Generating mutant renal cell lines using CRISPR technologies / Nuria Perretta-Tejedor.

Format:

Journal/Periodical

Author/Creator:

Perretta-Tejedor, Nuria, author.

Language:

English

Subjects (All):

Genetics--Periodicals.

Genetics.

Physical Description:

1 online resource

Place of Publication:

New York : Humana, 2020.

Summary:

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.

Notes:

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