CRISPR helps find new genetic suspects behind ALS/FTD

Accounting for nearly 40 percent of inherited cases of ALS and 25 percent of inherited FTD cases, disease-causing mutations in C9orf72 insert extra sequences of DNA, called hexanucleotide repeats, into the gene. These repeats produce potentially toxic RNA and protein molecules that kill neurons resulting in problems with movement and eventually paralysis for ALS patients and language and decision-making problems for FTD patients.

Led by Aaron D. Gitler, Ph.D., and Michael C. Bassik, Ph.D., the researchers used CRISPR to disable each gene, one-by-one, in a line of human leukemia cells and then tested whether the cells would survive exposure to toxic proteins derived from the hexanucleotide repeats, called DPRs. Any disabled genes that caused cells to live longer or die faster than normal were considered suspects in DPR toxicity. They confirmed that genes that control the movement of molecules in and out of a cell's nucleus may be partners. They also identified several new players, including genes that modify chromosomes and that help cells assemble proteins passing through a maze-like structure called the endoplasmic reticulum (ER). A second CRISPR search conducted on mouse brain cells confirmed the initial results. Disabling the top 200 genes identified in the leukemia cells helped neurons survive DPR exposure.

Finally, further experiments highlighted the importance of the ER genes, especially one called TMX2. For instance, the researchers could cause neurons derived from the skin cells of ALS patients with C9orf72 to live longer than normal when they silenced the TMX2 gene, suggesting it could be exploited in designing novel therapies for ALS. Decreasing TMX2 in cells caused an increase in the production of "survival proteins" that the authors hypothesized protected the cells against DPR toxicity.

Previously such studies needed a few months to find candidate genes and could only be performed on yeast, worm, and fly genomes. With CRISPR, the researchers in this study needed just about two weeks to conduct a complete search of the human genome. The results suggest that this faster and more comprehensive approach may be used to rapidly identify genes that may be involved in other neurological disorders.

This study was supported by the NIH (NS097263, NS097850, NS069375, HD084069); the National Human Genome Research Institute Training Grant; the National Science Foundation Graduate Research Fellowship; the Department of Defense (W81XWH-15-1-0187); the Robert Packard Center for ALS Research at Johns Hopkins; Target ALS; the Stanford Brain Rejuvenation Project of the Stanford Neurosciences Institute; the Muscular Dystrophy Association; and the New York Stem Cell Foundation.