fuw dcas9 plasmid  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole ( Cynoglossus semilaevis ) Testis Cells

doi: 10.3390/ijms25147637

Figure Lengend Snippet: Schematic overview of the CRISPR/dCas9- dnmt3a editing system for the emx2 promoter region of Chinese tongue sole testis cells. ( A ) Edit system schematic diagram. ( B ) dCas9- dnmt3a plasmid structure diagram and sgRNA plasmid structure diagram. ( C ) The sequence of the sgRNAs. ( D ) Bisulfite sequencing positions and sgRNA sites of emx2 ; lollipop chart on the left represents bisulfite sequencing of the CpGs’ position. The arrows indicate the target sequence of the designed sgRNAs, with the direction of the arrows aligning with the target sequence.

Article Snippet: The dnmt3a sequence and the fuw-dCas9 plasmid (Addgene #84476, Watertown, MA, USA) were digested with HamI (NEB) and EcoRI (NEB) enzymes at 37 °C for 2 h, respectively.

Techniques: CRISPR, Plasmid Preparation, Sequencing, Methylation Sequencing

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole ( Cynoglossus semilaevis ) Testis Cells

doi: 10.3390/ijms25147637

Figure Lengend Snippet: CRISPR/dCas9-mediated DNA methylation editing on emx2 gene. DNA methylation status of the emx2 promoter region with the ( A ) blank control; ( B ) dCas9- dnmt3a ; ( C ) dCas9- dnmt3a and sgRNA1; ( D ) dCas9- dnmt3a and sgRNA2; ( E ) dCas9- dnmt3a and sgRNA3; ( F ) dCas9- dnmt3a and sgRNA4; ( G ) dCas9- dnmt3a and sgRNA5; ( H ) dCas9- dnmt3a and sgRNA6; ( I ) dCas9- dnmt3a and sgRNA1+3; ( J ) Percentage of DNA methylation levels at emx2 promoter region with different treatments; ( K ) Relative expression of emx2 after transfection of dCas9- dnmt3a and different sgRNAs. Filled (black) circles correspond to methylated CpGs, unfilled (white) circles correspond to unmethylated CpGs. Upper black lines indicate statistical difference among blank control and sgRNA groups, while lines below the graph highlight that the sgRNAs were co-transfected with the CRISPR/dCas9- dnmt3a vector. The asterisks (*) are used to denote significant differences among groups using two-way ANOVA and Tukey’s multiple comparisons. *** p < 0.001, **** p < 0.0001.

Article Snippet: The dnmt3a sequence and the fuw-dCas9 plasmid (Addgene #84476, Watertown, MA, USA) were digested with HamI (NEB) and EcoRI (NEB) enzymes at 37 °C for 2 h, respectively.

Techniques: CRISPR, DNA Methylation Assay, Control, Expressing, Transfection, Methylation, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole ( Cynoglossus semilaevis ) Testis Cells

doi: 10.3390/ijms25147637

Figure Lengend Snippet: Off-target effects of CRISPR/dCas9- dnmt3a system. DNA methylation of the promoter region of ( A ) slc20a2 ; ( B ) ddx17 ; ( C ) katb6 and ( D ) xpa and relative expression of potential off-target genes ( E ) slc20a2 ; ( F ) ddx17 ; ( G ) katb6 and ( H ) xpa . Upper black lines indicate statistical difference between the blank control and sgRNA3 groups, while lines below the graph highlight that the sgRNAs were co-transfected with the CRISPR/dCas9- dnmt3a vector. The asterisks (*) are used to denote significant differences between groups using two-way ANOVA and Tukey’s multiple comparisons. *** p < 0.001.

Article Snippet: The dnmt3a sequence and the fuw-dCas9 plasmid (Addgene #84476, Watertown, MA, USA) were digested with HamI (NEB) and EcoRI (NEB) enzymes at 37 °C for 2 h, respectively.

Techniques: CRISPR, DNA Methylation Assay, Expressing, Control, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole ( Cynoglossus semilaevis ) Testis Cells

doi: 10.3390/ijms25147637

Figure Lengend Snippet: Effects on downstream genes after the emx2 promoter region methylated. Relative expression of downstream genes ( A ) myc , ( B ) klf4 , and ( C ) wnt1 . Upper black lines indicate statistical difference among blank control and sgRNA groups for myc expression, while lines below the graph highlight that the sgRNAs were co-transfected with the CRISPR/dCas9- dnmt3a vector. The asterisks (*) are used to denote significant differences between groups using two-way ANOVA and Tukey’s multiple comparisons. *** p < 0.001.

Article Snippet: The dnmt3a sequence and the fuw-dCas9 plasmid (Addgene #84476, Watertown, MA, USA) were digested with HamI (NEB) and EcoRI (NEB) enzymes at 37 °C for 2 h, respectively.

Techniques: Methylation, Expressing, Control, Transfection, CRISPR, Plasmid Preparation