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Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons.

Heman-Ackah SM, Bassett AR, Wood MJ - Sci Rep (2016)

Bottom Line: The application of CRISPR/Cas9 to mammalian systems is revolutionizing the utilization of genome editing technologies in the study of molecular contributors to the pathogenesis of numerous diseases.Here, we demonstrate that CRISPRa and CRISPRi can be used to exert precise modulations of endogenous gene expression in fate-committed iPSC-derived neurons.This highlights CRISPRa/i as a major technical advancement in accessible tools for evaluating the specific contributions of critical neurodegenerative disease-related genes to neuropathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3QX, UK.

ABSTRACT
The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and the subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Neurons differentiated from patient-specific iPSCs provide a means to recapitulate molecular phenotypes of neurodegenerative diseases in vitro. However, it remains challenging to conduct precise manipulations of gene expression in iPSC-derived neurons towards modeling complex human neurological diseases. The application of CRISPR/Cas9 to mammalian systems is revolutionizing the utilization of genome editing technologies in the study of molecular contributors to the pathogenesis of numerous diseases. Here, we demonstrate that CRISPRa and CRISPRi can be used to exert precise modulations of endogenous gene expression in fate-committed iPSC-derived neurons. This highlights CRISPRa/i as a major technical advancement in accessible tools for evaluating the specific contributions of critical neurodegenerative disease-related genes to neuropathogenesis.

No MeSH data available.


Related in: MedlinePlus

Effect of SNCA TSS2-1 sgRNA-Mediated CRISPRi on TSS Isoform-Specific mRNA Expression Levels.(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.
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f2: Effect of SNCA TSS2-1 sgRNA-Mediated CRISPRi on TSS Isoform-Specific mRNA Expression Levels.(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.

Mentions: We next tested whether CRISPRi could induce efficient silencing of SNCA via inhibition of transcription initiation. Alpha-synuclein is expressed from at least four different promoters (Fig. 2a) based on EST sequences, cap analysis of gene expression (CAGE) from the FANTOM5 consortium4142 and annotation in the Eukaryotic Promoter Database (EPDnew)43. We designed sgRNAs to the three TSSs annotated in EPDnew (human version 003). Specifically, sgRNAs were designed to align directly over each TSS, immediately upstream or immediately downstream on the non-template (NT) strand (Fig. 1a). We screened these sgRNAs for repressive activity in HEK293T cells and found the TSS2-1 (NT) sgRNA (henceforth TSS2-1) to have strong repressive activity when transfected alone, or within any combination of SNCA TSS-targeting sgRNAs (Fig. 1b). This is consistent with analysis of CAGE tags (Fig. 2a) and our own qRT-PCR data (Fig. 2b), which suggest that in HEK293T cells, transcription initiation is predominantly driven from this TSS. Analysis of CAGE tags from other tissues further indicates that SNCA transcription initiation in human brain tissue, neural stem cells and neurons is primarily from this TSS (Supplementary Table S3). Interestingly, despite sgRNA targeting to only one annotated TSS, the TSS2-1 sgRNA induced efficient silencing of spliced transcripts derived from all SNCA TSSs (Fig. 2c). Similar silencing of all TSS isoforms was observed when we analyzed nascent transcription by qRT-PCR with primers spanning specific exon-intron boundaries (Fig. 2d), suggesting coupling of expression from the three TSSs. The repressive effect on nascent transcripts further indicates that silencing by CRISPRi occurs at the level of transcription initiation, or the transition from initiation to elongation, rather than any post-transcriptional effect on the mRNA.


Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons.

Heman-Ackah SM, Bassett AR, Wood MJ - Sci Rep (2016)

Effect of SNCA TSS2-1 sgRNA-Mediated CRISPRi on TSS Isoform-Specific mRNA Expression Levels.(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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920027&req=5

f2: Effect of SNCA TSS2-1 sgRNA-Mediated CRISPRi on TSS Isoform-Specific mRNA Expression Levels.(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.
Mentions: We next tested whether CRISPRi could induce efficient silencing of SNCA via inhibition of transcription initiation. Alpha-synuclein is expressed from at least four different promoters (Fig. 2a) based on EST sequences, cap analysis of gene expression (CAGE) from the FANTOM5 consortium4142 and annotation in the Eukaryotic Promoter Database (EPDnew)43. We designed sgRNAs to the three TSSs annotated in EPDnew (human version 003). Specifically, sgRNAs were designed to align directly over each TSS, immediately upstream or immediately downstream on the non-template (NT) strand (Fig. 1a). We screened these sgRNAs for repressive activity in HEK293T cells and found the TSS2-1 (NT) sgRNA (henceforth TSS2-1) to have strong repressive activity when transfected alone, or within any combination of SNCA TSS-targeting sgRNAs (Fig. 1b). This is consistent with analysis of CAGE tags (Fig. 2a) and our own qRT-PCR data (Fig. 2b), which suggest that in HEK293T cells, transcription initiation is predominantly driven from this TSS. Analysis of CAGE tags from other tissues further indicates that SNCA transcription initiation in human brain tissue, neural stem cells and neurons is primarily from this TSS (Supplementary Table S3). Interestingly, despite sgRNA targeting to only one annotated TSS, the TSS2-1 sgRNA induced efficient silencing of spliced transcripts derived from all SNCA TSSs (Fig. 2c). Similar silencing of all TSS isoforms was observed when we analyzed nascent transcription by qRT-PCR with primers spanning specific exon-intron boundaries (Fig. 2d), suggesting coupling of expression from the three TSSs. The repressive effect on nascent transcripts further indicates that silencing by CRISPRi occurs at the level of transcription initiation, or the transition from initiation to elongation, rather than any post-transcriptional effect on the mRNA.

Bottom Line: The application of CRISPR/Cas9 to mammalian systems is revolutionizing the utilization of genome editing technologies in the study of molecular contributors to the pathogenesis of numerous diseases.Here, we demonstrate that CRISPRa and CRISPRi can be used to exert precise modulations of endogenous gene expression in fate-committed iPSC-derived neurons.This highlights CRISPRa/i as a major technical advancement in accessible tools for evaluating the specific contributions of critical neurodegenerative disease-related genes to neuropathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3QX, UK.

ABSTRACT
The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and the subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Neurons differentiated from patient-specific iPSCs provide a means to recapitulate molecular phenotypes of neurodegenerative diseases in vitro. However, it remains challenging to conduct precise manipulations of gene expression in iPSC-derived neurons towards modeling complex human neurological diseases. The application of CRISPR/Cas9 to mammalian systems is revolutionizing the utilization of genome editing technologies in the study of molecular contributors to the pathogenesis of numerous diseases. Here, we demonstrate that CRISPRa and CRISPRi can be used to exert precise modulations of endogenous gene expression in fate-committed iPSC-derived neurons. This highlights CRISPRa/i as a major technical advancement in accessible tools for evaluating the specific contributions of critical neurodegenerative disease-related genes to neuropathogenesis.

No MeSH data available.


Related in: MedlinePlus