Knockout Of NODAL Gene In MDA-MB-231 Breast Cancer Cell Line By CRISPR/Cas9 Technology

Objective: Breast cancer is the most common disease in women and the mortality rate ranks the second place.Beyond that,the incidence of breast cancer in China is increasing year by year.Thus,the research on the pathogenesis of breast cancer is particularly important.As a cytokine,NODAL protein plays an important role in facilitating the undifferentiation of stem cells,in taking shape of the left-right asymmetry and so on.Categorized into embryonic Protein,NODAL is not expressed usually in normal tissues.However,recent studies have found that the high expression of NODAL in a few types of cancer cells seems to be closely bound up with the occurrence and development of tumors.But it is still not well understood for the detailed molecular mechanism on tumor promotion of NODAL molecules.Given this situation,this research intends to knock out the NODAL genes of triple-negative breast cancer cell line(MDA-MB-231)through gene-editing technique known as CRISPR/Cas9 and establish a cell model for NODAL genes knockout.We aim to lay a foundation for the further demonstration of the molecular mechanism of NODAL genes that functions in breast cancer cells.In addition,we also look forward to building a gene-editing technique platform through this research.Methods:1.Designed a gene knockout planTarget cell lines were determined based on the relevant literatures and preliminary experimental results,and then the target sites of NODAL gene were determined by bioinformatics analysis of the structural characteristics of NODAL gene and related literature analysis.This research formulated two designs for NODAL genes knockout,of which Plan A tended to completely knock out the three exons of NODAL genes and Plan B was focused on working on the second exon of them.2.Constructed Cas9/sgRNA plasmidsThe target sites of the two schemes were verified by PCR amplification and sequencing.In accordance with the target sites determined by the knockout designs and sgRNA(single guide RNA)design principle,7 sgRNAs on the exon 1 and the same amount on the exon 3 were put in place in Plan A.When it cames to Plan B,7 sgRNAs and 4 sgRNAs were respectively arranged on the 5'as well as the 3'end of the exon 2 of NODAL genes.Double-stranded sgRNA oligo was obtained through denaturation-annealing process,which will be linked with the plasmid of CRISPR that contains Cas9 genes after restriction enzyme digestion.The construction of Cas9/sgRNA plasmid was successfully constructed by enzyme digestion and sequencing.With the activity detection of the established Cas9/sgRNA plasmids through SSA(Single Strand Annealing)Reporter Assay,Cas9/sgRNA plasmids with the best activity and strongest specificity were to be selected.3.Constructed targeting plasmidsBased on the design principles of target sites and homologous arms of target vectors designed by two sets of knockout schemes,two homologous arms of NODAL genes were cloned in the position of 5' end of the target site upstream as well as 3 ' end downstream respectively by conventional molecular cloning techniques.Then the targeting plasmids were to be constructed with one-step kit.4.Identified genotypes and detected protein expressionAs for the two designs,the targeting plasmids were transferred into MDA-MB-231 cell line by electroporation in combination with two kinds of Cas9/sgRNA plasmids.Then cells were screened by Puromycin and selected monoclonal cells.Later on,multiple pairs of PCR primer were designed to conduct such genotype identifications as homologous recombination,frameshift mutation and large fragment deletion.Combined with gene sequencing,the single-cell clone was used to comprehensively determine whether the cells with positive knockout of NODAL gene were cloned.Finally,western blot was applied to identify the expression of NODAL protein in monoclonal cell lines.Results:1.Cas9/sgRNA plasmids with high activity and specificity were successfully constructed.The target sites determined by the two knockout schemes were completely consistent with NODAL genes RefSeq sequence in the NCBI database by sequencing and BLAST alignment.Two designs constructed a total of 25 Cas9/sgRNA plasmids,which were well done with the confirmation by sequencing.Next,the activity of Cas9/sgRNA plasmid was detected by SSA Reporter Assay technology.pCS-gRNA1 and pCS-sgRNA9 as well as pCS-sgRNA1 and pCS-sgRNA8 were screened respectively by Plan A and Plan B,all of which were Cas9/sgRNA plasmids with high activity and specificity that acted on the corresponding target sites.2.Targeting vector was successfully constructedHomologous fragments of NODAL gene at both ends were constructed by molecular cloning technology and the target plasmid containing the puromycin gene.It was successfully constructed by DNA sequencing.3.Genotype identificationPCR amplification and sequencing were performed,in plan A,homologous recombination occurred in 4 single-cell clones,and large fragment deletion was detected in 3 of the 4 single-cell clones,with the deletion length of about 8.6kb.Besides,no frame-shift mutation and wild-type fragment were detected in these single-cell clones.While,as for plan B,homologous recombination occurred on 8 single-cell clones,4 of which were found large fragment deletion with a deletion length of about 0.8kb,1 of which was detected frameshift mutation and 4 of which wild-type fragments.4.Identification of protein expressionThe detection results by western blot revealed that there was a lower NODAL protein expression of D3 single-cell clone,the other three single-cell clones were not decreased,but increased to varying degrees in plan A.As for plan B,no NODAL protein expression was found on A4 and B6 single-cell clones.The expression of NODAL protein in B4,F3 and E4 single-cell clones decreased to different degrees,while the expression of NODAL protein in other single-cell clones not significant changes.Conclusions:Taken together,with the help of CRISPR/Cas9 gene-editing technique and the mechanisms of homologous recombination and non-homologous recombination repair after DNA damage,A4 and B6 were finally obtained as single cell clones with knockout genotypes and protein expression identification,so as to further explore the function of NODAL molecule in breast cancer cells.we found that some single-cell clones were identified as positive genotypes,the reason why the expression of NODAL protein increased instead of decreasing remains to be further studied.Finally,through the completion of the two gene knockout schemes in this study,a gene editing technology platform was established for our laboratory.