Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72

Mutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72. Overall design: RNA-seq: RNA was extracted from undifferentiated and PMA-treated control and C9KO U937 cells using the PureLink RNA Mini Kit (Life Technologies) according to the manufacturer's protocol, with on-column PureLink DNase treatment. Total RNA concentration and quality control was determined using the RNA 6000 Nano assay kit (Agilent) on the Agilent 2100 Bioanalyzer System for all samples. mRNA libraries were prepared for Illumina paired-end sequencing using the Agilent SureSelect Strand Specific RNA-Seq Library Preparation kit on the Agilent Bravo Automated Liquid Handling Platform. Libraries were sequenced on an Illumina HiSeq 4000 sequencer. Alignment of RNA-sequencing reads to the transcriptome was performed using STAR with ENCODE standard options, read counts were generated using rsem, and differential expression analysis was performed in R using DESeq2 package (Love et al., 2014). All bioinformatics analyses were performed on Sherlock, a Stanford HPC cluster. CRISPR screen: The lentiviral genome-wide sgRNA library was produced and infected separately into control and C9KO U937 cells stably expressing EF1a-Cas9-Blast as previously described (Haney et al., 2018). Control and C9KO infected populations were maintained in separate spinner flasks. Briefly, ~300 million control cells and ~300 million C9KO cells were infected with the 10 guide/gene genome-wide sgRNA library at a MOI < 1. Infected cells underwent puromycin selection (1ug/mL) for 5 days. Puromycin was then removed and cells were resuspended in normal growth media without puromycin. After selection, sgRNA infection was measured by flow cytometry confirming that > 90% of cells were mCherry+. Sufficient sgRNA library representation at the starting point of the screen was confirmed by Illumina sequencing after selection. Cells were maintained for 10 days at 1000× coverage (~1000 cells containing each sgRNA) at a concentration of 500,000 cells/mL, after which ~450 million control and ~450 million C9KO cells were removed for 50nM PMA differentiation in three 15cm plates as described above. At the end of the 5 day differentiation, genomic DNA was extracted for all screen populations separately according to the protocol included with QIAGEN Blood Maxi Kit. sgRNA sequences were amplified and prepared for deep-sequencing by two sequential PCR reactions as described previously (Morgens et al., 2016). Final PCR products were sequenced using an Illumina NextSeq.