Generating Mutant Renal Cell Lines Using CRISPR Technologies.

Generating Mutant Renal Cell Lines Using CRISPR Technologies.
Methods Mol Biol. 2020;2067:323-340
Authors: Perretta-Tejedor N, Freke G, Seda M, Long DA, Jenkins D
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.
PMID: 31701460 [PubMed - in process]

Source: Mol Biol Cell - November 10, 2019 Category: Molecular Biology Authors: Perretta-Tejedor N, Freke G, Seda M, Long DA, Jenkins D Tags: Methods Mol Biol Source Type: research