CRISPR/Cas9 genome editing technology is one of the most outstanding scientific breakthroughs in recent years, revolutionizing basic and medical research by enabling site-specific genome engineering of cell lines. ATCC has previously employed CRISPR/Cas9 technology to generate isogenic cell lines with disease-relevant mutations, reporter cell lines that provide real-time status of epithelial-mesenchymal transition, and STAT1 knockout cell lines capable of producing high-titer viral stocks. We now offer custom genome editing services that span from assay design to cell line development, and we will work with you to develop a cell line that best meets your research needs.
ATCC has mastered the science and art of CRISPR gene-editing. We take highly authenticated cell lines from our collection and introduce the disease-relevant mutations using single guide RNAs (sgRNAs) that are designed to guide Cas9 to the targeted regions. The parental cell lines are cotransfected with sgRNA and Cas9. The transfected cells are sorted into single cells and expanded for testing. The gene-edited isogenic cell clones are then rigorously screened.
Genetic and transcript validation
Protein expression and function validation:
Below is an example of the genomic and transcriptional validation of ATCC isogenic cell lines, using the NRAS Mutant-A375 cell line. The chromatograms show via Sanger sequencing the point mutation of glutamine to lysine at position 61 (Figure 1). These results were confirmed by NGS (data not shown) and inhibitor studies.
Figure 1. Validation of point mutation at genomic and transcript level. A) Screening for the NRASQ61K point mutation in the genome of the edited clones was carried out using PCR primers as shown left. Introduction of the NRASQ61K point mutation was then confirmed via sequencing shown on the right. Boxed in red is the expected C>A mutation. B) Validating the transcripts of NRASQ61K point mutation in the edited clones was carried out via cDNA generation from cells and PCR (red arrows; top image). The bottom left is the gel image of the PCR products. The bottom right is the Sanger sequencing chromatogram. Boxed in red is the expected point mutation.
The Rat Sarcoma (RAS) proto-oncogene encodes for proteins such as KRAS and NRAS that belong to the small GTPase superfamily. RAS proteins recruit and activate downstream effectors, such as those of the AKT and ERK pathways that in turn affect cell growth, differentiation, and survival. Mutations in the RAS gene have been identified in melanoma, and may be predictive of a very poor response to BRAF inhibitors.
CRISPR/Cas9 gene editing was used to create mutants in the A375 (ATCC® CRL-1619™) cell line, one of the most commonly used in vitro models of melanoma. We mutated glycine position 13 to aspartic acid in KRAS (KRASG13D) in A375 cells to create the KRAS mutant-A375 Isogenic Cell Line (ATCC® CRL-1619IG-2™). The NRAS mutant-A375 Isogenic Cell Line (ATCC® CRL-1619IG-2™) was created by replacing the glutamine at position 61 with a lysine (NRASQ61K). To assess the biofunctional responses of the KRAS- and NRAS-mutant Isogenic Cell Lines, we challenged them with the BRAF inhibitors.
Figure 2. The KRAS isogenic cell line is more resistant to BRAF inhibitors than the parental A375 cell line. A375 and KRAS Mutant-A375 Isogenic Cells were treated with the indicated concentrations of either A) dabrafenib or B) vemurafinib for five days. Cell survival was monitored via cell viability assay.
Figure 3. The NRAS isogenic cell line is more resistant to BRAF inhibitors than the parental A375 cell line. A375 and NRAS Mutant-A375 Isogenic Cells were treated with the indicated concentrations of either A) dabrafenib or B) vemurafinib for three days. Cell survival was monitored via cell viability assay.References
CRISPR enables precise modifications in the genomes of eukaryotic cells. Learn more about how ATCC creates and validates isogenic cell lines, and explore our new custom genome editing service.
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