Journal: bioRxiv

Article Title: MSH2 is not required for either maintenance of DNA methylation or repeat contraction at the FMR1 locus in fragile X syndrome

doi: 10.1101/2024.12.20.629815

Figure Lengend Snippet: (A) A schematic showing the creation of MSH2 null fragile X syndrome human embryonic stem cell lines (FXS hESCs). WCMC 37F FXS ESCs were transfected with plasmids expressing nCas9 and dual guide RNAs targeting MSH2 exon 3. Edited clones were screened using western blot and DNA sequencing, and MSH2 null clones were selected for further study. (B) Western blot showing MSH2 expression in two cell lines each for FXS MSH2 WT and FXS MSH2 KO. β-actin used as loading control. Western blot of all clones assayed is shown in Figure S1. (C) Immunofluorescence imaging for MSH2 (green) shows a strong, nuclear signal in FXS MSH2 WT-2 and the absence of MSH2 in FXS MSH2 KO-2 cells. Scale bars represent 300 µm.

Article Snippet: Gibson assembly ( ) was used to construct the plasmid pMLM3636-Puro-dCas9-TET1 using NEBuilder®HiFi DNA Assembly Master Mix (New England Biolabs, E2621L) and fragments derived from the following plasmids: PuroR from pSpCas9(BB)-2A-Puro (PX459) V2.0, a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ) , dCas9 from Inducible Caspex expression, a gift from Steven Carr & Samuel Myers (Addgene plasmid # 97421; http://n2t.net/addgene:97421 ) , and TET1CD from pPlatTET-gRNA2, a gift from Izuho Hatada (Addgene plasmid # 82559 ; http://n2t.net/addgene:82559 ) ( ) and vector backbone from pMLM3636 (a gift from Keith Joung (Addgene plasmid # 43860 ; http://n2t.net/addgene:43860 ).

Techniques: Transfection, Expressing, Clone Assay, Western Blot, DNA Sequencing, Control, Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: MSH2 is not required for either maintenance of DNA methylation or repeat contraction at the FMR1 locus in fragile X syndrome

doi: 10.1101/2024.12.20.629815

Figure Lengend Snippet: Transient transfection of FXS MSH2 WT-2 and FXS MSH2 KO-2 cell lines with plasmid pMLM3636-Puro-dCas9-TET1-CGG. (A) FMR1 mRNA levels are shown for FXS MSH2 WT-2 and FXS MSH2 KO-2 cells untransfected (UT) and at diderent days post transfection with plasmid encoding dCas9-TET1 and gRNA targeting the CGG repeats. Data shown are an average of two independent experiments and error bars represent standard deviation. The dashed line represents FMR1 mRNA levels in H1 ESCs with typical number of CGG repeats at 94% of GUSB . (B) Methylation-sensitive qPCR results for the FMR1 promoter after dCas9-TET1-CGG 6 CG transfection in FXS MSH2 WT-2 and FXS MSH2 KO-2 cells. GAPDH is used as an unmethylated region for HpaII digestion control. Data shown are an average of two independent experiments and error bars represent standard deviation. ** p = 0.001. (C) CGG repeat PCR on DNA from two diderent transfections (T1 and T2) in FXS MSH2 WT-2 and FXS MSH2 KO-2 cells with plasmid dCas9-TET1-CGG. HpaII digested DNA lanes show amplification of alleles that are fully methylated while undigested samples amplify both methylated and unmethylated alleles. The FMR1 gene in FXS ESCs carries 400 repeats resulting in a PCR product of about 1.4 kb. FXS MSH2 WT-2 and FXS MSH2 KO-2 cells show CGG repeat contractions indicated by discrete bands and smears below 1 kb. FXS MSH2 KO cells show almost complete loss of methylated full- length alleles in both transfection replicates. (D) Small pool PCR on the same DNA sample used for PCRs in panel C shows full length PCR product in untransfected (UT) cells and contracted alleles in both MSH2 WT and MSH2 KO cells transfected with plasmid dCas9- TET1-CGG.

Article Snippet: Gibson assembly ( ) was used to construct the plasmid pMLM3636-Puro-dCas9-TET1 using NEBuilder®HiFi DNA Assembly Master Mix (New England Biolabs, E2621L) and fragments derived from the following plasmids: PuroR from pSpCas9(BB)-2A-Puro (PX459) V2.0, a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ) , dCas9 from Inducible Caspex expression, a gift from Steven Carr & Samuel Myers (Addgene plasmid # 97421; http://n2t.net/addgene:97421 ) , and TET1CD from pPlatTET-gRNA2, a gift from Izuho Hatada (Addgene plasmid # 82559 ; http://n2t.net/addgene:82559 ) ( ) and vector backbone from pMLM3636 (a gift from Keith Joung (Addgene plasmid # 43860 ; http://n2t.net/addgene:43860 ).

Techniques: Transfection, Plasmid Preparation, Standard Deviation, Methylation, Control, Amplification

Journal: bioRxiv

Article Title: MSH2 is not required for either maintenance of DNA methylation or repeat contraction at the FMR1 locus in fragile X syndrome

doi: 10.1101/2024.12.20.629815

Figure Lengend Snippet: Transient transfection of FXS MSH2 WT-2 and FXS MSH2 KO-2 cell lines with plasmid pMLM3636-Puro-dCas9-TET1-PRM and pMLM3636-Puro-dCas9-TET1. (A) FMR1 mRNA levels in FXS MSH2 WT-2 and FXS MSH2 KO-2 cells are shown as a percentage of GUSB mRNA in untransfected (UT) cells and at day 7 (7d) and day 24 (24d) after transient transfections with plasmid dCas9-TET1-PRM with 2 gRNAs directed to the FMR1 promoter or with plasmid dCas9-TET1 with no gRNA. Data shown are an average of two independent experiments and error bars represent standard deviation. (B) Methylation-sensitive qPCR for the FMR1 promoter at day 7 (7d) and day 24 (24d) after transient transfection with plasmids expressing dCas9-TET1 and gRNAs targeted to the FMR1 promoter or dCas9-TET1 with no guide. GAPDH is used as an unmethylated region for HpaII digestion control. Data shown are an average of two independent experiments and error bars represent standard deviation. (C) CGG repeat PCR results for DNA from untransfected (UT) cells and replicate 1 (T1) at 7d and 24d for cells transfected with plasmid dCas9-TET1-PRM and plasmid dCas9-TET1 without any gRNAs. HpaII-digested DNA lanes show amplification of alleles that are fully methylated while undigested samples amplify both methylated and unmethylated alleles. The FMR1 gene in FXS ESCs carries 400 repeats resulting in a PCR product of about 1.4 kb. FXS MSH2 WT and FXS MSH2 KO cell lines transfected with dCas9- TET1-PRM and dCas9-TET1 with no guide show CGG repeat sizes similar to untransfected (UT) cells at day 7 (7d) and day 24 (24d). The results of CGG repeat PCR for a replicate transfection (T2) are shown in Figure S5. (D) Small pool PCR on the same DNA sample used for PCRs in panel C shows full length PCR product in both MSH2 WT and MSH2 KO cells transfected with plasmid dCas9-TET1-PRM.

Article Snippet: Gibson assembly ( ) was used to construct the plasmid pMLM3636-Puro-dCas9-TET1 using NEBuilder®HiFi DNA Assembly Master Mix (New England Biolabs, E2621L) and fragments derived from the following plasmids: PuroR from pSpCas9(BB)-2A-Puro (PX459) V2.0, a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ) , dCas9 from Inducible Caspex expression, a gift from Steven Carr & Samuel Myers (Addgene plasmid # 97421; http://n2t.net/addgene:97421 ) , and TET1CD from pPlatTET-gRNA2, a gift from Izuho Hatada (Addgene plasmid # 82559 ; http://n2t.net/addgene:82559 ) ( ) and vector backbone from pMLM3636 (a gift from Keith Joung (Addgene plasmid # 43860 ; http://n2t.net/addgene:43860 ).

Techniques: Transfection, Plasmid Preparation, Standard Deviation, Methylation, Expressing, Control, Amplification