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Sensitive Detection of KRAS Mutations by Clustered Regularly Interspaced Short Palindromic Repeats
Huifen Zhou1, Jen-Hui Tsou1, Qixin Leng1
1Department of Pathology, University of Maryland School of Medicine, 10 S. Pine St., Baltimore, MD 21201, USA.
Abstract:
Kirsten rat sarcoma viral oncogene (KRAS) is the isoform most frequently mutated in human tumors. Testing for activating KRAS mutations has important implications for diagnosis and the personalized medicine of cancers. The current techniques for detecting KRAS mutations have moderate sensitivity. The emerging clustered regularly interspaced short palindromic repeats (CRISPR) system shows great promise in the detection of nucleic acids and is revolutionizing medical diagnostics. This study aimed to develop CRISPR-Cas12a as a sensitive test to detect KRAS mutations. Serially diluted DNA samples containing KRAS mutations are subjected to CRISPR-Cas12a and polymerase chain reaction (PCR). CRISPR-Cas12a and PCR can specifically detect 0.01% and 0.1% mutant KRAS DNA in the presence of wild-type KRAS DNA, respectively. Twenty pairs of lung tumor and noncancerous lung tissues are tested by CRISPR-Cas12a, PCR, and direct sequencing. CRISPR-Cas12a could identify the G12C mutation in five of 20 tumor tissues, while both PCR and direct sequencing discovered the KRAS mutation in three of the five tumor tissues. Furthermore, the results of CRISPR-Cas12a for testing the mutation could be directly and immediately visualized by a UV light illuminator. Altogether, CRISPR-Cas12a has a higher sensitivity for the detection of KRAS mutations compared with PCR and sequencing analysis, and thus has diagnostic and therapeutic implications. Nevertheless, the technique needs to be validated for its clinical significance in a large and prospective study.
Insights
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a offers a highly sensitive method for detecting Kirsten rat sarcoma viral oncogene (KRAS) mutations in cancer. This CRISPR-based test shows improved accuracy over traditional methods for diagnosing KRAS mutations.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Kirsten rat sarcoma viral oncogene (KRAS) mutations are frequent in human tumors, impacting cancer diagnosis and personalized medicine.
- Current methods for detecting KRAS mutations possess moderate sensitivity, necessitating more advanced diagnostic tools.
- The clustered regularly interspaced short palindromic repeats (CRISPR) system presents a promising technology for nucleic acid detection and medical diagnostics.
Purpose of the Study:
- To develop and evaluate the CRISPR-Cas12a system for sensitive detection of KRAS mutations.
- To compare the sensitivity and specificity of CRISPR-Cas12a against established methods like polymerase chain reaction (PCR) and direct sequencing.
Main Methods:
- CRISPR-Cas12a assay combined with polymerase chain reaction (PCR) amplification.
- Testing serially diluted DNA samples with known KRAS mutations.
- Analysis of 20 pairs of human lung tumor and noncancerous tissue samples.
- Direct visualization of results using a UV light illuminator.
Main Results:
- CRISPR-Cas12a detected 0.01% mutant KRAS DNA, outperforming PCR's 0.1% sensitivity.
- In clinical samples, CRISPR-Cas12a identified KRAS G12C mutations in 5 out of 20 tumor tissues.
- PCR and direct sequencing detected KRAS mutations in only 3 of the 5 positive tumor tissues identified by CRISPR-Cas12a.
- CRISPR-Cas12a results were directly visualized under UV light.
Conclusions:
- CRISPR-Cas12a demonstrates superior sensitivity for detecting KRAS mutations compared to PCR and sequencing.
- The enhanced sensitivity and visual output of CRISPR-Cas12a have significant diagnostic and therapeutic implications for cancer management.
- Further validation in large, prospective clinical studies is warranted to establish the full clinical utility of CRISPR-Cas12a for KRAS mutation testing.
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