| Background:Early diagnosis of lung cancer is essential to prolong the patients’life and improve the quality of life.Serum biomarkers and the technologies for detecting gene mutations to early diagnosis of lung cancer still have some limitations because of lower sensitivity and specificity.Consequently,it is necessary to develop some new methods to detect gene mutations with higher sensitivity and specificity for early diagnosis of lung cancer.Objective:To establish a enrichment method for detecting EGFR exon19 deletion mutations of ctDNA in the blood of lung cancer patients.This technology bases on the CRISPR/Cas9 gene editing system to target wide-type fragments and PCR amplification,and then mutant fragments can be detected by Sanger sequencing or next generation sequencing.The technology can increase the detection sensitivity of mutant sites of peripheral blood in lung cancer patients.Method:The transcription template of sgRNA was synthesized by overlap PCR technology,which utilized four overlapping primers and connected adjacent fragments with overlapped regions;The targeting efficiency and specificity sgRNA were verified in a reaction mixture that contained Cas9 enzyme、sgRNA、DNA and digested buffer,and then analysing the cleavage efficiency according to the analysis of agarose gel;Wild-type and deletion mutant DNA sequence of EGFR-exon19 were searched on the website of NCBI and COSMIC database,and then constructing the plasmids that harbor wild-type or mutant DNA sequence as the target sites using molecular cloning technology;Various ratios of wild-type and mutant DNA fragments were mixed in 10:1、100:1、1000:1、10000:1、100000:1,optimizing the condition of Cas9/sgRNA digestion and PCR amplication,and then the PCR products were send to sanger sequencing and next generation sequencing to evaluate the enrichment effectiveness;Furthermore,two samples from patients with NSCLC and two samples from healthy controls were collected,using the established method to detect whether the patients generate EGFRexon19 deletion mutations.Results:We successfully established a method of synthesizing sgRNA transcript template without amplification template,only using four overlapping primers to extend each primer,and we verified the sgRNA with Cas9 enzymen could target DNA fragment in high efficiency.We also constructed wild-type and nine plasmids harboring different deletion mutation fragments according to website of NCBI and COSMIC database.The result of in vitro cleavage assay showed that wild-type DNA sequence was digested efficiently by Cas9/sgRNA complex and showed low cleavage efficiency toward all mutant fragments,indicating the high specificity and target efficiency of sgRNA.We established the detected method that utilized Cas9 enzyme to selectively target wild-type fragments and further mutant type was amplified by PCR technology.Different ratios of wide and mutant-type were detected by the method and the result of Sanger sequencing showed that a low concentration of mutant DNA fragments(0.01%)could be detected after enrichment.The result of NGS showed a thousand-folds signal was increased after enrichment.We further detected cfDNA of plasma from two patients with non-small cell lung cancer and two healthy people.The result of Sanger sequencing indicated that the two patients occurred EGFR exon 19 deletion mutations and the result of NGS indicated a thousand-folds signal was increased after enrichment.Conclusion:In this study,we established a rapid method of synthesizing the transcription template of sgRNA by four overlapping primers and verified the efficiency of sgRNA.We established a detected technology to enrich EGFR deletion mutation fragments in blood that could increase the sensitivity of diagnosis combining Cas9/sgRNA system with DNA polymerase.The method established in the study has the potential application value in the early diagnosis of lung cancer and the guidance to target drug. |
