SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [CRISPR]

In this study, we identified SFSWAP as a negative regulator of O-GlcNAc transferase (OGT) intron 4 detention and global pre-mRNA splicing. To do this, we performed CRISPR knockout screens using the Brunello knockout library on Thiamet G (TG)-treated HCT116 cells containing an O-GlcNAc responsive GFP reporter (GFP-β-OGT) integrated at the AAVS1 locus. Knockout of negative regulatory factors which regulate OGT intron 4 detention lead to increased GFP expression. Using replicate screens and further biochemical validation, we identified SFSWAP as one of the top hits affecting OGT intron 4 detention. CRISPR knockout screens were performed in biological triplicates. Reporter lines were infected with lentiviral particles encoding the Brunello knockout library at 0.3 MOI and selected on puromycin for 8 days starting at day 2 post infection (total 10 days). Unselected cells were used to estimate library representation (sample 'library'). Cells were treated with 1 μM TG for 24 hours (replenished every 12 hours) on day 9 and sorted for high GFP expression at the end of day 10 ('sorted' samples). Unsorted cells were used as control ('input' samples). The pilot screen (biological replicate 1) was performed at 100X library coverage and replicate screens (biological replicates 2 and 3) were performed at 300X library coverage.