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Brad's Most Recent Paper of the Day
Category:   Cool Techniques Genome Editing Molecular Biology Cell Biology Transcription

Robustness of Catalytically-Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells

Journal: Molecular Therapy Nucleic Acids  Publication year: 2020

The original application of the CRISPR/Cas9 system was to generate precisely targeted double-strand breaks that could be repaired by non-homologous end joining or by homology-directed repair when a suitable repair template was provided. Numerous tweaks to this initial system have led to a wide variety of additional applications. The focus of this paper is on the use of catalytically dead Cas9 (dCas9)  fused to transcriptional activators, which can be recruited to specific genomic locations by single-guide RNAs to activate locus-specific transcription. The authors compare first-generation dCas9-VP64 and second-generation dCas9-SAM and dCas9-SunTag to induce gene expression in human pluripotent stem cells and human mesenchymal stem cells. The authors find that all systems induced specific and potent gene expression, but the 2nd-generation systems yielded higher and more consistent increases in expression of target genes.

Category:   Cool Techniques Epigenetics Transcription Cell Biology

ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions

Journal: Molecular Cell      Publication year: 2020

This is another new technique for investigating chromatin accessibility by using an ATAC-based approach. The cool new alteration to the technique is the incorporation of DNA methylation analysis, which allows for the analysis of chromatin accessibility and DNA methylation from single library preparations. Intriguingly, this technique revealed a disconnect between chromatin accessibility, methylation status and gene activity when it was used to investigate chromatin and transcriptional changes occurring during the differentiation of monocytes to macrophages over a defined time course.

Category:   Cool Techniques Molecular Biology Childhood genetic diseases Cell Biology

Extracellular nanovesicles for packaging of CRISPRCas9 protein and sgRNA to induce therapeutic exon skipping

Journal: Nature Communications    Publication year: 2020

In this paper, the authors describe an elegant system for packaging CRISPRCas9 protein and sgRNAs into extracellular nanovesicles. The extracellular nanovesicle system is termed NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo). The system employs chemically induced dimerization of FKBP12 and FRB(T2098L) domains with the rapamycin analogue AP21967 to recruit FRB-SpCas9 to an FKBP12-membrane anchoring protein. HIV Tat and Psi+ are used to actively package  ribozyme-sgRNA-ribozyme sequences into the nanovesicles. The authors show that their system is capable of efficiently editing hard-to-transfect cells such as neurons and myoblasts and can successfully induce permanent genomic exon skipping in mouse models (including a the mdx model of muscular dystrophy), which suggests that this system may be of great utility for the therapeutic delivery of genome editing machinery to cells and tissues.

Category:   Cell Biology Development Cancer

Of Cell Shapes and Motion: The Physical Basis of Animal Cell Migration

Journal:  Developmental Cell  Publication year: 2020

I have been working with cells in culture for many years and am used to viewing them under a light microscope, where cells like fibroblasts or embryonic stem cells appear to be more-or-less stationary over minutes of viewing. When the same cells are viewed over a time-course by using time-lapse imaging, it becomes very clear that individual cells within a population are anything but stationary! The processes by which cells move about on cell culture plates, extracellular matrix, or other surfaces are beginning to be understood at increasingly detailed levels. In this review, the authors provide a comprehensive overview of the mechanisms employed by metazoan cells to confer motility. The authors discuss the implications of different modes of cellular migration in normal development and in pathological events such as cancer cell metastasis.

Category:   Cancer Transcription Post-transcriptional Regulation Epigenetics Cell Biology

m6A Modification in Coding and Non-coding RNAs: Roles and Therapeutic Implications in Cancer

Journal:  Cancer Cell   Publication year: 2020

N6-Methyladenosine (m6A) RNA modification is a reversible modification that occurs on mRNA and lncRNA transcripts. This review describes the enzymes responsible for methylating and demethylating m6A, "readers" of m6A-marked RNA, and the impact m6A modification can have on transcript function. For those studying transcriptional and post-transcriptional regulation this review serves as an excellent resource to help one catch up on what is known about m6A modification of coding and non-coding RNAs.

Category:   Cool Techniques Molecular Biology Genome Editing

Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

Journal: Cell Reports   Publication year: 2020

This is a "cool techniques" paper that provides an easy and quick way with which to detect precision genome editing events. The authors' DTECT (Dinucleotide signaTurE CapTure) technique uses the type IIS restriction enzyme AcuI as a tool with which to evaluate target sequences in their wild-type and genome-edited states. This is a great addition to the toolbox of those employing precision genome editing in their research.

Category:   Stem Cells Regenerative Medicine Wnt Signaling Development Proteomics Cell Biology

Proteomic profiling of stem cell tissues during regeneration of deer antler - a model of mammalian organ regeneration

Journal: Journal of Proteome Research Publication year: 2020

I was only made aware of the regenerative properties of deer antler late last year at the Till & McCulloch Stem Cell meeting in Montreal. Deer antlers regenerate themselves annually and studying the details of this process could provide insights to guide new regenerative medicine strategies, especially for tissues generated from neural crest-derived stem cells. In this paper, the authors use a label-free proteomic approach to compare the protein expression profiles in different stem cell tissues of the antler. The datasets the authors generated are readily available and will be a useful resource for those studying regeneration. For those interested in Wnt and Hippo signaling, these pathways featured prominently in the data obtained from "activated" antler stem cells.

Category:   Stem Cells Cell Biology Wnt Signaling Transcription

Ascl2-Dependent Cell Dedifferentiation Drives Regeneration of Ablated Intestinal Stem Cells

Journal: Cell Stem Cell    Publication year: 2020

In this paper, the authors use lineage tracing and selective ablation of Lgr5(+) intestinal stem cells to show that intestinal stem cell regeneration occurs through dedifferentiation of recently spawned stem cell descendants. This finding challenges the previously held view that +4 reserve stem cells were responsible for replacing ablated Lgr5(+) stem cells of the intestinal crypt. The authors highlight the importance of the transcription factor Ascl2 in mediating the dedifferentiation required to give rise to nascent replacement stem cells.

Category:   Cancer Stem Cells Wnt Signaling Cell Biology Cool Techniques

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer

Journal:  Cell Stem Cell   Publication year: 2020

This is another cool paper that is highly relevant to those studying cancer stem cell (CSC) biology, especially in cases where Lgr5-positive CSCs are involved. The authors examine the progression of colorectal cancer (CRC) in a mouse model comprising tamoxifen-inducible Villin-driven Cre, LoxP-flanked APC, KRAS-(LSL-G12D), TP53(KO/KO), Lgr5(DTR-eGFP), and R26R-confetti, which allows for the visualization of LGR5-positive cancer stem cells and selective ablation of these CSCs by using diphtheria toxin. By using their elegant system, the authors find that the tumour cells that migrate, disseminate, and colonize metastases are mainly Lgr5-negative, and Lgr5-negative metastases have the ability to develop an epithelial hierarchy, due to their intrinsic phenotypic plasticity. The authors also use human-mouse xenografts to substantiate the relevance of their mouse model findings to human CRC. This paper has important implications for treatment strategies focused on CSCs and CSC-maintaining niche factors, which do not generally consider endogenous cellular plasticity during the metastatic program.

Category:   Cool Techniques Cell Biology Molecular Biology Post-transcriptional regulation Transcription

The biogenesis, biology and characterization of circular RNAs

Journal: Nature Reviews Genetics     Publication year: 2019

Based on some recent discoveries in the lab, I have become very interested in non-coding RNAs, especially long-non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). This is a recent review on circular RNAs that provides a nice overview of our current understanding of the biology of circular RNAs and the ways in which circular RNAs are studied. Circular RNAs are important regulators of cellular function and are known to be dysregulated in several human diseases. Their circular nature makes them somewhat difficult to study, but new bioinformatic tools specifically designed for circRNA characterization and quantitation are helping researchers investigate these molecules. I highly recommend this review for those unfamiliar with circRNAs.

Category:   Cool Techniques Wnt Signaling Cancer Development Epigenetics Transcription

Aberrant Development Corrected in Adult-Onset Huntington’s Disease iPSC-Derived Neuronal Cultures via WNT Signaling Modulation

Journal: Stem Cell Reports    Publication year: 2020

This paper contains many of my favourite things: Wnt signaling, stem cells, developmental biology, single-cell sequencing, and mechanisms of human disease. The authors used differentiation assays in which induced pluripotent stem cells were directed to generate cultures comprising primarily medium spiny neurons, the most affected cell type in Huntington's disease. By using single-cell and bulk transcription and epigenomic analyses, the authors found that iPSCs derived from adult-onset Huntington's disease patients did not differentiate properly into medium spiny neurons but rather produced a population of persistent cyclin D1-positive neural stem cells. Inhibiting Wnt signaling by using ICG-001, which prevents the interaction between β-catenin and the transcriptional activator, CREB-binding protein, preserved neuronal differentiation but abrogated the persistence of neural stem cells. Taken together, the data in this paper set the stage for the development of potential therapeutic strategies for adult-onset Huntington's disease that target β-catenin-mediated transcription.

Category:   Cell Biology Development Epigenetics Post-translational modifications

SUMO Chains Rule on Chromatin Occupancy

Journal: Frontiers in Cell and Developmental Biology Publication year: 2020

I have recently become very interested in sumoylation, a post-translational modification similar to ubiquitination, in which SUMO polypeptides are added to target proteins on target lysine residues. This is a nice mini-review that I found to be very useful in providing me with a quick overview of the role of sumoylation in chromatin occupancy. More SUMO-related reviews will no doubt show up in my list of "papers of the day" as I'll be reading many of them to get up to speed on what is currently known about sumoylation in contexts of relevance to my lab's research.

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