A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V...

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• KRAS mutations are critical biomarkers for targeted therapy and drug resistance in colorectal cancer.
• Accurate detection of KRAS mutations is vital for clinical decision-making.
• Current detection methods, especially next-generation sequencing (NGS), lack adequate quality control materials.

Purpose of the Study:

• To develop a novel quality control material for KRAS mutation detection.
• To address the deficiency of control materials that include both mutant genes and matched background DNA.
• To validate the utility of the developed material for various detection platforms.

Main Methods:

• Utilized clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to create a KRAS G12V mutant cell line.
• Validated the cell line's suitability as quality control material using amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS.

Main Results:

• Successfully generated a cell line with a specific KRAS G12V mutation.
• Demonstrated high consistency in mutation detection across ARMS, Sanger sequencing, dPCR, and NGS.
• Quantified mutation allele fractions in three screened cell lines, ranging from 17.29% to 82.06%.

Conclusions:

• The novel KRAS G12V cell line is a suitable quality control material for current detection methods.
• This development offers a new solution for quality control in KRAS mutation analysis.
• Facilitates accurate distinction between germline polymorphisms and somatic mutations in cancer diagnostics.