2.4. Generation of CRISPR/Cas9 knock out (KO) U2OS cell lines

The following guide RNAs (sgRNAs) were designed by the CRISPR design tool (http://crispr.mit.edu): SUMO1 5’-TCCCTCCTCCCTGCGCGAAG-3’; SUMO2 5’- CCTCACCTGTCGTTCACAAT-3’; SUMO3 5’- TGATGAAGGCCTACTGCGAG-3’. sgRNAs were cloned into pSpCas9(BB)-2A-GFP vectors by BpiI enzyme sites, and vectors were transiently transfected into U2OS cells using X-tremeGENE HP reagent (Cat#XTGHP-RO, Roche) according to the manufacturer’s protocol. Transfected cells expressing GFP were sorted into 96-well plates with a single cell/well by FACS using the Flow Cytometry and Immunology core in the Johns Hopkins University Bloomberg School of Public Health. Genotyping of each SUMO knockout clone was performed to examine genetic deletion status. Total genomic DNA was extracted using the GeneJET genomic DNA purification kit (Cat#K0721, ThermoFisher Scientific, Waltham, MA), followed by PCR amplification with SUMO- specific primers. PCR amplicons were cloned into the pJet1.2 vector (Cat#K1231, ThermoFisher Scientific, Waltham, MA) and analyzed using Sanger DNA sequencing. RT-PCR determined RNA expression levels of each SUMO paralogue with SUMO-specific primers (data not shown). SUMO1 and SUMO2/3 protein expression levels were determined via western blotting and immunofluorescence microscopy.