px330a dcas9 lsd1 vector  (Addgene inc)

Journal: Metabolic engineering

Article Title: Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells.

doi: 10.1016/j.ymben.2021.04.014

Figure Lengend Snippet: Fig. 2. Effect of dCas9-DNMT3A on FUT8, ST6GAL1 and BFP expression and the effect of CMV promoter deletion in CHO–K1_BFP cells. A) Efficiency of FUT8 knock- down after targeting dCas9-DNMT3A with different gRNA mixes (Supplementary Table 2) to the FUT8 promoter. (Bonferroni’s test, n = 3, mean ± SD) analyzed by lectin staining ten days after transfection. B) ST6GAL1 knock-down by dCas9-DNMT3A and mixed gRNAs (gRNAs 2, 4 and 6) analyzed by lectin staining ten days after transfection (Bonferroni’s test, n = 3, mean ± SD). C) BFP expression of CHO–K1_BFP cells analyzed by flow cytometry after targeting the CMV promoter with dCas9-DNMT3A and mixed gRNAs (gRNAs 1–5) (Dunnet’s test, n = 3, mean ± SD). D) BFP expression of CHO–K1_BFP cells after deletion of the CMV promoter with active Cas9 and different paired gRNAs (Bonferroni’s test, n = 3, mean ± SD) ten days after transfection. Expression levels were compared to the parental cell line (wt). E) Illustration of the SunTag-dCas9-complex. The dCas9 is fused to a GCN4 peptide array that serves as an antigen of a single chain variable fragment (scFv) coupled to an effector domain (ED). Targeting EDs to promoter regions can result in transcriptional activation or repression.

Article Snippet: pSPgRNA was a gift from Charles Gersbach, pCAG-dCas95xPlat2AflD and pCAG-scFvGCN4sfGFPTET1CD were gifts from Izuho Hatada, pdCas9-DNMT3A-EGFP was a gift from Vlatka Zoldoš, pSpCas9 (BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138), pET28-Dnmt3a3L-sc27 was a gift from Albert Jeltsch (Addgene plasmid # 71827), pHR-SFFV-dCas9-BFP-KRAB was a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid # 46911), pX330a dCas9- LSD1 was a gift from Tatjana Sauka-Spengler (Addgene plasmid # 92362).

Techniques: Expressing, Knockdown, Staining, Transfection, Flow Cytometry, Peptide Microarray, Activation Assay

Journal: Metabolic engineering

Article Title: Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells.

doi: 10.1016/j.ymben.2021.04.014

Figure Lengend Snippet: Fig. 3. Effect of dCas9-DNMT3A3L on FUT8 and ST6GAL1 expression and methylation after targeting the respective promoters. A) - D) and G) Percentage of expression based on lectin staining of FUT8 (Dyelight-649-LCA, red bars) and ST6GAL1 (Cy5-SNA, purple bars) and BFP flow cytometric analysis four, ten and 21 days after transfection with dCas9 and different effector domains. Expression levels are compared to the parental cell line (wt) (Dunnet’s test, n = 3; mean ± SD). On the right, results from CHO–K1_BFP cells targeted with CMVgRNAmix (B), targeted with BFPgRNAmix (D) and targeted with individual gRNAs against the CMV promoter or BFP gene (G) are shown. E) FUT8 fold change in methylation ten days after transfection as determined by MSR-qPCR after HpaII-digest of gDNA. Compared to wt gDNA (Dunnet’s test, n = 3; mean ± SD). F) Fold change in FUT8 mRNA expression of FUT8 promoter targeted cells on day 10 after transfection (Student’s t-test, n = 3; mean ± SD) H) Schematic representation of different effector domains used and their epigenetic function. The applied EDs and the respective amino acid residues are displayed.

Article Snippet: pSPgRNA was a gift from Charles Gersbach, pCAG-dCas95xPlat2AflD and pCAG-scFvGCN4sfGFPTET1CD were gifts from Izuho Hatada, pdCas9-DNMT3A-EGFP was a gift from Vlatka Zoldoš, pSpCas9 (BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138), pET28-Dnmt3a3L-sc27 was a gift from Albert Jeltsch (Addgene plasmid # 71827), pHR-SFFV-dCas9-BFP-KRAB was a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid # 46911), pX330a dCas9- LSD1 was a gift from Tatjana Sauka-Spengler (Addgene plasmid # 92362).

Techniques: Expressing, Methylation, Staining, Transfection

Journal: Metabolic engineering

Article Title: Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells.

doi: 10.1016/j.ymben.2021.04.014

Figure Lengend Snippet: Fig. 4. FACS and analysis of cell pools with active and silenced genes. A) Responding and non-responding cell populations to dCas9-treatment with either DNMT3A3L (FUT8 and CMV) or TET1 (ST6GAL1) were sorted 10 days after transfections. ST6GAL1 expressing populations were sorted from CHO-DuxB11_ST6 cell pools (purple) and compared to the natively non-expressing parental cell line CHO-DuxB11 (ST6_parental). Above: histograms before sorting with the applied sorting gates. Below: Cell populations analyzed by flow cytometry 14 days after cell sorting. B)-D) Fold change in (FUT8 and BFP) or relative (ST6GAL1) mRNA expression levels of the respective gene in sorted pools and parental cell lines of ST6GAL1, FUT8 and BFP. E) Fold change in methylation of CpG −179 within the CMV promoter sequence of CHO–K1_BFP cell pools (n = 3). gDNA was digested with the SsiI restriction enzyme. Samples for analysis in B)-E) were taken 16 days after sorting and 26 days post-transfection. ST6_pos expression was compared to ST6_parental, F8_neg to F8_pos and CMV_neg to CMV_pos (Student’s t-test,n = 3; mean ± SD).

Article Snippet: pSPgRNA was a gift from Charles Gersbach, pCAG-dCas95xPlat2AflD and pCAG-scFvGCN4sfGFPTET1CD were gifts from Izuho Hatada, pdCas9-DNMT3A-EGFP was a gift from Vlatka Zoldoš, pSpCas9 (BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138), pET28-Dnmt3a3L-sc27 was a gift from Albert Jeltsch (Addgene plasmid # 71827), pHR-SFFV-dCas9-BFP-KRAB was a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid # 46911), pX330a dCas9- LSD1 was a gift from Tatjana Sauka-Spengler (Addgene plasmid # 92362).

Techniques: Transfection, Expressing, Flow Cytometry, FACS, Methylation, Sequencing

Journal: Metabolic engineering

Article Title: Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells.

doi: 10.1016/j.ymben.2021.04.014

Figure Lengend Snippet: Fig. 5. Percent input of ChIP samples after immunoprecipitation of CHO–K1_F8, CHO-DuxB11_ST6, and CHO–K1_BFP cell pools. ChIP qPCR data of the respective promoters: A) Enrichment at the ST6GAL1 promoter of the parental non-expressing population and the ST6GAL1 activated population by targeted demethylation. B) Enrichment at the FUT8 promoter of the parental expressing population, the sorted expressing (F8_pos) and sorted non-expressing (F8_neg) population after dCas9- DNMT3A3L treatment. C) Enrichment at the CMV promoter of the parental expressing population, the sorted expressing (CMV_pos) and sorted non-expressing (CMV_neg) population after targeting dCas9-DNMT3A3L to the CMV promoter. D) Enrichment at the CMV promoter of cell populations after dCas9-DNMT3A3L targeting with a mix of BFP gRNAs (1g–3g) or a single gRNA (BFP gRNA 1g) to the BFP gene. (Student’s t-test, n.s. = not significant, n = 3, mean ± SD; for H3K9me3 of CMV_pos and CMV_neg and data from D: n = 2; mean ± SD).

Article Snippet: pSPgRNA was a gift from Charles Gersbach, pCAG-dCas95xPlat2AflD and pCAG-scFvGCN4sfGFPTET1CD were gifts from Izuho Hatada, pdCas9-DNMT3A-EGFP was a gift from Vlatka Zoldoš, pSpCas9 (BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138), pET28-Dnmt3a3L-sc27 was a gift from Albert Jeltsch (Addgene plasmid # 71827), pHR-SFFV-dCas9-BFP-KRAB was a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid # 46911), pX330a dCas9- LSD1 was a gift from Tatjana Sauka-Spengler (Addgene plasmid # 92362).

Techniques: Immunoprecipitation, ChIP-qPCR, Expressing