Generating mutant renal cell lines using CRISPR technologies

Gene-editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalised cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles (‘indels’) at the desired locus via the non-homologous end-joining pathway, or in conjunction with a homology directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene-editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.