. CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nat Genet. 2018 Apr;50(4):603-612. Epub 2018 Mar 5 PubMed.


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  1. This is another great paper by the Gitler and Bassik groups, using the power of genetic screens to identify previously unanticipated modifiers of dipeptide toxicity from C9ORF72 repeat expansion. The beauty of their approach is that it immediately provides a target for drug screening, and a therapeutic avenue toward the clinic. It will be very exciting to see whether manipulating TMX2 can be used for therapeutic intervention in C9ORF72 ALS/FTD in the future.

    View all comments by Robert Baloh
  2. The group of Aaron Gitler is the first one to successfully perform a genome-wide gene knockout screen using CRISPR–Cas9 in a human cells system to search for suppressors and enhancers of C9ORF72 DPR toxicity.

    First of all, this study provides clear proof of principle that such a CRISPR-Cas9 screen in the context of ALS is feasible in human cells, as well as in mouse primary cortical neurons. Moreover, and very importantly, the obtained hits were unique for the DPR-induced toxicity and didn’t overlap with hits of other unrelated toxicity screens.

    Second, it is very encouraging that there is considerable overlap between the hits in the PR and GR toxicity screen. This indicates that similar pathways are involved in the induction of toxicity, which is in line with what one could expect.

    Third, these CRISPR-Cas9 screens could also provide interesting new insights into the pathways playing a role in the toxicity induced by synthetic arginine-containing DPRs or PR translated from a codon-optimized construct. At present, the screen using the human cell system especially confirms the importance of a number of pathways that have already been linked to DPR-induced toxicity in other systems, which is also very encouraging. These include nucleocytoplasmic transport, endoplasmic reticulum (ER) stress, proteasomal dysfunction, involvement of RNA-processing as well as of chromatin modifications. A very interesting observation is the involvement of RAB7A and the endosomal pathway in the uptake of DPRs. This could have important implications for the cell-to-cell spreading of DPRs.

    Finally, it is very interesting that one modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and also improved survival of human induced motor neurons (iMNs) from patients with C9ORF72 ALS. Moreover, the major importance of ER stress could also be confirmed pharmacologically.

    In conclusion, it is very clear that the information obtained from these genome-wide screens will be an important source of modifiers that can be tested for their effect in other systems, and that could lead to the development of new therapeutic strategies. Moreover, there is no doubt that more genome-wide CRISPR-Cas9-related screens will follow in other cell systems and in other toxicity paradigms related to ALS, given the fact that the feasibility and the potential success is convincingly shown by the current study.

    View all comments by Ludo Van Den Bosch
  3. This work merits attention. It again truly shows that CRISPR/Cas9 is not only a tool for therapeutic genome editing but, compared with other genome-editing approaches, has the unique capacity for genome-wide screens and biological discovery. One can hope that the results of this work point to general mechanisms of ALS pathology that translate to other genotypes and sporadic cases, such that we can begin to consider genome editing or other approaches to develop a broad therapy for this devastating disease.

    View all comments by J. David Schaffer
  4. Debate over dipeptide repeats (DPRs) continues, but undoubtedly they are toxic in culture. This important study from Aaron Gitler and Michael Bassik describes the first genome-wide screen for modifiers of DPR toxicity in a mammalian cell line. The genome-wide section of the work utilized K562 cells exposed to exogenous poly proline-arginine (PR) or poly glycine-arginine (GR). Interestingly, there was significant correlation between hits in response to both proteins (but zero overlap with a ricin toxicity screen), suggesting a common pathway for toxicity. This is not a “given” and will help to direct future model design centered on DPRs.

    Hits from the K562 screen were validated in a number of ways, including exposing mouse primary cortical neurons to exogenous PR or using a lentivirus to induce endogenous PR expression. Interestingly, the method of PR delivery appeared to have a significant effect—both models validated hits from the original screen but the overlap was relatively small. This is particularly interesting when it is considered that neither system included RAN translation, which is the method by which DPR are produced in patients.

    Top hits, which were extensively validated, implicate the ER stress response and, in the context of extracellular PR, the intracellular trafficking of PR. A number of other novel and established biological pathways were highlighted including nucleocytoplasmic transport.

    The authors rightly highlight the current lack of supporting evidence of DPR toxicity in postmortem studies but in vitro, the field is making good progress towards therapeutic targets.

    View all comments by Johnathan Cooper-Knock
  5. Here are my thoughts on the paper.

    1. This screen is specifically trying to identify toxicity from two DPRs: PR and, to a lesser extent, GR. It does not speak to toxicity from other DPRs or from the repeats as RNA and it does not look at modifiers of RAN translation, per se.
    2. A strength of the paper is that they performed screens in two different cell types and with delivery of PR and GR dipeptide repeat proteins through two different modalities: exogenous application of synthetic peptides or expression of PR50 from a lentiviral construct (of note, this construct does not contain the GGGGCC repeat). Their initial genome-wide screen in K562 cells revealed a lot of hits. They advanced their top 200 to mouse neurons and were able to validate about 10 percent of these (17 total) with synthetic peptide delivery and 6 percent (13) in mouse neurons expressing PR5 from a lentiviral construct. Only one gene was positive in all three screens, suggesting that exogenous peptide delivery and endogenous production of the peptides drive toxicity through somewhat different mechanisms. Both could be in play in the disease state, but presumably endogenous expression is more likely to be a primary driver of pathogenicity.
    3. There was pretty good evidence before this paper that DPRs (PR and GR in particular) can activate ER stress and the unfolded protein response and suppress global protein synthesis. This paper supports those findings for PR and provides some evidence that ISRIB, which suppresses the stress response, can partially mitigate toxicity from at least synthetic PR peptides. They then provide pretty strong data to support a role for ER stress in PR toxicity by knocking down TMX2, which was a lead hit from their screen and is involved in the UPR.
    4. The data in iPSC-derived motor neurons, while modest in effect, is really a key finding in this the paper. It is the only part of the manuscript done at endogenous levels in the setting of the GGGGCC repeat and further supports the argument that DPRs, and PR and GR in particular, are drivers of toxicity in the disorder.
    5. Given that this effect is presumably separate from RAN translation activation elicited by ER stress (as shown by our paper and Shuying Sun’s paper a few months back), this paper provides more evidence suggesting that targeting the stress response pathway is a sensible path forward in therapeutic development as we attempt to mitigate C9 repeat-associated toxicity. Whether a more targeted approach such as targeting TMX2 or a factor specifically required for RAN translation or a broad approach such as application of ISRIB to block the whole pathway is more sensible will be a point of discussion in the field going forward.

    View all comments by Peter Todd

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  1. CRISPR Screen Pulls Down Fresh Targets for C9ORF72 ALS