dcas9  (Addgene inc)

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

Article Snippet: The doxycycline-inducible dCas9-DNMT3A AML12 cell line was constructed by transfecting wild-type AML12 cells with a piggyBac transposase constructs 137 and 138-dCas9-DNMT3a (Addgene 84570) and selecting with hygromycin for 7 days.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic of designed sgRNAs targeting the Pcsk9 promoter and Pyro-seq assay area in yellow. B Pcsk9 expression in AML12 cells after DNA methylation editing by dCas9-DNMT3A with sgRNAs in ( A ). ( n = 5 biological replicates per group, one way ANOVA (Dunnett’s test vs Scr), sgRNA-2 vs Scr P = 0.0058, sgRNA-1 + 2 vs Scr P = 0.00147). C DNA methylation status of the Pcsk9 promoter after DNA methylation editing by dCas9-DNMT3A in AML12 cells measured by pyrosequencing. Pcsk9 sgRNAs refers to editing with sgRNAs 1 + 2. ( n = 3 biological replicates per group, one-way ANOVA with Tukey’s multiple comparisons per CpG, * P < 0.05, exact P -values provided in source data). D Scheme for in vivo repression of Pcsk9 by targeted methylation of the Pcsk9 promoter in the liver. Created in BioRender. Liu, S. (2025) https://BioRender.com/m5iikln . E DNA methylation status of the Pcsk9 promoter after 6 weeks from liver tissue isolated from dCas9-DNMT3A mice injected with AAV containing Cre and Scr or Pcsk9 targeting sgRNAs measured by pyrosequencing of area ( A and B ). ( n = 3 (PBS), n = 9 (Scr), n = 10 ( Pcsk9 ) mice, one-way ANOVA (Tukey’s test exact P values in source data). F Pcsk9 transcript expression in livers from ( D ). ( n = 5 (Scr) and n = 6 ( Pcsk9 ) mice, two-sided t test, P = 0.0292). G Western blot of LDLR and PCSK9 protein expression levels in liver tissue in ( D ). H Quantification of LDLR and PCSK9 protein levels from western blot in ( G ). ( n = 6 (Scr) and n = 7 ( Pcsk9 ) mice, two-sided t test, LDLR P = 0.0292, PCSK9 P = 0.040). I Representative immunofluorescent staining of LDLR in livers from Scr and Pcsk9 targeted livers. Scale bar: 50 μm. J Quantification of % area stained of LDLR in livers from Scr and Pcsk9 targeted livers. ( n = 4 mice per group, two-sided t test, P = 0.0093). K Serum LDL cholesterol measured biweekly in dCas9-DNMT3A mice injected with Scr or Pcsk9 targeting sgRNAs. ( n = 9 mice per group, two-way ANOVA repeated measures, P = 0.000021). L Quantification of serum PCSK9 levels at study endpoint from Scr and Pcsk9 targeted livers. ( n = 5 (Scr) and n = 6 ( Pcsk9 ) mice, two-sided t test, Absolute p = 0.0032, Relative p = 0.00078).

Article Snippet: The doxycycline-inducible dCas9-DNMT3A AML12 cell line was constructed by transfecting wild-type AML12 cells with a piggyBac transposase constructs 137 and 138-dCas9-DNMT3a (Addgene 84570) and selecting with hygromycin for 7 days.

Techniques: Expressing, DNA Methylation Assay, In Vivo, Methylation, Isolation, Injection, Western Blot, Staining

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A IGV browser track of anti-Cas9 ChIP-seq data demonstrating the specific binding of dCas9-DNMT3A to the Pcsk9 promoter in DNA methylation edited AML cells. B Representative IGV track of WGBS data of Scr and Pcsk9 sgRNA injected livers at the Pcsk9 locus. The differentially methylated region (DMR) at the Pcsk9 promoter is highlighted in red. C Quantification of average methylation at the DMR identified at the Pcsk9 promoter in Scr and Pcsk9 targeted livers. D DNA methylation of the dCas9-DNMT3A binding sites in Scr and Pcsk9 targeted livers measured by whole genome bisulfite sequencing (WGBS). Pcsk9 is labeled by a red dot and other sites are labeled by black dots. The dashed lines represent a 20% change of DNA methylation level. E RNA-seq of Scr sgRNA and Pcsk9 sgRNA injected livers. Pcsk9 is labeled by a red dot and other genes associated with dCas9-TET1 binding sites are labeled by black dots. The dashed lines represent a twofold change in expression between the conditions.

Article Snippet: The doxycycline-inducible dCas9-DNMT3A AML12 cell line was constructed by transfecting wild-type AML12 cells with a piggyBac transposase constructs 137 and 138-dCas9-DNMT3a (Addgene 84570) and selecting with hygromycin for 7 days.

Techniques: ChIP-sequencing, Binding Assay, DNA Methylation Assay, Injection, Methylation, Methylation Sequencing, Labeling, RNA Sequencing, Expressing