Construction Of A Stable Knockout Cell Line Of NLRP3 Gene Based On CRISPR/Cas9 Technology

CRISPR/Cas9 technology is a genetic editing technology modified from the adaptive immune system CRISPR/Cas of bacteria and archaea.This technology has impacted various fields of life science research.CRISPR/Cas9 technology relies on sgRNA and Cas9 to edit target genes,which is easy to operate,short in experimental period and widely used.Mastering CRISPR/Cas9 technology can provide an efficient and powerful tool for future scientific research.NLRP3 inflammatory corpuscle is an important component of the immune system.In addition to inflammatory diseases,studies have shown that NLRP3 inflammatory bodies affect the occurrence and development of cancer.There have been studies using RNAi technology to demonstrate that NLRP3 inflammatory bodies are associated with breast cancer proliferation,invasion and metastasis,but due to the limitations of RNAi technology,the mechanism of NLRP3 inflammatory bodies associated with breast cancer has not been elucidated.In this study,CRISPR/Cas9 technology was used to construct MDA-MB-231 and MCF-7 cell lines stably knocked out by NLRP3 gene to study the relationship between NLRP3 inflammatory bodies and breast cancer cells.At the same time,HEK-293 T cells are commonly used tool cells,construct NLRP3 knockout HEK-293 T cell line can deeply study the operation of CRISPR/Cas9 technology and lay the foundation for studying the relationship between NLRP3 inflammatory bodies and HEK-293 T cells.In this study,NCBI,Uniprot website and sgRNA sequence design website(http//crispr.mit.edu./)were used to design sgRNA,and four CRISPR/Cas9 recombinant plasmids were constructed by selecting four sgRNAs.The CRISPR/Cas9 system used in this experiment is a PX459 plasmid containing a puromycin resistance marker.The minimum screening concentration of puromycin in MDA-MB-231,MCF-7,and HEK-293 T cells was determined before cell transfection.The minimum concentration of puromycin in MDA-MB-231 was 0.6 ?g/mL,the concentration of the lowest puromycin in MCF-7 was 1.5 ?g/mL,and the concentration of the lowest puromycin in HEK-293 T was 1.5 ?g/mL..The CRISPR/Cas9 recombinant plasmid was transfected into cells,and the successfully transfected cells were screened by puromycin medium and verified by T7E1 digestion.It was revealed that MDA-MB-231 and HEK-293 T mixed cloned cells with genetically modified cells were obtained.The obtained MDA-MB-231 and HEK-293 T mixed clonal cells were cultured in a single cell by limiting dilution method and colony formation method,and finally 35 monoclonal cell lines were obtained from MDA-MB-231 cells,and 2 single cells were identified.The cloned cell line produced a mutation in the NLRP3 gene,and a monoclonal cell line was obtained from MCF-7 cells.No mutation was identified.HEK-293 T cells obtained 23 monoclonal cell lines,including 12 monoclonal cells.The strain produced a gene mutation,and eight monoclonal cell lines developed a frameshift mutation.In this experiment,MDA-MB-231 and HEK-293 T cell lines with NLRP3 gene mutation were successfully obtained.At the same time,MDA-MB-231,MCF-7 and HEK-293 T cells were transfected with CRISPR/Cas9 plasmid PX458 plasmid with PX459 plasmid structure and size but containing EGFP marker,and the fluorescence intensity of three cells was compared.It can be seen that the transfection efficiency of HEK-293 T is much higher than that of MDA-MB-231 and MCF-7 cells.In this study,we compared the transfection efficiency of MDA-MB-231,MCF-7,and HEK-293 T cells with the obtained ratio of the obtained monoclonal antibody strain of NLRP3 gene mutation,and speculated that the higher the transfection efficiency,the greater the possibility of obtaining a genetically modified cell line.This provides research ideas for other researchers who will use CRISPR/Cas9 technology for scientific research in the future.In summary,this paper used CRISPR/Cas9 technology to construct MDA-MB-231 and HEK-293 T cell lines with NLRP3 gene mutations.Eight stable NLRP3 knockout cell lines were obtained from HEK-293 T cells.This laid the foundation for the future study of the relationship between NLRP3 inflammatory bodies and MDA-MB-231,HEK-293 T cells.