Rapid and ultra-sensitive early detection of cervical cancer using CRISPR/Cas12-based assay based on methylated SEPT9

Cervical cancer is the fourth most common tumor in women with high mortalities. Early diagnosis can improve the survival rate. Nevertheless, there is still a lack of specific biomarkers and efficient methods for the diagnosis of cervical cancer with excellent sensitivity and specificity. In this work, we identified the methylated SEPT9 (mSEPT9) as a specific biomarker for the diagnosis of cervical cancer. We developed a novel method using methylation-sensitive restriction endonuclease (MSRE) combined with recombinase polymerase amplification (RPA)-based CRISPR/Cas12a system (MeCRISPR) for rapid and ultrasensitive detection of mSEPT9. We demonstrated the MeCRISPR enabled to detect 1 copy/μL of mSEPT9 and distinguishes 0.01% mSEPT9 from large amounts of gDNA. This method could be completed in 60 min without any complicated instruments, which is favorable for point-of-care testing (POCT). We applied MeCRISPR to detect mSEPT9 in clinical cervical cancer patients. The sensitivity and specificity were 100% and 92.3% respectively. Our results demonstrate that mSEPT9 is a promising biomarker for cervical cancer. The MeCRISPR is a sensitive and simple method to analyze the methylated gene which holds great potential for screening cancer in a low-resource setting.