Journal: Cell

Article Title: Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene

doi: 10.1016/j.cell.2018.01.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Cas9 (IF staining) Active Motif Cat#61577 (7A9-3A3) Mouse monoclonal anti-Cas9 (ChIP) Active Motif Cat#61757 (8C1-F10) Chicken polyclonal anti-GFP Aves Labs Cat#GFP-1020 Chicken polyclonal anti-MAP2 Encor Biotech Cat# CPCA-MAP2 Rabbit polyclonal anti-FMRP (WB, ICC, IHC) Cell Signaling Cat#4317 Mouse monoclonal anti-FMRP (ICC, IHC) Biolegend Cat#834701 Mouse monoclonal anti-FMR1PolyG EMD Millipore Cat#MABN784 Goat polyclonal anti-mCherry SICGEN Cat#AB0040-200 Mouse monoclonal anti-RNA Polymerase II Abcam Cat#ab817 Rabbit polyclonal anti-H3K27Ac Abcam Cat#ab4729 Rabbit polyclonal anti-H3K9me3 Abcam Cat#ab8898 Mouse monoclonal anti-H3K27me3 Abcam Cat#ab6002 Rabbit polyclonal anti-H3K4me3 Millipore Cat#07-473 Chemicals Doxycycline hyclate Sigma Aldrich Cat#D9891-100G Critical Commercial Assays EpiTect Bisulfite Kit Qiagen Cat#59104 EpiNext™ High-Sensitivity Bisulfite-Seq kit EPIGENTEK Cat#P-1056 EpiNext™ NGS Barcode Set-12 EPIGENTEK Cat#P-1060 DNeasy Blood & Tissue Kit Qiagen Cat#69504 Zymoclean Gel DNA Recovery Kit Zymo Research Cat#D4002 DNA Clean & Concentrator-5 Zymo Research Cat#D4013 X-tremeGENE™ 9 DNA Transfection Reagent Sigma Aldrich Cat#6365809001 Direct-zol RNA Miniprep Zymo Research Cat#R2050 SuperScript III First-Strand Synthesis SuperMix Life Technologies Cat#18080400 Fast SYBR Green Master Mix Life Technologies Cat#4385618 Deposited Data Raw data files for sequencing NCBI Gene Expression Omnibus Database: {"type":"entrez-geo","attrs":{"text":"GSE102655","term_id":"102655"}} GSE102655 , {"type":"entrez-geo","attrs":{"text":"GSE102684","term_id":"102684"}} GSE102684 , {"type":"entrez-geo","attrs":{"text":"GSE108171","term_id":"108171"}} GSE108171 , {"type":"entrez-geo","attrs":{"text":"GSE108498","term_id":"108498"}} GSE108498 , {"type":"entrez-geo","attrs":{"text":"GSE108577","term_id":"108577"}} GSE108577 Experimental Models: Organisms/Strains Mouse: NOD- scid IL2Rg null , Jackson Laboratories Stock No: 005557 Experimental Models: Cell Lines FX52 iPSCs ( Urbach et al., 2010 ) N/A FXS iPSC (135.3) ( Brick et al., 2014 ) N/A FXS iPSC (FXS_SW) ( Xie et al., 2016 ) N/A Recombinant DNA Fuw-dCas9-Tet1-P2A-BFP This paper N/A pgRNA This paper N/A Fuw-AcrIIA4-P2A-GFP This paper N/A See “ Plasmid design and construction ” in METHOD DETAILS section Sequence-Based Reagents See Table S4 , S5 , S6 for primer sequences This paper N/A Software and Algorithms MACS (ChIP-seq algorithms) Wu et al., 2014b http://liulab.dfci.harvard.edu/MACS/ ImageJ (Fiji) NIH http://imagej.net/Fiji Open in a separate window

Techniques: Staining, Bisulfite Sequencing, Transfection, SYBR Green Assay, Sequencing, Expressing, Recombinant, Plasmid Preparation, Software

Journal: Cell

Article Title: Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene

doi: 10.1016/j.cell.2018.01.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Cas9 (IF staining) Active Motif Cat#61577 (7A9-3A3) Mouse monoclonal anti-Cas9 (ChIP) Active Motif Cat#61757 (8C1-F10) Chicken polyclonal anti-GFP Aves Labs Cat#GFP-1020 Chicken polyclonal anti-MAP2 Encor Biotech Cat# CPCA-MAP2 Rabbit polyclonal anti-FMRP (WB, ICC, IHC) Cell Signaling Cat#4317 Mouse monoclonal anti-FMRP (ICC, IHC) Biolegend Cat#834701 Mouse monoclonal anti-FMR1PolyG EMD Millipore Cat#MABN784 Goat polyclonal anti-mCherry SICGEN Cat#AB0040-200 Mouse monoclonal anti-RNA Polymerase II Abcam Cat#ab817 Rabbit polyclonal anti-H3K27Ac Abcam Cat#ab4729 Rabbit polyclonal anti-H3K9me3 Abcam Cat#ab8898 Mouse monoclonal anti-H3K27me3 Abcam Cat#ab6002 Rabbit polyclonal anti-H3K4me3 Millipore Cat#07-473 Chemicals Doxycycline hyclate Sigma Aldrich Cat#D9891-100G Critical Commercial Assays EpiTect Bisulfite Kit Qiagen Cat#59104 EpiNextTM High-Sensitivity Bisulfite-Seq kit EPIGENTEK Cat#P-1056 EpiNextTM NGS Barcode Set-12 EPIGENTEK Cat#P-1060 DNeasy Blood & Tissue Kit Qiagen Cat#69504 Zymoclean Gel DNA Recovery Kit Zymo Research Cat#D4002 DNA Clean & Concentrator-5 Zymo Research Cat#D4013 X-tremeGENETM 9 DNA Transfection Reagent Sigma Aldrich Cat#6365809001 Direct-zol RNA Miniprep Zymo Research Cat#R2050 SuperScript III First-Strand Synthesis SuperMix Life Technologies Cat#18080400 Fast SYBR Green Master Mix Life Technologies Cat#4385618 Deposited Data Raw data files for sequencing NCBI Gene Expression Omnibus Database: {"type":"entrez-geo","attrs":{"text":"GSE102655","term_id":"102655"}} GSE102655 , {"type":"entrez-geo","attrs":{"text":"GSE102684","term_id":"102684"}} GSE102684 , {"type":"entrez-geo","attrs":{"text":"GSE108171","term_id":"108171"}} GSE108171 , {"type":"entrez-geo","attrs":{"text":"GSE108498","term_id":"108498"}} GSE108498 , {"type":"entrez-geo","attrs":{"text":"GSE108577","term_id":"108577"}} GSE108577 Experimental Models: Organisms/Strains Mouse: NOD- scid IL2Rg null , Jackson Laboratories Stock No: 005557 Experimental Models: Cell Lines FX52 iPSCs ( Urbach et al., 2010 ) N/A FXS iPSC (135.3) ( Brick et al., 2014 ) N/A FXS iPSC (FXS_SW) ( Xie et al., 2016 ) N/A Recombinant DNA Fuw-dCas9-Tet1-P2A-BFP This paper N/A pgRNA This paper N/A Fuw-AcrIIA4-P2A-GFP This paper N/A See “ Plasmid design and construction ” in METHOD DETAILS section Sequence-Based Reagents See Table S4 , S5 , S6 for primer sequences This paper N/A Software and Algorithms MACS (ChIP-seq algorithms) Wu et al., 2014b http://liulab.dfci.harvard.edu/MACS/ ImageJ (Fiji) NIH http://imagej.net/Fiji Open in a separate window

Techniques: Staining, Bisulfite Sequencing, Transfection, SYBR Green Assay, Sequencing, Gene Expression, Recombinant, Plasmid Preparation, Software

Journal: Cell

Article Title: Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene

doi: 10.1016/j.cell.2018.01.012

Figure Lengend Snippet: (A) Methylation edited FX52 iPSCs were differentiated into neuronal processor cells (NPCs) and post-mitotic neurons. Scale bar: 500 um. (B) Expression levels of linage-specific markers in neurons described in A measured by qPCR. Shown is the mean percentage ± SD of two biological replicates. (C) FMR1 expression level of neurons in A measured by qPCR. Shown is the mean percentage ± SD of two biological replicates. (D) Neurons in A were subject to immunofluorescence staining. Scale bar: 100 um. Stained in green for FMRP, red for Cas9, blue for DNA, and grey for MAP2. (E) RNA-seq of mock and dCas9-Tet1/CGG sgRNA expressing FX52 neurons described in A. Red circles highlight 43 genes with a change of methylation larger than 10% in dCas9-Tet1/CGG sgRNA expressing neurons (Table S2). FMR1 is labeled with a green dot. The dashed red lines mark a 4-fold difference between the samples. (F) Multi-electrode array (MEA) of wild-type WIBR1 neurons and FX52 mock, dCas9-Tet1/CGG sgRNA or dCas9-dTet1/CGG sgRNA expressing neurons. Shown is the mean ± SD of biological triplicates for each type of neurons. (G) Schematic representation of engraftment of 1:1 mixed cells containing mock FX52 NPCs labeled with GFP and methylation edited FX52 NPCs labeled with RFP into the P1 mouse brain and harvested for analysis one month post-engraftment. (H) Representative confocal micrographs of cells engrafted in the mouse brain at one month post-transplantation. Scale bar: 50 μm. Stained in red for RFP, green for GFP, magenta for FMRP, and blue for DNA. A total of 496 RFP-positive neurons and 149 GFP-positive neurons from three mice after one month were counted. 56% ± 9% of RFP neurons were FMRP-positive, whereas all GFP positive neurons were FMRP-negative. A total of 203 RFP-positive neurons and 117 GFP-positive neurons from two mice after three months were counted. 57% ± 3% of RFP neurons were FMRP-positive, whereas all GFP positive neurons were FMRP-negative.

Article Snippet: Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Cas9 (IF staining) Active Motif Cat#61577 (7A9-3A3) Mouse monoclonal anti-Cas9 (ChIP) Active Motif Cat#61757 (8C1-F10) Chicken polyclonal anti-GFP Aves Labs Cat#GFP-1020 Chicken polyclonal anti-MAP2 Encor Biotech Cat# CPCA-MAP2 Rabbit polyclonal anti-FMRP (WB, ICC, IHC) Cell Signaling Cat#4317 Mouse monoclonal anti-FMRP (ICC, IHC) Biolegend Cat#834701 Mouse monoclonal anti-FMR1PolyG EMD Millipore Cat#MABN784 Goat polyclonal anti-mCherry SICGEN Cat#AB0040-200 Mouse monoclonal anti-RNA Polymerase II Abcam Cat#ab817 Rabbit polyclonal anti-H3K27Ac Abcam Cat#ab4729 Rabbit polyclonal anti-H3K9me3 Abcam Cat#ab8898 Mouse monoclonal anti-H3K27me3 Abcam Cat#ab6002 Rabbit polyclonal anti-H3K4me3 Millipore Cat#07-473 Chemicals Doxycycline hyclate Sigma Aldrich Cat#D9891-100G Critical Commercial Assays EpiTect Bisulfite Kit Qiagen Cat#59104 EpiNextTM High-Sensitivity Bisulfite-Seq kit EPIGENTEK Cat#P-1056 EpiNextTM NGS Barcode Set-12 EPIGENTEK Cat#P-1060 DNeasy Blood & Tissue Kit Qiagen Cat#69504 Zymoclean Gel DNA Recovery Kit Zymo Research Cat#D4002 DNA Clean & Concentrator-5 Zymo Research Cat#D4013 X-tremeGENETM 9 DNA Transfection Reagent Sigma Aldrich Cat#6365809001 Direct-zol RNA Miniprep Zymo Research Cat#R2050 SuperScript III First-Strand Synthesis SuperMix Life Technologies Cat#18080400 Fast SYBR Green Master Mix Life Technologies Cat#4385618 Deposited Data Raw data files for sequencing NCBI Gene Expression Omnibus Database: {"type":"entrez-geo","attrs":{"text":"GSE102655","term_id":"102655"}} GSE102655 , {"type":"entrez-geo","attrs":{"text":"GSE102684","term_id":"102684"}} GSE102684 , {"type":"entrez-geo","attrs":{"text":"GSE108171","term_id":"108171"}} GSE108171 , {"type":"entrez-geo","attrs":{"text":"GSE108498","term_id":"108498"}} GSE108498 , {"type":"entrez-geo","attrs":{"text":"GSE108577","term_id":"108577"}} GSE108577 Experimental Models: Organisms/Strains Mouse: NOD- scid IL2Rg null , Jackson Laboratories Stock No: 005557 Experimental Models: Cell Lines FX52 iPSCs ( Urbach et al., 2010 ) N/A FXS iPSC (135.3) ( Brick et al., 2014 ) N/A FXS iPSC (FXS_SW) ( Xie et al., 2016 ) N/A Recombinant DNA Fuw-dCas9-Tet1-P2A-BFP This paper N/A pgRNA This paper N/A Fuw-AcrIIA4-P2A-GFP This paper N/A See “ Plasmid design and construction ” in METHOD DETAILS section Sequence-Based Reagents See Table S4 , S5 , S6 for primer sequences This paper N/A Software and Algorithms MACS (ChIP-seq algorithms) Wu et al., 2014b http://liulab.dfci.harvard.edu/MACS/ ImageJ (Fiji) NIH http://imagej.net/Fiji Open in a separate window

Techniques: Methylation, Expressing, Immunofluorescence, Staining, RNA Sequencing, Labeling, Transplantation Assay

Journal: Cell

Article Title: Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene

doi: 10.1016/j.cell.2018.01.012

Figure Lengend Snippet: (A) Schematic representation of targeting the CGG repeats of FMR1 by dCas9-Tet1 with a CGG sgRNA to erase methylation and activate FMR1 expression. (B) A previously reported FXS iPSC line (FX52) was infected with lentiviruses expressing dCas9-Tet1-P2A-BFP (dC-T) with a mCherry-expressing sgRNA targeting the CGG repeats “GGCGGCGGCGGCGGCGGCGGNGG” (CGG sgRNA) or lentiviruses expressing dCas9 fused with a catalytically dead Tet1 (dC-dT) with the same sgRNA. Double positive (BFP+; mCherry+) cells were isolated by FACS after infection. The expression level of FMR1 were quantified by qPCR analysis, and shown as the mean of relative percentages to the one in WIBR1 hESCs ± SD of three biological replicates. (C) Cells in B were cultured on feeder MEFs and subject to immunofluorescence staining. Scale bar: 100 um. Stained in green for FMRP, red for Cas9, and blue for DNA. (D) Cells in B were subject to western blot analysis. The protein level of FMRP was quantified by ImageJ and shown as the mean of relative percentages to the one in WIBR1 hESCs ± SD of two biological replicates. (E) Methylation levels of the CGG repeats in the FMR1 locus. Shown is the mean percentage of two biological replicates. (F) Bisulfite sequencing of cells described in B. (G) Methylation levels of individual CpGs in the FMR1 promoter region. Shown is the mean percentage ± SD of two biological replicates. See also Figure S1.

Article Snippet: Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Cas9 (IF staining) Active Motif Cat#61577 (7A9-3A3) Mouse monoclonal anti-Cas9 (ChIP) Active Motif Cat#61757 (8C1-F10) Chicken polyclonal anti-GFP Aves Labs Cat#GFP-1020 Chicken polyclonal anti-MAP2 Encor Biotech Cat# CPCA-MAP2 Rabbit polyclonal anti-FMRP (WB, ICC, IHC) Cell Signaling Cat#4317 Mouse monoclonal anti-FMRP (ICC, IHC) Biolegend Cat#834701 Mouse monoclonal anti-FMR1PolyG EMD Millipore Cat#MABN784 Goat polyclonal anti-mCherry SICGEN Cat#AB0040-200 Mouse monoclonal anti-RNA Polymerase II Abcam Cat#ab817 Rabbit polyclonal anti-H3K27Ac Abcam Cat#ab4729 Rabbit polyclonal anti-H3K9me3 Abcam Cat#ab8898 Mouse monoclonal anti-H3K27me3 Abcam Cat#ab6002 Rabbit polyclonal anti-H3K4me3 Millipore Cat#07-473 Chemicals Doxycycline hyclate Sigma Aldrich Cat#D9891-100G Critical Commercial Assays EpiTect Bisulfite Kit Qiagen Cat#59104 EpiNextTM High-Sensitivity Bisulfite-Seq kit EPIGENTEK Cat#P-1056 EpiNextTM NGS Barcode Set-12 EPIGENTEK Cat#P-1060 DNeasy Blood & Tissue Kit Qiagen Cat#69504 Zymoclean Gel DNA Recovery Kit Zymo Research Cat#D4002 DNA Clean & Concentrator-5 Zymo Research Cat#D4013 X-tremeGENETM 9 DNA Transfection Reagent Sigma Aldrich Cat#6365809001 Direct-zol RNA Miniprep Zymo Research Cat#R2050 SuperScript III First-Strand Synthesis SuperMix Life Technologies Cat#18080400 Fast SYBR Green Master Mix Life Technologies Cat#4385618 Deposited Data Raw data files for sequencing NCBI Gene Expression Omnibus Database: {"type":"entrez-geo","attrs":{"text":"GSE102655","term_id":"102655"}} GSE102655 , {"type":"entrez-geo","attrs":{"text":"GSE102684","term_id":"102684"}} GSE102684 , {"type":"entrez-geo","attrs":{"text":"GSE108171","term_id":"108171"}} GSE108171 , {"type":"entrez-geo","attrs":{"text":"GSE108498","term_id":"108498"}} GSE108498 , {"type":"entrez-geo","attrs":{"text":"GSE108577","term_id":"108577"}} GSE108577 Experimental Models: Organisms/Strains Mouse: NOD- scid IL2Rg null , Jackson Laboratories Stock No: 005557 Experimental Models: Cell Lines FX52 iPSCs ( Urbach et al., 2010 ) N/A FXS iPSC (135.3) ( Brick et al., 2014 ) N/A FXS iPSC (FXS_SW) ( Xie et al., 2016 ) N/A Recombinant DNA Fuw-dCas9-Tet1-P2A-BFP This paper N/A pgRNA This paper N/A Fuw-AcrIIA4-P2A-GFP This paper N/A See “ Plasmid design and construction ” in METHOD DETAILS section Sequence-Based Reagents See Table S4 , S5 , S6 for primer sequences This paper N/A Software and Algorithms MACS (ChIP-seq algorithms) Wu et al., 2014b http://liulab.dfci.harvard.edu/MACS/ ImageJ (Fiji) NIH http://imagej.net/Fiji Open in a separate window

Techniques: Methylation, Expressing, Infection, Isolation, Cell Culture, Immunofluorescence, Staining, Western Blot, Methylation Sequencing

Journal: Cell

Article Title: Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene

doi: 10.1016/j.cell.2018.01.012

Figure Lengend Snippet: (A) FX52 iPSCs were infected with lentiviruses expressing dCas9-Tet1-P2A-BFP (dC-T) and a CGG sgRNA, and were harvested for qPCR analysis of FMR1 expression at various time points. Shown is the mean expression level relative to that in a CGG deletion FX52 iPSC line (± SD of two biological replicates). This CGG deletion FX52 iPSC line was previously reported to restore FMR1 expression (Park et al., 2015a). (B) 293T cells were transfected with dC-T alone, with dC-T/CGG sgRNA, or with dC-T/CGG sgRNA and AcrIIA4. The cells were subject to an anti-Cas9 ChIP experiment and the enrichment of dC-T at the FMR1 and BDNF loci was examined by qPCR analysis. Shown is the mean of relative binding normalized to the input ± SD from three biological replicates. (C) Cells on Day-23 (labeled as Meth edited) in A were infected with lentivirus expressing AcrIIA4 and harvested for qPCR analysis at various time points. Shown is the mean FMR1 (red bar) and AcrIIA4 (blue bar) expression levels relative to the levels in the CGG deletion FX52 iPSC line (CGGΔ) and the cells on Day-3 after AcrIIA4 infection (D3), respectively (± SD of two biological replicates). (D) Pyro-seq of cells in A and C. Shown is the mean of percentage ± SD of two biological replicates.

Article Snippet: Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Cas9 (IF staining) Active Motif Cat#61577 (7A9-3A3) Mouse monoclonal anti-Cas9 (ChIP) Active Motif Cat#61757 (8C1-F10) Chicken polyclonal anti-GFP Aves Labs Cat#GFP-1020 Chicken polyclonal anti-MAP2 Encor Biotech Cat# CPCA-MAP2 Rabbit polyclonal anti-FMRP (WB, ICC, IHC) Cell Signaling Cat#4317 Mouse monoclonal anti-FMRP (ICC, IHC) Biolegend Cat#834701 Mouse monoclonal anti-FMR1PolyG EMD Millipore Cat#MABN784 Goat polyclonal anti-mCherry SICGEN Cat#AB0040-200 Mouse monoclonal anti-RNA Polymerase II Abcam Cat#ab817 Rabbit polyclonal anti-H3K27Ac Abcam Cat#ab4729 Rabbit polyclonal anti-H3K9me3 Abcam Cat#ab8898 Mouse monoclonal anti-H3K27me3 Abcam Cat#ab6002 Rabbit polyclonal anti-H3K4me3 Millipore Cat#07-473 Chemicals Doxycycline hyclate Sigma Aldrich Cat#D9891-100G Critical Commercial Assays EpiTect Bisulfite Kit Qiagen Cat#59104 EpiNextTM High-Sensitivity Bisulfite-Seq kit EPIGENTEK Cat#P-1056 EpiNextTM NGS Barcode Set-12 EPIGENTEK Cat#P-1060 DNeasy Blood & Tissue Kit Qiagen Cat#69504 Zymoclean Gel DNA Recovery Kit Zymo Research Cat#D4002 DNA Clean & Concentrator-5 Zymo Research Cat#D4013 X-tremeGENETM 9 DNA Transfection Reagent Sigma Aldrich Cat#6365809001 Direct-zol RNA Miniprep Zymo Research Cat#R2050 SuperScript III First-Strand Synthesis SuperMix Life Technologies Cat#18080400 Fast SYBR Green Master Mix Life Technologies Cat#4385618 Deposited Data Raw data files for sequencing NCBI Gene Expression Omnibus Database: {"type":"entrez-geo","attrs":{"text":"GSE102655","term_id":"102655"}} GSE102655 , {"type":"entrez-geo","attrs":{"text":"GSE102684","term_id":"102684"}} GSE102684 , {"type":"entrez-geo","attrs":{"text":"GSE108171","term_id":"108171"}} GSE108171 , {"type":"entrez-geo","attrs":{"text":"GSE108498","term_id":"108498"}} GSE108498 , {"type":"entrez-geo","attrs":{"text":"GSE108577","term_id":"108577"}} GSE108577 Experimental Models: Organisms/Strains Mouse: NOD- scid IL2Rg null , Jackson Laboratories Stock No: 005557 Experimental Models: Cell Lines FX52 iPSCs ( Urbach et al., 2010 ) N/A FXS iPSC (135.3) ( Brick et al., 2014 ) N/A FXS iPSC (FXS_SW) ( Xie et al., 2016 ) N/A Recombinant DNA Fuw-dCas9-Tet1-P2A-BFP This paper N/A pgRNA This paper N/A Fuw-AcrIIA4-P2A-GFP This paper N/A See “ Plasmid design and construction ” in METHOD DETAILS section Sequence-Based Reagents See Table S4 , S5 , S6 for primer sequences This paper N/A Software and Algorithms MACS (ChIP-seq algorithms) Wu et al., 2014b http://liulab.dfci.harvard.edu/MACS/ ImageJ (Fiji) NIH http://imagej.net/Fiji Open in a separate window

Techniques: Infection, Expressing, Transfection, Binding Assay, Labeling

Journal: Cell

Article Title: Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene

doi: 10.1016/j.cell.2018.01.012

Figure Lengend Snippet: (A) FX52 neurons were infected with lentiviruses expressing dCas9-Tet1 (dC-T) and the CGG sgRNA or dCas9-dTet1 (dC-dT) with the same sgRNA. Infected neurons were subject to immunofluorescence staining. Scale bar: 50 um. Stained in green for FMRP, red for Cas9, blue for DNA, and grey for MAP2. (B) The expression level of FMR1 in A was quantified by qPCR analysis, and shown as the mean relative to the level in WIBR1 hESCs ± SD of two biological replicates. (C) Methylation levels of the CGG repeats in the FMR1 locus. Shown is the mean percentage ± SD of two biological replicates. (D) Bisulfite sequencing of the FMR1 promoter in cells described in A. Shown is the mean percentage ± SD of two biological replicates. (E) MEA assay of the neurons in A. Shown is the mean percentage ± SD of two biological replicates.

Article Snippet: Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Cas9 (IF staining) Active Motif Cat#61577 (7A9-3A3) Mouse monoclonal anti-Cas9 (ChIP) Active Motif Cat#61757 (8C1-F10) Chicken polyclonal anti-GFP Aves Labs Cat#GFP-1020 Chicken polyclonal anti-MAP2 Encor Biotech Cat# CPCA-MAP2 Rabbit polyclonal anti-FMRP (WB, ICC, IHC) Cell Signaling Cat#4317 Mouse monoclonal anti-FMRP (ICC, IHC) Biolegend Cat#834701 Mouse monoclonal anti-FMR1PolyG EMD Millipore Cat#MABN784 Goat polyclonal anti-mCherry SICGEN Cat#AB0040-200 Mouse monoclonal anti-RNA Polymerase II Abcam Cat#ab817 Rabbit polyclonal anti-H3K27Ac Abcam Cat#ab4729 Rabbit polyclonal anti-H3K9me3 Abcam Cat#ab8898 Mouse monoclonal anti-H3K27me3 Abcam Cat#ab6002 Rabbit polyclonal anti-H3K4me3 Millipore Cat#07-473 Chemicals Doxycycline hyclate Sigma Aldrich Cat#D9891-100G Critical Commercial Assays EpiTect Bisulfite Kit Qiagen Cat#59104 EpiNextTM High-Sensitivity Bisulfite-Seq kit EPIGENTEK Cat#P-1056 EpiNextTM NGS Barcode Set-12 EPIGENTEK Cat#P-1060 DNeasy Blood & Tissue Kit Qiagen Cat#69504 Zymoclean Gel DNA Recovery Kit Zymo Research Cat#D4002 DNA Clean & Concentrator-5 Zymo Research Cat#D4013 X-tremeGENETM 9 DNA Transfection Reagent Sigma Aldrich Cat#6365809001 Direct-zol RNA Miniprep Zymo Research Cat#R2050 SuperScript III First-Strand Synthesis SuperMix Life Technologies Cat#18080400 Fast SYBR Green Master Mix Life Technologies Cat#4385618 Deposited Data Raw data files for sequencing NCBI Gene Expression Omnibus Database: {"type":"entrez-geo","attrs":{"text":"GSE102655","term_id":"102655"}} GSE102655 , {"type":"entrez-geo","attrs":{"text":"GSE102684","term_id":"102684"}} GSE102684 , {"type":"entrez-geo","attrs":{"text":"GSE108171","term_id":"108171"}} GSE108171 , {"type":"entrez-geo","attrs":{"text":"GSE108498","term_id":"108498"}} GSE108498 , {"type":"entrez-geo","attrs":{"text":"GSE108577","term_id":"108577"}} GSE108577 Experimental Models: Organisms/Strains Mouse: NOD- scid IL2Rg null , Jackson Laboratories Stock No: 005557 Experimental Models: Cell Lines FX52 iPSCs ( Urbach et al., 2010 ) N/A FXS iPSC (135.3) ( Brick et al., 2014 ) N/A FXS iPSC (FXS_SW) ( Xie et al., 2016 ) N/A Recombinant DNA Fuw-dCas9-Tet1-P2A-BFP This paper N/A pgRNA This paper N/A Fuw-AcrIIA4-P2A-GFP This paper N/A See “ Plasmid design and construction ” in METHOD DETAILS section Sequence-Based Reagents See Table S4 , S5 , S6 for primer sequences This paper N/A Software and Algorithms MACS (ChIP-seq algorithms) Wu et al., 2014b http://liulab.dfci.harvard.edu/MACS/ ImageJ (Fiji) NIH http://imagej.net/Fiji Open in a separate window

Techniques: Infection, Expressing, Immunofluorescence, Staining, Methylation, Methylation Sequencing

Journal: Cell

Article Title: Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene

doi: 10.1016/j.cell.2018.01.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Cas9 (IF staining) Active Motif Cat#61577 (7A9-3A3) Mouse monoclonal anti-Cas9 (ChIP) Active Motif Cat#61757 (8C1-F10) Chicken polyclonal anti-GFP Aves Labs Cat#GFP-1020 Chicken polyclonal anti-MAP2 Encor Biotech Cat# CPCA-MAP2 Rabbit polyclonal anti-FMRP (WB, ICC, IHC) Cell Signaling Cat#4317 Mouse monoclonal anti-FMRP (ICC, IHC) Biolegend Cat#834701 Mouse monoclonal anti-FMR1PolyG EMD Millipore Cat#MABN784 Goat polyclonal anti-mCherry SICGEN Cat#AB0040-200 Mouse monoclonal anti-RNA Polymerase II Abcam Cat#ab817 Rabbit polyclonal anti-H3K27Ac Abcam Cat#ab4729 Rabbit polyclonal anti-H3K9me3 Abcam Cat#ab8898 Mouse monoclonal anti-H3K27me3 Abcam Cat#ab6002 Rabbit polyclonal anti-H3K4me3 Millipore Cat#07-473 Chemicals Doxycycline hyclate Sigma Aldrich Cat#D9891-100G Critical Commercial Assays EpiTect Bisulfite Kit Qiagen Cat#59104 EpiNextTM High-Sensitivity Bisulfite-Seq kit EPIGENTEK Cat#P-1056 EpiNextTM NGS Barcode Set-12 EPIGENTEK Cat#P-1060 DNeasy Blood & Tissue Kit Qiagen Cat#69504 Zymoclean Gel DNA Recovery Kit Zymo Research Cat#D4002 DNA Clean & Concentrator-5 Zymo Research Cat#D4013 X-tremeGENETM 9 DNA Transfection Reagent Sigma Aldrich Cat#6365809001 Direct-zol RNA Miniprep Zymo Research Cat#R2050 SuperScript III First-Strand Synthesis SuperMix Life Technologies Cat#18080400 Fast SYBR Green Master Mix Life Technologies Cat#4385618 Deposited Data Raw data files for sequencing NCBI Gene Expression Omnibus Database: {"type":"entrez-geo","attrs":{"text":"GSE102655","term_id":"102655"}} GSE102655 , {"type":"entrez-geo","attrs":{"text":"GSE102684","term_id":"102684"}} GSE102684 , {"type":"entrez-geo","attrs":{"text":"GSE108171","term_id":"108171"}} GSE108171 , {"type":"entrez-geo","attrs":{"text":"GSE108498","term_id":"108498"}} GSE108498 , {"type":"entrez-geo","attrs":{"text":"GSE108577","term_id":"108577"}} GSE108577 Experimental Models: Organisms/Strains Mouse: NOD- scid IL2Rg null , Jackson Laboratories Stock No: 005557 Experimental Models: Cell Lines FX52 iPSCs ( Urbach et al., 2010 ) N/A FXS iPSC (135.3) ( Brick et al., 2014 ) N/A FXS iPSC (FXS_SW) ( Xie et al., 2016 ) N/A Recombinant DNA Fuw-dCas9-Tet1-P2A-BFP This paper N/A pgRNA This paper N/A Fuw-AcrIIA4-P2A-GFP This paper N/A See “ Plasmid design and construction ” in METHOD DETAILS section Sequence-Based Reagents See Table S4 , S5 , S6 for primer sequences This paper N/A Software and Algorithms MACS (ChIP-seq algorithms) Wu et al., 2014b http://liulab.dfci.harvard.edu/MACS/ ImageJ (Fiji) NIH http://imagej.net/Fiji Open in a separate window

Techniques: Staining, Bisulfite Sequencing, Transfection, SYBR Green Assay, Sequencing, Gene Expression, Recombinant, Plasmid Preparation, Software