Abstract
Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized 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 nonhomologous 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.
Highlights
The concept of gene editing began in the late 1990s with the development of zinc-finger nucleases (ZFNs) [1] and subsequently with transcription-like effector nucleases (TALENs) [2–4]
We describe a procedure which we have adapted from the Zhang Lab at the Broad Institute [9], in order to generate frameshift and point mutations in renal cell lines
Key features of this protocol include the following: (1) sorting of cells co-transfected with GFP together with the Cas9/synthetic guide RNA (sgRNA)-expressing plasmid enhances the overall targeting efficiency
Summary
There has been huge growth in the application of gene-editing technologies. The concept of gene editing began in the late 1990s with the development of zinc-finger nucleases (ZFNs) [1] and subsequently with transcription-like effector nucleases (TALENs) [2–4] Both of these technologies are limited by their relatively low targeting efficiencies, the relative rarity of suitable ZFN cut sites within the genome, and the need for complex cloning procedures to generate TALEN DNA-binding oligonucleotides. We describe a procedure which we have adapted from the Zhang Lab at the Broad Institute [9], in order to generate frameshift and point mutations in renal cell lines Key features of this protocol include the following: (1) sorting of cells co-transfected with GFP together with the Cas9/sgRNA-expressing plasmid enhances the overall targeting efficiency. The improved phenotypic consistency of these cellular models means that the CRISPR/Cas system is invaluable for studies of disease mechanisms and for drug screening approaches
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