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Budagyan K , Chernoff J
A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background
Methods Mol Biol. 2021 ;2262 :323-334
PMID: 33977487   
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Oncogenic KRAS mutations are common in colorectal cancer (CRC), found in ~50% of tumors, and are associated with poor prognosis and resistance to therapy. There is substantial diversity of KRAS mutations observed in CRC. Importantly, emerging clinical and experimental analysis of relatively common KRAS mutations at amino acids G12, G13, A146, and Q61 suggest that each mutation differently influences the clinical properties of a disease and response to therapy. Although clinical evidence suggests biological differences between mutant KRAS alleles, these differences and the mechanisms underlying them are not well understood, and further exploration of allele-specific differences may provide evidence for individualized therapeutics. One approach to study allelic variation involves the use of isogenic cell lines that express different endogenous KRAS mutants. Here we developed an assay using fluorescent co-selection for CRISPR-driven gene editing to generate various Kras mutations in an isogenic murine colon epithelial cell line background. This assay involves generation of a cell line stably expressing Cas9 linked to BFP and simultaneous introduction of single-guide RNAs (sgRNAs) to two different gene loci resulting in double-editing events. Single-stranded donor oligonucleotides are introduced for a GFP gene and a Kras mutant allele of our choice as templates for homologous recombination (HDR). Cells that successfully undergo HDR are GFP-positive and have a higher probability of containing the desired Kras mutation. Therefore, selection for GFP-positive cells allows us to identify those with phenotypically silent Kras edits. Ultimately, this method allows us to toggle between different mutant alleles and preserve the wild-type allele while maintaining an isogenic background.
1940-6029 Budagyan, Konstantin Chernoff, Jonathan Journal Article United States Methods Mol Biol. 2021;2262:323-334. doi: 10.1007/978-1-0716-1190-6_20.