RNA toxicity, and/or protein toxicity from the DPRs (37), it has remained unclear which molecular drivers induce the ER stress response. The ATF4-driven ER stress response we observed in primary neuron cultures expressing PR 50 and the enrichment of genes functioning at the ER as hits in our CRISPR screens suggest that this stress response may be a major driver of pathogenesis and, importantly, is likely driven by the production of arginine-rich DPRs instead of by loss of C9orf72 function or toxicity from repeat-containing RNAs.
Given that the main transcriptional stress response to endogenous and exogenous DPR treatment was the ER stress response, we chose to further investigate TMX2, a gene encoding a poorly characterized ER-resident protein that was one of the strongest protective hits in both the PR and GR genome-wide CRISPR screens in K562 cells and in the targeted primary neuron screen expressing PR 50 . We first confirmed that the sgRNAs targeting TMX2 effectively knocked out the gene in K562 cells and primary neurons cultured from the Cas9 mouse ( Fig. 4A,C).
Strikingly, reduction of TMX2 levels was sufficient to markedly suppress C9orf72 DPR toxicity in both K562 cells (Fig. 4B) and primary mouse neurons (Fig. 4D).
TMX2 is a transmembrane protein that localizes to the mitochondrial-associated membrane (MAM) of the ER (38). It is a member of the protein disulfide isomerase (PDI) family, but it is unclear whether it is catalytically active since it harbors an SXXC motif instead of the canonical CXXC motif thought to be necessary to reduce disulfide bonds (39). To define the mechanism by which TMX2 reduction mitigates DPR toxicity we performed RNA-seq on primary Cas9 + neurons infected with control sgRNAs or sgRNAs targeting TMX2, with or without PR 50 expression ( Fig. 4E-G, S7). Upon PR 50 expression, Tmx2 KO neurons upregulated a distinct set of pro-survival unfolded protein response (UPR) pathway genes and downregulated calcium binding and apoptotic genes, compared to primary neurons expressing control sgRNAs ( Fig. 4G). We validated by qRT-PCR one of the upregulated UPR genes in the Tmx2 KO neurons, Atf3, which has been previously shown to be protective upon overexpression in mouse models of ALS (40) and neuronal models of excitotoxicity (41) (Fig. 4H). These results suggest that TMX2 KO may be protective by modulating the ER stress response elicited by the DPRs.
Here, we used comprehensive CRISPR-Cas9 screens in human cells with further validation screens in primary neurons to discover modifiers of C9orf72 DPR toxicity. We not only identified nucleocytoplasmic transport machinery, confirming previous studies in model organisms, but also identified new genes that point to ER stress as critical for c9FTD/ALS pathogenesis. The gene knockouts that mitigated toxicity, such as TMX2, could represent therapeutic targets. It is notable that TMX2 KO was able to confer near complete protection from PR toxicity. More generally, we anticipate that these types of CRISPR screens in human cells and primary neurons will be a powerful addition to the experimental toolbox to study mechanisms of neurodegenerative disease.    Each measurement was performed in triplicate with mean and SD plotted by error bars (p-value <.005, two-tailed t-test).

Lentivirus production
HEK293T cells were cultured under standard conditions (DMEM + 10% FBS + Penicillin-Streptomycin.) and used to package lentiviral particles following standard protocols with 3rd generation packaging plasmids. Lentiviral containing media was harvested after 48hrs., centrifuged at 300g for 5 min. to remove cellular debris, and concentrated 10-fold using Lenti-X concentrator (Clontech) before adding to cell cultures.

Immunocytochemistry and antibodies
Cells were grown on poly-L-lysine coated glass coverslips (0.1% w/v) in standard multiwell cell culture plates and were stained using standard immunocytochemistry techniques. Briefly, cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% normal goat serum, and stained with the following antibodies: mouse monoclonal anti-FLAG Images were acquired using either a Leica DMI6000B inverted fluorescence microscope with a 40X oil immersion objective or a confocal Zeiss LSM710 microscope with a 63X oil immersion objective.

Cytotoxicity Assays
K562 cell viability after DPR treatment was measured by flow cytometry using a BD Accuri C6 flow cytometer by measuring number of events in forward scatter and side scatter gates .
Cytotoxicity in primary neuron cultures was measured both by lactose dehydrogenase (LDH) release assays (Promega, CytoTox 96® Non-Radioactive Cytotoxicity Assay) and by NeuN+ quantification of surviving neurons using immunocytochemistry. To quantify numbers of NeuN+ neurons, cells were fixed and stained as described above, and quantified using ImageJ. Briefly, thresholds were applied to image stacks in order to detect NeuN positive neuronal nuclei, which were then automatically counted using built in ImageJ plugins.   Table  1). (C) Volcano plot of effect vs. confidence scores for all human genes in the PR 20 screen. Colored in blue are all the genes conferring resistance to PR 20 when knocked out (10%FDR) and colored in red are all the genes conferring sensitivity to PR 20 when knocked out (10% FDR).    Table 3).  Table 4). (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 1.5µM PR 20 (Supplemental Table 5).  Table 7).