Crisprv2pspcas9 bb 2a puro px459 v2

Full length human TAOK2 was PCR amplified from pCMV-Sp6-TAOK2 plasmid described previously (Ultanir et al., 2014) , and inserted in vector sfGFP-C1 (Addgene #54579) using restriction sites HindIII and MfeI. Domain dissection mutants were subcloned from sfGFP-TAOK2 using restriction enzymes HindIII and MfeI (New England Biolabs). All resultant plasmids were verified by sequencing. GST-TAOK2-(1187 -1235) was subcloned from the sfGFP-TAOK2 into the pGEX4T1 vector using sites SalI and NotI. TAOK2 knockout cell line was generated using CRISPR/Cas9 genome editing in HEK293T cells. Four independent guides were designed using Synthego guide design tool (https://www.synthego.com) to target coding exon 2. Two plasmids were made by adding their respective guides into CrisprV2pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene Plasmid #62988). Cells were passaged in single cell suspension and plated at 50% confluence. Cultures were then transfected with lipofectamine 2000 reagent (Invitrogen 11668-030) and 2mg of each respective plasmid. Non-Homology End Joining (NHEJ) repair created a deletion around the gRNA cutting site. Cells were selected with Puromycin for 2 days. Genomic DNA was extracted and the region around the cutting site was PCR amplified. Knockout of the gene TAOK2 was confirmed by Sanger sequencing analysis, and absence of encoded protein was validated using western blot.