Genetic interaction screen for FXN in K562 cells, to identify genes that are synthetic sick/buffering in the absence of robust Fe-S cluster synthesis

Friedreich’s ataxia (FA) is the most common monogenic mitochondrial disease. FA is caused by a depletion of the mitochondrial protein frataxin (FXN), an iron-sulfur (Fe-S) cluster biogenesis factor. To better understand the cellular consequences of FA, we performed genetic interaction mapping in control or FXN edited cells. This screen identified impaired mitochondrial translation downstream of FXN loss, and specifically highlighted the methyltransferase-like protein METTL17 as a candidate effector. To investigate the genetic interactions of FXN, K562 cells were infected with pRDA_186 lentiviral vectors, which express sgRNA against a control locus or FXN, blasticidin resistance from the PGK promoter, and a 2A site-expressing EGFP. Cells were sorted for low GFP expression and expanded. For the screen, the all-in-one Brunello barcoded library was utilized. This library contains 77,441 sgRNA; an average of 4 guides per gene and 1000 non–targeting control guides. Infections were performed in distinct duplicate at a predetermined MOI of ~0.5 in 12-well plates with 5µg/mL polybrene supplementation. Cells were infected for 2h under centrifugation at 1000 x g, incubated for 22h under standard culturing conditions and pooled 24 h post-centrifugation. Infections were performed with 6.5x107 cells per replicate, in order to achieve a representation of at least 300 cells per sgRNA following puromycin selection. 48 hours after infection, cells were selected with puromycin for 2 days to remove uninfected cells. Cells were passaged in fresh media every 2–3 days. Cells were harvested 11 days after initiation of treatment.