4.6. Generation of C2C12 LRRC8A Knockout Cell Lines Using CRISPR/Cas9 Technology

To create LRRC8A knockout C2C12 cells using the CRISPR/Cas9 technology, the targeting sgRNA sequences (5′-GCCCCGGAAGGAGTCGTTGCAGG-3′) was cloned into the px459-V.2 vector and transfected into C2C12 cells. Two days post-transfection, transfected cells were selected by treatment with 10 µg/mL puromycin for two days before single clone expansion by dilution to statistically 0.5 cells per well in 96-well format. Monoclonal cell lines were expanded and tested for sequence alterations using target-site-specific PCR with primers 5′-CATGTATGTCTCACTACACCTAACTTGTAG-3′ and 5′-CCAGGAAGATGAGGGTGTGCA-3′ on genomic DNA followed by Sanger sequencing.