Journal: Scientific Reports

Article Title: Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons

doi: 10.1038/srep28420

Figure Lengend Snippet: ( a ) Schematic representation of SNCA exon- and TSS-targeting sgRNA positions. ( b ) qRT-PCR screening of total alpha-synuclein mRNA in HEK293T cells transfected with SNCA exon- and TSS-targeting sgRNAs and dCas9. Data are represented as mean ± SEM. ( c ) HA ChIP of HEK293T cells transfected with empty dCas9 vector or TSS2-1, TSS2-2, or TSS2-3 sgRNA co-expressing dCas9 vectors. Data are represented as mean ± SEM. ( d ) qRT-PCR for total alpha-synuclein mRNA in HEK293T, BE(2)-M17 and SH-SY5Y cells transfected with TSS2-1 sgRNA co-expressing dCas9 vector. Data are represented as mean ± SEM. ( e ) ELISA for alpha-synuclein protein in HEK293T, BE(2)-M17 and SH-SY5Y cells transfected with TSS2-1 sgRNA co-expressing dCas9 vector. Data are represented as mean ± SEM. *p ≤ 0.05 compared to control, **p ≤ 0.01 compared to control. SD = splice donor, SA = splice acceptor.

Article Snippet: For CRISPRa, SNCA TSS2-2 sgRNA and modified F + E sgRNA backbone were cloned with a human U6 promoter by overlap PCR into pGEM-Teasy (Promega) and co-transfected with the SP-dCas9-VPR vector, which was a gift from George Church (Addgene #63798) .

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons

doi: 10.1038/srep28420

Figure Lengend Snippet: ( a ) SNCA promoter region. Predicted transcripts (from UCSC Genome Browser) and transcription start sites (TSS) are indicated, along with the position of the TSS-targeting sgRNAs (sgRNAs). Cap analysis of gene expression (CAGE) data from the FANTOM5 consortium shows the relative TSS usage, averaged across all samples, and positions of predicted promoter regions (Promoters, p1-14@SNCA). The promoter associated chromatin signatures, H3K27Ac, H3K4me3 and DNaseI hypersensitivity, show data from the Encyclopedia of DNA Elements (ENCODE) project. Basewise conservation (PhyloP) and repeats (RepeatMasker) are also shown. ( b ) qRT-PCR analysis of TSS usage in HEK293T cells demonstrates that TSS2-1 is the predominant TSS. The TSS3 transcript isoform was undetectable. Data are represented as mean ± SEM. ( c ) Expression of TSS isoform-specific mRNAs following TSS2-1 sgRNA-mediated CRISPRi. Analysis of expression levels of total SNCA mRNA and spliced transcripts specific to TSS1, TSS2-1 and TSS2-2 by qRT-PCR in dCas9/TSS2-1 sgRNA-transfected HEK293T cells (red bars, dCas9) relative to controls (black bars, control). Data are represented as mean ± SEM. ( d ) Analysis of nascent transcript expression following TSS2-1 sgRNA-mediated CRISPRi. Analysis of nascent transcripts was conducted on dCas9/TSS2-1 sgRNA transfected HEK293T cells (red bars, dCas9) relative to controls (black bars, control). Expression of nascent transcripts was estimated by qRT-PCR using primers spanning the exon 1:intron boundaries specific to transcripts deriving from TSS1, TSS2-1, and TSS2-2 or at the exon 2:intron and exon 3:intron boundaries. Data are represented as mean ± SEM. *p ≤ 0.05 compared to control, **p ≤ 0.01 compared to control, ***p ≤ 0.001 compared to control.

Article Snippet: For CRISPRa, SNCA TSS2-2 sgRNA and modified F + E sgRNA backbone were cloned with a human U6 promoter by overlap PCR into pGEM-Teasy (Promega) and co-transfected with the SP-dCas9-VPR vector, which was a gift from George Church (Addgene #63798) .

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Transfection, Control

Journal: Scientific Reports

Article Title: Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons

doi: 10.1038/srep28420

Figure Lengend Snippet: ( a ) CRISPRa-mediated activation of alpha-synuclein in NAS iPSC-derived neurons with TSS2-2 sgRNA and dCas9-VPR transcriptional activator. mRNA levels were quantified by qRT-PCR in biological triplicate, and data are represented as mean ± SEM. Protein levels were quantified by ELISA in biological triplicate, and data are represented as mean ± SEM. ( b ) CRISPRi-mediated repression of alpha-synuclein in AST iPSC-derived neurons with TSS2-1 sgRNA and dCas9-KRAB transcriptional repressor. mRNA levels were quantified by qRT-PCR in biological triplicate, and data are represented as mean ± SEM. Protein levels were quantified by ELISA in biological triplicate, and data are represented as mean ± SEM. Micrographs demonstrate the neuronal identity of iPSC derivatives as confirmed by co-immunostaining with the pan-neuronal markers, MAP2 and TUJ1 (scalebars are 100 μm). Schematic diagrams represent the intended outcome of CRISPRa/i-mediated gene expression modulation. *p ≤ 0.05 compared to control, **p ≤ 0.01 compared to control, ***p ≤ 0.001 compared to control. NAS = normal alpha synuclein; AST = alpha-synuclein triplication.

Article Snippet: For CRISPRa, SNCA TSS2-2 sgRNA and modified F + E sgRNA backbone were cloned with a human U6 promoter by overlap PCR into pGEM-Teasy (Promega) and co-transfected with the SP-dCas9-VPR vector, which was a gift from George Church (Addgene #63798) .

Techniques: Activation Assay, Derivative Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunostaining, Gene Expression, Control