ULK1/2 Restricts the Formation of Inducible SINT-Speckles, Membraneless Organelles Controlling the Threshold of TBK1 Activation

Saul, Vera Vivian; Seibert, Markus; Krüger, Marcus; Jeratsch, Sylvia; Kracht, Michael; Schmitz, Michael Lienhard

In order to generate SINTBAD-and/or AZI2-deficient U2OS cells, CRISPR/Cas9-mediated genome editing technology was performed as described (Ran et al., 2013) . The target site for human SINTBAD was designed as an anti-sense sgRNA (5´-CGTAGACTTTGAGGCGGCGT-3´) within the first exon of the SINTBAD gene. The target site for sgAZI2 was designed within the second exon as 5´-GGCCTATCATGCATATCGAG-3´. Oligos were ligated into px459 V2.0 vector (Addgene plasmid #62988) using standard protocols and verified by sequencing. U2OS cells, seeded in a 6 cm dish, were transfected with 1 µg of empty px459 or px459-sgRNA vector. One day after transfection, cells were selected for 30 h using 1 µg/ml Puromycin (Invivogen), diluted and further grown to allow the formation of single-cell clones. These clones were picked and analyzed for SINTBAD, AZI2