RESUMEN
Objective: To explore the feassibility to edit human p53 and PTEN, two tumor suppressor genes, by CRISPR-Cas9 technology and to evaluate the editing efficiency in vitro, and to provide the experimental study tools for transforming primary healthy cells into malignant tumor cells and establishment of humanized mouse models with human oncogenesis in vivo Methods: The smgle-gmde 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 plasrmd to construct sgRNA-Cas9-P2A-GFP vector that co-expressed sgRNA, Cas9 and GFP. The 293T cells in logarithmic growth phase were transfected with the sgRNA-Cas9-P2A-GFP vector (experimental group) or PBS (control group) by Lipofectamine 2000. After two-week expansion, the GFP-positive 293T 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 82% of the sgRNA-Cas9-P2A-GFP transfected cells were found to express GFP gene after flow sorting in 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 length of the amplified fragments containing the p53 mutation site was 612 bp, while the lengths of the amplified fragments containing the PTEN-1/PTEN-2 mutation site were 667 and 947 bp. The results of sequencing showed that the efficiency of editing induced by p53-l, p53-2 and PTEN-2 sgRNA were 54.5%, 45.5% and 33.3%, respectively. Conclusion: The sgRNAs p53-1, p53-2 and PTEN-2 designed for p53 and PTEN can successfully guide Cas9-mediated site-specific genome editing with high efficiency at the genome level.
RESUMEN
Objective:To explore the feassibility to edit human p53 and PTEN,two tumor suppressor genes,by CRISPR-Cas9 technology and to evaluate the editing efficiency in vitro,and to provide the experimental study tools for transforming primary healthy cells into malignant tumor cells and establishment of humanized mouse models with human oncogenesis in vivo.Methods:The 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 293T cells in logarithmic growth phase were transfected with the sgRNA-Cas9-P2A-GFP vector(experimental group)or PBS(control group)by Lipofectamine 2000.After two-week expansion,the GFP-positive 293T 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 82% of the sgRNA-Cas9-P2A-GFP transfected cells were found to express GFP gene after flow sorting in 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 length of the amplified fragments containing the p53 mutation site was 612 bp,while the lengths of the amplified fragments containing the PTEN-1/PTEN-2 mutation site were 667 and 947 bp.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:The sgRNAs p53-1,p53-2 and PTEN-2 designed for p53 and PTEN can successfully guide Cas9-mediated site-specific genome editing with high efficiency at the genome level.