Generation of SPHKs knock-out cell lines

For knocking out SPHK1 and SPHK2, we used sgRNA sets designed by SYNTHEGO (3 sgRNA per target) and an additional set of control nontargeting sgRNAs (TRAC). The following sgRNA sequences were used: SPHK1 5′-UUUUCUCAGCGGGCGGCCCC-3′, 5′-GCAAGGCCUUGCAGCUCUUC-3′ and 5′-ACCAGUGAGCAUCAGCGUGA-3′; SPHK2 5′-CGCAGGCCCUGCACAUACAG-3′, 5′-UGGCCUGGUCCCGUUGGCCG-3′ and 5′-CCGCUGAGUCUGAGGGGCUG-3′. Briefly, sgRNAs were coupled with Cas9 2NLS nuclease (S. pyogenes) (SYNTHEGO) forming ribonucleoprotein (RNP) complexes, and cells were transfected using Lipofectamine CRISPRMAX Cas9 Transfection Reagent (Thermo Fisher, Sweden). U2OS cells were seeded on top of the transfection mixes (30,000 cells/well) in 12-well plates. Single-cell clones were generated and screened for finding single knock-outs of SPHK1 and SPHK2. For producing the double knock-out, SPHK1-KO selected clones were transfected with sgRNAs targeting SPHK2 as already stated and vice versa for SPHK2-KO clones. Then, single clones were generated and screened. The same procedure was done using cells transfected with nontargeting sgRNAs as a control.