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Uncovering mechanisms of early human lineage specification by CRISPR/Cas9-mediated genome editing

ABSTRACT: Uncovering mechanisms of early human lineage specification by CRISPR/Cas9-mediated genome editing

PROVIDER: PRJNA392661 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5710350_1.fastq.gz	Fastqsanger.gz
	SRR5710350_2.fastq.gz	Fastqsanger.gz
	SRR5710360_1.fastq.gz	Fastqsanger.gz
	SRR5710360_2.fastq.gz	Fastqsanger.gz
	SRR5710370_1.fastq.gz	Fastqsanger.gz

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

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 | GSE100118 | GEO

Uncovering mechanisms of early human lineage specification by CRISPR/Cas9-mediated genome editing

Project description:This SuperSeries is composed of the SubSeries listed below.

2017-09-21 | GSE100120 | GEO

Targeted DNA-seq of Human HepG2 cells following editing with CRISPR/Cas9

Project description:We performed a large-scale genome-wide characterisation of indels generated following editing with CRISPR/Cas9. We used pools of sgRNAs and performed targeted capture and sequencing of the edited regions in HepG2 cells.

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Increase genome editing efficiency by Cas9 transcriptional activator and histone acetyltransferase activator

Project description:The CRISPR/Cas9 system shows diverse levels of genome editing activities on eukaryotic genomic DNA targets, and experiments desire high-efficiency targets. Here we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing activity. However, the strategy that fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR/Cas9 targets in Lenti-X 293T cells. Because histone acetylation increases eukaryotic chromatin accessibility, we further improve genome editing by elevating histone acetylation. We demonstrate that promoting histone acetylation using histone acetyltransferase (HAT) activator YF-2 can improve genome editing from Cas9 and more robustly from the Cas9 transcriptional activator. This provides a strategy to improve CRISPR/Cas9 genome editing activity and enables broader gRNA target choices in eukaryotes.

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Project description:CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2,500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

2022-06-07 | GSE178440 | GEO

Genome-wide specificity of genome editing by both CRISPR-Cas9 and TALEN in the model plant Physcomitrium patens

Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.

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