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

Journal: Nature

Article Title: Base-editing mutagenesis maps alleles to tune human T cell functions

doi: 10.1038/s41586-023-06835-6

Figure Lengend Snippet: a , Schematic of arrayed validation by co-electroporation of ABE mRNA and synthetic sgRNA. b , ABE base editing at a positive VAV1 ( VAV1 pos sgRNA) site and AAVS1 control site verified by deep amplicon sequencing and analysed with Crispresso2 54 . Predicted editing window in green, guide sequence in grey, and NGG PAM in dark grey. c , Representative flow cytometry plots for indicated cytokines in control ( AAVS1 ) or PIK3CD pos sgRNA-edited T cells, gated on CD4 + T cells. d , log 2 FC of intracellular expression of indicated cytokines over control (mean of two AAVS1 guide RNAs) measured by flow cytometry. Negative sgRNAs in the original screen are in blue, positive sgRNAs are in red. n = 6; * P < 0.05, ** P < 0.01, *** P < 0.001. pos and neg indicate sgRNAs with positive and negative effects on T cell activation responses, respectively. e , Cytokine secretion in culture supernatants for base-edited T cells measured by Luminex. The heat map represents log 2 FC over mean of cells edited with two AAVS1 controls. n = 4. f , Cytotoxicity (A375 cell killing) of antigen-specific T cells base edited with control, positive or negative guide RNAs targeting DGKZ or PIK3CD measured by Incucyte imaging over time. n = 6. g , Area under the curve (AUC) of A375 cell counts over time when co-cultured with base edited T cells ( x axis) at an effector:target (E:T) ratio of 4. n = 6 donors in technical duplicates. h , Imaging of A375 cells co-cultured with base-edited, antigen-specific T cells using indicated guides and E:T ratios after 120 h. i , Change in production of indicated cytokines in arrayed validation for nine PIK3CD guides relative to AAVS1 control in CD4 + T cells; colour indicates the guide log 2 FC value in the original ABE TNF screen. n = 6. j , Sequencing of base edits clustering around Y524 (green) show distinct mutations. All n values refer to the number of human donors. Data are mean ± s.e.m. Two-tailed independent two-sample t -test.

Article Snippet: Two micrograms of ABE mRNA mixed with 1.5 μg synthetic modified sgRNA (Synthego) was added per 20 μl cells, not exceeding 25 μl total per reaction.

Techniques: Biomarker Discovery, Functional Assay, Electroporation, Control, Amplification, Sequencing, Flow Cytometry, Expressing, Activation Assay, Luminex, Imaging, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Base-editing mutagenesis maps alleles to tune human T cell functions

doi: 10.1038/s41586-023-06835-6

Figure Lengend Snippet: a , Structural model of the PIK3CD–PIK3R1 complex (Protein Data Bank: 7JIS), with residues coloured by log 2 FC of TNF production from the NG PAM screen. The catalytic loop (residues 890–905) is marked by negative base-editing effects (top left); two other domains—the PIK3R1-interaction region (residues 566–586) (bottom right) and residues 515–535 (bottom left) are marked by positive base-editing effects. b , Flow cytometry plots of CD4 + T cells edited with ABE mRNA and synthetic sgRNA targeting PIK3R1 or AAVS1 (control). c , log 2 FC in cytokine production for CD4 + or CD8 + T cells edited with positive PIK3R1 sgRNA or AAVS1 controls in arrayed format. n = 6 human donors. Data are normalized to controls and show mean ± s.e.m.

Article Snippet: Two micrograms of ABE mRNA mixed with 1.5 μg synthetic modified sgRNA (Synthego) was added per 20 μl cells, not exceeding 25 μl total per reaction.

Techniques: Functional Assay, Flow Cytometry, Control

Journal: Nature

Article Title: Base-editing mutagenesis maps alleles to tune human T cell functions

doi: 10.1038/s41586-023-06835-6

Figure Lengend Snippet: a , Scatter plots showing LFC (log2-fold changes) of pairwise donor-to-donor correlations (high/low bins) for each screen. Three human donors were used for all screens except the IFNγ-ABE and CD25-CBE screens, where two were used for analyses. Pearson correlation coefficient is given for each comparison. b , Comparison of sgRNA level effect sizes between CD25 and PD1 screens (left) or CD25 and TNFα screens (right), shown as LFC (log2-fold changes, high/low bins).

Article Snippet: Two micrograms of ABE mRNA mixed with 1.5 μg synthetic modified sgRNA (Synthego) was added per 20 μl cells, not exceeding 25 μl total per reaction.

Techniques: Comparison

Journal: Nature

Article Title: Base-editing mutagenesis maps alleles to tune human T cell functions

doi: 10.1038/s41586-023-06835-6

Figure Lengend Snippet: a , Editing by ABE mRNA and synthetic sgRNA co-electroporation for each sgRNA chosen for validation, assessed by deep amplicon sequencing and analyzed with Crispresso2 54 . Guide sequences are in gray, predicted editing window in green, and PAM in dark gray. b , LFC (log2-fold changes) of levels of the indicated cytokines over control (mean of 2 AAVS1 control gRNAs) measured by intracellular staining and flow cytometry are plotted. n = 6 human donors; mean ± SE; *p < 0.05, **p < 0.01, ***p < 0.001. P-values were derived using a two-tailed independent two-sample t-test. c , Cytokine secretion in culture supernatants for T cells with the indicated base edits were measured by Luminex. Heatmap represents LFC (log2-fold changes) over the mean of cells edited with two AAVS1 control sgRNAs. n = 4 human donors.

Article Snippet: Two micrograms of ABE mRNA mixed with 1.5 μg synthetic modified sgRNA (Synthego) was added per 20 μl cells, not exceeding 25 μl total per reaction.

Techniques: Biomarker Discovery, Electroporation, Amplification, Sequencing, Control, Staining, Flow Cytometry, Derivative Assay, Two Tailed Test, Luminex

Journal: Nature

Article Title: Base-editing mutagenesis maps alleles to tune human T cell functions

doi: 10.1038/s41586-023-06835-6

Figure Lengend Snippet: a , Predicted distribution of fraction of editable residues, within the original 385 genes in the NGG-PAM Cas9 screen, using either WT nCas9 (NGG PAM) or SpG Cas9 (NG PAM) for ABE. Box plots show median, center quartiles, and extremes within 1.5 * IQR. b , Distribution of surface protein expression levels for CD5 or CD7 in pan T cells gated for CD4+ (right) or CD8 + (left) base edited with one (CD5) or two (CD7) ABE NGG sgRNAs targeting each gene and one ABE NG sgRNA targeting each gene plus AAVS1 control. c , LFC (log2 fold changes) in the TNFα screens using WT nCas9 (NGG, y-axis) vs. using SpG Cas9 (NG, x-axis) for 13,334 overlapping guides between the two screens. The average knockout effect of specific genes present in both screens is shown with overlaid blue/red dots and labels. Knockout effects were calculated using the top 3 predicted knockout guides. d , Scatter plots showing LFC (log2-fold changes) of pairwise donor-to-donor correlations for each NG screen. Two human donors were used for all NG screens.

Article Snippet: Two micrograms of ABE mRNA mixed with 1.5 μg synthetic modified sgRNA (Synthego) was added per 20 μl cells, not exceeding 25 μl total per reaction.

Techniques: Expressing, Control, Knock-Out