Screening And Efficiency Verification Of CRISPR/Cas9 Single Guide RNA Against Human Tumor Suppressor Gene P53 And PTEN

Background and objective:Tumors have always been one of the main threats to human health and human life.With the advancement of clinical medicine in recent years,more and more methods for treatment of tumors have emerged,and each method needs a large number of animal experiments to verify its effectiveness and safety before being applied to the clinic.The early construction of animal tumor models was mainly induced by physical and chemical factors,but there was a big difference between the occurrence and development of human tumors.Therefore,more researches are currently focused on the methods of transplanting primary tumor cells or inducing intracellular gene mutations to make experimental animals sick by biotechnological means.To explore the strategies to edit human p53 and PTEN,two famous tumor suppressor genes,by CRISPR-Cas9 technology and to evaluate the editing efficiency in vitro,and to provide experimental study tools for transforming primary healthy cells into malignant tumor cells and establishment of humanized mouse models with human oncogenesis in vivo.Methods:Single-guide RNA(sgRNA)sequences were designed to target the common exon regions of p53 and PTEN mRNA isoforms based on software analysis,that could predict their gene editing efficiency.The sgRNAs with high scores were selected and cloned into Cas9-P2A-GFP plasmid to construct sgRNA-Cas9-P2A-GFP vector that co-expressed sgRNA,Cas9 and GFP.The 293 T cells in logarithmic growth phase were transfected with the sgRNA-Cas9-P2A-GFP vector(experimental group)or PBS(control group)by Lipo-fectamine2000.After two-week expansion,the GFP-positive 293 T cells were purified by flow cytometric sorter,whose genomic DNA was extracted for further analysis.The DNA fragments containing the sgRNA targeting site were amplified from the extracted genomic DNA by PCR and purified by gel extraction.Then,they were linked into the pEASY-Blunt Zero cloning vector and transformed into competent E.coli cells.The single colonies formed by pEASY-Blunt Zero vector transformed cells were used to extract the plasmid for DNA sequencing.And the sequencing results of control group and experimental group were compared to judge the gene editing efficiency.Results:Over 80% of the sgRNA-Cas9-P2A-GFP transfected cells were found to express GFP gene after flow sorting in the experimental group,which was significantly higher than that of the pre-sorted cells(P <0.05).Genomic DNA was extracted from the sorted cells after expansion and used as PCR template.The results of sequencing showed that the efficiency of editing induced by p53-1,p53-2 and PTEN-2 sgRNA were 54.5%,45.5% and 33.3%,respectively.Conclusion:Based on CRISPR-Cas9,the sgRNAs we designed,p53-1,p53-2 and PTEN-2,can successfully and site-specifically guide Cas9-mediated genome editing with high efficiency at the genome level.