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A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V
Shiyu Jia1,2,3, Rui Zhang2,3, Guigao Lin2,3
1Peking University Fifth School of Clinical Medicine, National Center for Clinical Laboratories, National Center of Gerontology, Beijing Hospital, Beijing, China.
Background:
KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations.
Methods:
We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS.
Results:
We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively.
Conclusion:
The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.
Insights
A novel KRAS G12V mutant cell line was developed using CRISPR/Cas9 technology. This cell line serves as essential quality control material for accurate KRAS mutation detection in colorectal cancer diagnostics.
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.
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