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Uncovering mechanisms of early human lineage specification by CRISPR/Cas9-mediated genome editing [RNA-seq]

ABSTRACT: Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR?Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocys t development was established, but maintenance was compromised. We conclude that CRISPR?Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE100118 | GEO | 2017/09/21

SECONDARY ACCESSION(S): PRJNA392971

REPOSITORIES: GEO

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Genome editing reveals a role for OCT4 in human preimplantation development [MiSeq]

Project description:Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR?Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocys t development was established, but maintenance was compromised. We conclude that CRISPR?Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

2017-09-21 | GSE100119 | GEO

Cas9 interactions with host RNAs and transposable elements impair neurodevelopment (irCLIP-seq)

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Cas9 interactions with host RNAs and transposable elements impair neurodevelopment (RNA-seq)

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Cas9 interactions with host RNAs and transposable elements impair neurodevelopment (CUT&Tag)

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High-fidelity base editing mediated by Cpf1-cytidine deaminase fusion

Project description:C-to-T base editing mediated by CRISPR/Cas9 base editors (BEs) needs a G/C-rich PAM and the editing fidelity is compromised by unwanted indels and non-C-to-T substitutions. We developed CRISPR/Cpf1-based BEs to recognize a T-rich PAM and induce efficient C-to-T editing with few indels and/or non-C-to-T substitutions. The requirement of editing fidelity in therapeutic-related trials necessitates the development of CRISPR/Cpf1-based BEs, which also facilitates base editing in A/T-rich regions.

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rRNA 2'-O-methylation profile of SNORD71 knockdown cells

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2023-06-16 | E-MTAB-13009 | biostudies-arrayexpress

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Project description:CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce detectable cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics mRNA-Seq from muscles (9 samples; 3 mice x 3 conditions) and lymph nodes (9 samples; 3 mice x 3 conditions).

2016-07-29 | E-GEOD-84534 | biostudies-arrayexpress

The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation

Project description:Human primordial germ cells (hPGCs) are the first embryonic progenitors in the germ cell lineage, yet the molecular mechanisms required for hPGC formation are not well characterized. To identify regulatory regions in hPGC development, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to systematically characterize regions of open chromatin in hPGCs and hPGC-like cells (hPGCLCs) differentiated from human embryonic stem cells (hESCs). We discovered regions of open chromatin unique to hPGCs and hPGCLCs that significantly overlap with TFAP2C-bound enhancers identified in the naive ground state of pluripotency. Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity.

2018-12-27 | GSE120648 | GEO

RNA-seq of wild-type and KLF17-null hESCs during chemical resetting from primed to naive pluripotency

Project description:Bulk RNA-seq of H9 human embryonic stem cells undergoing conversion from primed to naive pluripotency using the chemical/epigenetic resetting method in tt2iL+Go-based media conditions. The dataset includes three wild-type clones (WT1-3) and two KLF17-null clones (KO1-2) generated through CRISPR-Cas9-mediated gene editing. Samples were collected at day 0 (primed cells in mTeSR1 (StemCell Technologies)), then throughout resetting at day 2 (cells in cRM-1), day 8 (cells in cRM-2+XAV939, immediately prior to the first passage), naive passage 5 (p5), 7 (p7) and 10 (p10) (cells in tt2iL+Go).

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