Study Of Quality Control Materials Based On CRISPR/Cas9 Technology For BRAF V600E Mutation

Objective:BRAF V600E mutation is closely related to the occurrence and development of cancer,such as colorectal cancer,malignant melanoma and thyroid papillary carcinoma.BRAF V600E mutations in metastatic colorectal cancer suggest the resistance of EGFR inhibitors.BRAF V600E mutations in patients with metastatic melanoma and non-small cell lung cancer can guide the use of BRAF inhibitors.Therefore,the accurate detection of BRAF V600E mutation is essential for the diagnosis of related diseases and the guidance of targeted therapy.However,many factors such as sensitivity of the detection method,sample type and so on may affect the consistency and accuracy of laboratory results.So,it is imperative to establish a complete internal quality control and external quality assessment system.However,there are no related quality control material,especially which can be used for different types of sample and detection methods.In this study,we using the CRISPR/Cas9 gene editing system generated a BRAF V600E mutation positive cell line that can be suitable for the quality control of different types of sample.Methods:CRISPR/Cas9 is a convenient and efficient gene editing technique developed in recent years.3 different groups of sgRNA were designed and were linked with pSpCas9(BB)-2A-GFP plasmids respectively to form expression vectors.The HEK293T cells were co-transfected with expression vectors and HR template.The cleavageefficiencies of 3 different groups of sgRNAs were evaluated by the T7EI assay.The BRAF V600E mutations were detected by ARMS-PCR.Single cells were sorted by flow cytometry and then were cultured.The screened BRAF V600E mutation positive cell lines were confirmed by Sanger sequencing.Results:The recombinant plasmids used to transfect HEK293T cells were successfully constructed.The designed 3 groups of sgRNA showed high transfection efficiency and cleavage activity.Finally,a BRAF V600E mutant positive cell line was screened and confirmed.Conclusions:In this study,using the CRISPR/Cas9 technology,we generated a novel BRAF V600E cell line that could be suitable as a quality control material for differences type of sample.It provides a new direction and method for the preparation of quality control material.