Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.

Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells

A functional variant on 20q13.33 related to glioma risk alters enhancer activity and modulates expression of multiple genes.

Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with glioma risk on 20q13.33, but the biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 20q13.33 impacted the activity of an enhancer, leading to an altered expression of nearby genes. To identify candidate functional SNPs, we identified all SNPs in linkage disequilibrium with the risk-associated SNP rs2297440 that mapped to putative enhancers. Putative enhancers containing candidate functional SNPs were tested for allele-specific effects in luciferase enhancer activity assays against glioblastoma multiforme (GBM) cell lines. An enhancer containing SNP rs3761124 exhibited allele-specific effects on activity. Deletion of this enhancer by CRISPR-Cas9 editing in GBM cell lines correlated with an altered expression of multiple genes, including STMN3, RTEL1, RTEL1-TNFRSF6B, GMEB2, and SRMS. Expression quantitative trait loci (eQTL) analyses using nondiseased brain samples, isocitrate dehydrogenase 1 (IDH1) wild-type glioma, and neurodevelopmental tissues showed STMN3 to be a consistent significant eQTL with rs3761124. RTEL1 and GMEB2 were also significant eQTLs in the context of early CNS development and/or in IDH1 wild-type glioma. We provide evidence that rs3761124 is a functional variant on 20q13.33 related to glioma/GBM risk that modulates the expression of STMN3 and potentially other genes across diverse cellular contexts

Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10-30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesisclos

Using CRISPR Gene Editing to Prevent Accumulation of Lipids in Hepatocytes

CRISPR gene editing is a molecular technology that can be used to silence gene expression. In this experiment, genes that are known to play a role in lipid accumulation in hepatocytes were targeted. Specifically, levels of fatty acid transport proteins 2 and 5 (FATP2 & 5) have been shown to be elevated in cases of non-alcoholic fatty liver disease. The goal of this experiment was to reduce expression of these genes by using a dead Cas9 (dCas9) protein with an attached inhibitory domain (KRAB) that acts on the promotor region. When measuring the mRNA expression, it was determined that the levels of the CRISPR-modified gene products were significantly reduced compared to the control. However, the same extent of inhibition was not consistently observed when conducting flow cytometry. Current work is aimed at discovering why lipid accumulation is not inhibited to the expected degree based on the results of mRNA expression

A Review of Defatting Strategies for Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease is a huge cause of chronic liver failure around the world. This condition has become more prevalent as rates of metabolic syndrome, type 2 diabetes, and obesity have also escalated. The unfortunate outcome for many people is liver cirrhosis that warrants transplantation or being unable to receive a transplant since many livers are discarded due to high levels of steatosis. Over the past several years, however, a great deal of work has gone into understanding the pathophysiology of this disease as well as possible treatment options. This review summarizes various defatting strategies including in vitro use of pharmacologic agents, machine perfusion of extracted livers, and genomic approaches targeting specific proteins. The goal of the field is to reduce the number of necessary transplants and expand the pool of organs available for use

Mitochondrial Role in Oncogenesis and Potential Chemotherapeutic Strategy of Mitochondrial Infusion in Breast Cancer

Triple negative breast cancer (TNBC) is one of the most aggressive cancers diagnosed amongst women with a high rate of treatment failure and a poor prognosis. Mitochondria have been found to be key players in oncogenesis and tumor progression by mechanisms such as altered metabolism, reactive oxygen species (ROS) production and evasion of apoptosis. Therefore, mitochondrial infusion is an area of interest for cancer treatment. Studies in vitro and in vivo demonstrate mitochondrial-mediated reduction in glycolysis, enhancement of oxidative phosphorylation (OXPHOS), reduction in proliferation, and an enhancement of apoptosis as effective anti-tumor therapies. This review focuses on mitochondrial dysregulation and infusion in malignancies, such as TNBC

Transcriptional and Epigenetic Regulation of <em>KIAA1199</em> Gene Expression in Human Breast Cancer

<div><p>Emerging evidence has demonstrated that upregulated expression of <em>KIAA1199</em> in human cancer bodes for poor survival. The regulatory mechanism controlling <em>KIAA1199</em> expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human <em>KIAA1199</em> promoter in terms of regulation of <em>KIAA1199</em> gene expression. A 3.3 kb fragment of human genomic DNA containing the 5′-flanking sequence of the <em>KIAA1199</em> gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic <em>KIAA1199</em> promoter containing a TATA-box close to the transcription start site. A combination of 5′-primer extension study with 5′RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human <em>KIAA1199</em> gene. Bioinformatics analysis suggested that the 1.4 kb <em>KIAA1199</em> promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for <em>KIAA1199</em> gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these <em>cis</em>- and <em>trans</em>-acting elements in controlling <em>KIAA1199</em> gene expression. Finally, we found that upregulated <em>KIAA1199</em> expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of <em>KIAA1199</em> expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling <em>KIAA1199</em> gene expression in human cancer.</p> </div