Dataset Information

Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes.

ABSTRACT: CRISPR/Cas9-based approaches have greatly facilitated targeted genomic deletions. Contrary to coding genes however, which can be functionally knocked out by frame-shift mutagenesis, non-coding RNA (ncRNA) gene knockouts have remained challenging. Here we present a universal ncRNA knockout approach guided by epigenetic hallmarks, which enables robust gene silencing even in provisionally annotated gene loci. We build on previous work reporting the presence of overlapping histone H3 lysine 4 tri-methylation (H3K4me3) and DNaseI hypersensitivity sites around the transcriptional start sites of most genes. We demonstrate that excision of this gene-proximal signature leads to loss of microRNA and lincRNA transcription and reveals ncRNA phenotypes. Exemplarily we demonstrate silencing of the constitutively transcribed MALAT1 lincRNA gene as well as of the inducible miR-146a and miR-155 genes in human monocytes. Our results validate a role of miR-146a and miR-155 in negative feedback control of the activity of inflammation master-regulator NF?B and suggest that cell-cycle control is a unique feature of miR-155. We suggest that our epigenetically guided CRISPR approach may improve existing ncRNA knockout strategies and contribute to the development of high-confidence ncRNA phenotyping applications.

SUBMITTER: Janga H

PROVIDER: S-EPMC5815609 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes.

Janga Harshavardhan H Aznaourova Marina M Boldt Fabian F Damm Katrin K Grünweller Arnold A Schulte Leon N LN

PloS one 20180216 2

CRISPR/Cas9-based approaches have greatly facilitated targeted genomic deletions. Contrary to coding genes however, which can be functionally knocked out by frame-shift mutagenesis, non-coding RNA (ncRNA) gene knockouts have remained challenging. Here we present a universal ncRNA knockout approach guided by epigenetic hallmarks, which enables robust gene silencing even in provisionally annotated gene loci. We build on previous work reporting the presence of overlapping histone H3 lysine 4 tri-me ...[more]

PMID: 29451908

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Project description:Ischial avulsion fractures classically occur in the pediatric population and are relatively uncommon. These injuries are treated conservatively; however, in cases where there is greater than 2 cm of displacement, surgical intervention is recommended. In some cases, displaced fractures are either misdiagnosed or proper treatment is neglected, and patients who transition into adulthood are left with chronic nonunions that can become a source of pain and disability. Here we present a surgical technique for a chronic ischial avulsion fracture nonunion that is excised, and the hamstring tendons are then primarily repaired to the ischium using suture anchors. Technique Video Video 1 This is a video for the surgical intervention for a patient with a chronic right sided ischial avulsion fracture. The patient is placed in the prone position with all bony prominences padded. After a standard sterile prep and drape of the right lower extremity, an 8 to 10 cm incision is made in the gluteal crease, overlying the right-sided ischium. Electrocautery is used to reveal the underlying gluteal fascia. The gluteal fascia is incised using dissecting scissors. A Schnitt is used to open the interval inferior to the gluteus maximus muscle, and a large retractor is placed. A sciatic neurolysis is performed. The chronic avulsion fracture is revealed using electrocautery. A combination of electrocautery and a cobb elevator is used to free the bony fragment. After removal, the eburnated surface of the ischium is revealed. A combination of curettes and osteotomes are used to create a bleeding bone bed on the native ischium. Next, two 2.8 mm Q-fix (Smith and Nephew, Andover, MA) double-loaded all-suture anchors are placed into the native ischium. From each anchor, 1 suture limb from 1 pair is passed through the hamstring tendon in a locking fashion while the other is left free. From the other suture pair from the same anchor, one limb is passed in a modified Mason-Allen technique, and the other limb is left free. This is repeated for the other anchor. Using the free limbs of the anchors, the tendon is delivered to the ischium. The corresponding suture limbs are tied, and the free ends are cut with a knife. After copious irrigation a multilayer closure is performed. Dermabond is placed on the skin and a dry sterile dressing. After surgery the patient is made flat foot weightbearing in a hip orthosis and is started on chemical anticoagulation. At the first postoperative visit the wound is evaluated, and an X-ray film is obtained. (Soundtrack: Morning Routine by Ghostrifter Official | https://soundcloud.com/ghostrifter-official Music promoted by https://www.chosic.com/free-music/all/Creative Commons CC BY-SA 3.0 https://creativecommons.org/licenses/by-sa/3.0/)

Dataset Information

Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes.

Publications

Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes.

Similar Datasets

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