Construction of Rho GDIa-sgRNAs plasmids by clustered regularly interspaced short palindromic repeats/associated protein 9 and the effect on migration of Hepa 1-6 cell line in mouse / 解剖学报

Objective To construct the recombinant plasmids of knocking down Rho guanine dissociation inhibitor α (GDIα ) gene by using clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) technique, and investigate the effect of Rho GDIα interference on the migration of Hepa 1-6 cells of mouse in order to provide the method of prevention and treatment of liver cancer. Methods To construct and identify the PX458-Rho GDIα-single guide (sg) RNAs by using CRISPR/Cas9 technique. And the Hepa 1-6 cells were transfected by liposomes with PX458-Rho GDIα-sgRNAs for 48 hours respectively, and cells treated with PX458 plasmids were used as control. The migration ability of Hepa 1-6 was checked by wound healing assay and Transwell assay, respectively. Results The expression of Rho GDIα was depressed in group of PX458-Rho GDIα-sgRNAl transfection which was detected by using RT-PCR. The migration distance of Hepa 1-6 in PX458-Rho GDIα-sgRNAl transfection group was significantly promoted comparing with the control group which was transfected with PX458 only, and the cell number of PX458-Rho GDIα-sgRNAl group was more than that in control group by using transwell assay, indicating concluded that knocking down of Rho GDIα promoted the migration ability of Hepal-6 cells. Conclusion The result is explicit that in vivo, Rho GDIα may inhibit the migration of Hepal-6 partially. Overexpression of Rho GDIα might be used as an important method to prevent the metastasize of carcinoma.

Construction of forkhead box J2 gene knockout plasmids by CRISPR / Cas9 and the effects on expression of transforming growth factor￣β/ Smads and proliferation in hepatocellular carcinoma / 解剖学报

Objective To construct the clustered regularly interspaced short palindromic repeats / associated protein 9 (CRISPR/ Cas9) plasmid targeting forkhead box J2 (FOXJ2) gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β) / Smads and proliferation in hepatocellular carcinoma cells of mouse. Methods Small guide RNA(sgRNA) sequence of FOXJ2 was designed, linked with PX458 vector and transfected into competent E. coli for proliferation. The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence. The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection, respectively. The empty vectors of PX458 were transfected as control group. After 48 hours, the expression of FOXJ2, TGF-β and Smads were obtained by RT-PCR and agarose gel electrophoresis, respectively. The cell proliferation was detected by methylthio tetrazole (MTT) method . Results The CRISPR/ Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed. The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β, Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only. And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group. Conclusion In hepatocellular carcinoma cells of mouse, FOXJ2 gene inhibits the expression of TGF-β, Smad2, Smad4 and cell proliferation partially, which indicates the relationship between FOXJ2 and TGF-β signal pathway. The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

Screening and efficiency verification of CRISPR-Cas9 single-guide RNA against human tumor suppressor gene p53 and PTEN / 吉林大学学报(医学版)

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.

Screening and efficiency verification of CRISPR-Cas9 single-guide RNA against human tumor suppressor gene p53 and PTEN / 吉林大学学报(医学版)

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.

Establishment and verification of a mouse model of gene H435Y mutation / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a mouse model of H435Y mutation of gene using CRISPR/Cas9- mediated gene targeting.</p><p><b>METHODS</b>The single-stranded guide RNA (sgRNA) specific to the H435Y loci of gene was designed based on the sequence of gene. After activity assessment, the active sgRNA and Cas9 were transcribed into RNA and microinjected along with the donor DNA fragment with point mutations into fertilized mouse eggs. The microinjected eggs were transferred into pseudopregnant mice to obtain the F0 generation mice with the target gene mutation confirmed by PCR and gene sequencing. gene mutations in the offsprings of the F0 generation mice were analyzed.</p><p><b>RESULTS</b>Gene sequencing confirmed the successful establishment of mouse models carrying H435Y mutation of gene in 4 of the F0 generation mice. The positive F0 generation mice were crossed with wild-type C57BL/6J mice to obtain the F1 generation mice, and PCR confirmed the presence of H435Y mutations of gene in 6 of the F1 mice. Then F2 generation mice were obtained by F1 generation matting with each other. PCR showed that H435Y mutation of gene in F2 mice was found, indicating the mousemodel of gene mutation in H435Y was established and propagated successfully.</p><p><b>CONCLUSIONS</b>We successfully established gene H435Y mutant mouse models using CRISPR/Cas9 technique.</p>