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Divergent transcription from lentiviral gene editing of cancer cells

ABSTRACT: Divergent transcription from lentiviral gene editing of cancer cells

PROVIDER: PRJNA776033 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16620500_1.fastq.gz	Fastqsanger.gz
	SRR16620500_2.fastq.gz	Fastqsanger.gz
	SRR16620501_1.fastq.gz	Fastqsanger.gz
	SRR16620501_2.fastq.gz	Fastqsanger.gz
	SRR16620502_1.fastq.gz	Fastqsanger.gz

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Divergent transcription from lentiviral gene editing of cancer cells

Project description:We explored a gene editing approach to depleting ephrinB2 from colorectal cancer cells using an inducible lentiviral vector encoding a short hairpin (sh) RNA for ephrinB2 silencing via RNA interference. We discovered that lentiviral integration in the host DNA frequently drives divergent gene transcription generating antisense reads by forcing splicing and read-through into the host DNA resulting in expression of chimeric vector/host transcripts.

2022-05-25 | GSE186796 | GEO

Lentiviral CRISPR Epigenome Editing of Inflammatory Receptors as a Gene Therapy Strategy for Disc Degeneration

Project description:Background: Degenerative disc disease (DDD) is a primary contributor to low back pain, a leading cause of disability. Progression of DDD is aided by inflammatory cytokines in the intervertebral disc (IVD), particularly TNF-α and IL-1β, but current treatments fail to effectively target this mechanism. The objective of this study was to explore the feasibility of CRISPR epigenome editing based therapy for DDD, by modulation of TNFR1/IL1R1 signaling in pathological human IVD cells. Methods: Human IVD cells from the nucleus pulposus of patients receiving surgery for back pain were obtained and the regulation of TNFR1/IL1R1 signaling by a lentiviral CRISPR epigenome editing system was tested. These cells were tested for successful lentiviral transduction/expression of dCas9-KRAB system and regulation of TNFR1/IL1R1 expression. TNFR1/IL1R1 signaling disruption was investigated via measurement of NF-κB activity, apoptosis, and anabolic/catabolic changes in gene expression post inflammatory challenge. Results: CRISPR epigenome editing systems were effectively introduced into pathological human IVD cells and significantly downregulated TNFR1 and IL1R1. This downregulation significantly attenuated deleterious TNFR1 signaling but not IL1R1 signaling. This is attributed to less robust IL1R1 expression downregulation, and IL-1β driven reversal of IL1R1 expression downregulation in a portion of patient IVD cells. Additionally, RNAseq data indicated a novel transcription factor targets, IRF1 and TFAP2C, as being a primary regulators of inflammatory signaling in IVD cells. Discussion: These results demonstrate the feasibility of CRISPR epigenome editing of inflammatory receptors in pathological IVD cells, but highlight a limitation in epigenome targeting of IL1R1. This method has potential application as a novel gene therapy for DDD, to attenuate the deleterious effect of inflammatory cytokines present in the degenerative IVD.

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Measuring gene expression and RNA editing in Drosophila adapting to divergent microclimates

Project description:We performed RNA-seq to profile gene expression in the heads and whole bodies of 32 isofemale fly lines from two divergent microclimates at 'Evolution Canyon' in Israel (16 fly lines from each microclimate). We also measured RNA editing levels in the head tissue of these flies.

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CTCF blocks anti-sense transcription initiation at divergent gene promoters [ChIP-Seq]

Project description:Transcription is typically divergent, initiating at closely spaced oppositely oriented core promoters to produce sense and unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and coordinated with sense transcription is largely unknown. Here by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters, the great majority of which bear proximal CTCF binding sites. Genome editing, chromatin conformation studies and 5’ transcript mapping revealed that CTCF directly suppresses uasTrx initiation in manner independent of its chromatin architectural function. Primary transcript RNA FISH revealed co-bursting of sense and anti-sense transcripts is disfavored, suggesting CTCF-regulated competition for transcription initiation. In sum, CTCF shapes the noncoding transcriptional landscape by suppressing upstream antisense transcription.

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Genetic stability in FA cells before and after gene editing and cell reprogramming.

Project description:Fibroblasts from a Fanconi anemia (FA) patient (FA-52) before and after correction by gene editing and transduction with a lentiviral vector expressing telomerase (geFA-52T fibroblasts). IPCs were generated from fibroblasts corrected by gene editing using STEMCCA LV (geFA-52T IPSCs clone 16) and finally the reprogramming cassette was excised (excised geFA-52T IPSCs clone 16.1) Four groups: Fibroblasts from a FA patient (FA-52) before and after correction by gene editing and transduction with a lentiviral vector expressing telomerase (geFA-52T) and gene editied IPSCs before and after excision of the reprogramming cassette (geFA-52T IPSCs clone 16 and excised geFA-52T IPSCs clone 16.1)

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Lentiviral CRISPR Epigenome Editing of Inflammatory Receptors as a Gene Therapy Strategy for Disc Degeneration

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