scholarly journals Using CRISPR to understand and manipulate gene regulation

Development ◽  
2021 ◽  
Vol 148 (9) ◽  
Author(s):  
Ersin Akinci ◽  
Marisa C. Hamilton ◽  
Benyapa Khowpinitchai ◽  
Richard I. Sherwood
Keyword(s):  
Gene Regulation ◽  
Cell Fate ◽  
Disease Modeling ◽  
Cell Types ◽  
Future Application ◽  
Gene Dysregulation ◽  
Epigenetic Code ◽  
Gene Regulatory ◽  
Correct Level ◽  
Biology Research

ABSTRACT Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.

Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation

2018 ◽  
Vol 24 (1) ◽  
pp. 41-54 ◽  
Author(s):  
Duncan M. Chadly ◽  
Andrew M. Oleksijew ◽  
Kyle S. Coots ◽  
Jose J. Fernandez ◽  
Shun Kobayashi ◽  
...  
Keyword(s):  
Cell Fate ◽  
Soft Lithography ◽  
Disease Modeling ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Adherent Cell ◽  
Auditory Neuron ◽  
Therapeutic Tools ◽  
On Chip ◽  
Full Factorial

Human pluripotent stem cells (hPSCs) are promising therapeutic tools for regenerative therapies and disease modeling. Differentiation of cultured hPSCs is influenced by both exogenous factors added to the cultures and endogenously secreted molecules. Optimization of protocols for the differentiation of hPSCs into different cell types is difficult because of the many variables that can influence cell fate. We present microfluidic devices designed to perform three- and four-factor, two-level full factorial experiments in parallel for investigating and directly optimizing hPSC differentiation. These devices feature diffusion-isolated, independent culture wells that allow for control of both exogenous and endogenous cellular signals and that allow for immunocytochemistry (ICC) and confocal microscopy in situ. These devices are fabricated by soft lithography in conjunction with 3D-printed molds and are operable with a single syringe pump, eliminating the need for specialized equipment or cleanroom facilities. Their utility was demonstrated by on-chip differentiation of hPSCs into the auditory neuron lineage. More broadly, these devices enable multiplexing for experimentation with any adherent cell type or even multiple cell types, allowing efficient investigation of the effects of medium conditions, pharmaceuticals, or other soluble reagents.

scholarly journals Specification of cell fate in the sea urchin embryo: summary and some proposed mechanisms

Development ◽  
1998 ◽  
Vol 125 (17) ◽  
pp. 3269-3290 ◽  
Author(s):  
E.H. Davidson ◽  
R.A. Cameron ◽  
A. Ransick
Keyword(s):  
Cell Fate ◽  
Sea Urchin ◽  
Cell Types ◽  
Expression Of Genes ◽  
Sea Urchin Embryo ◽  
Indirect Development ◽  
Genes Encoding ◽  
Stage Embryo ◽  
Gene Regulatory ◽  
Signaling Interactions

An early set of blastomere specifications occurs during cleavage in the sea urchin embryo, the result of both conditional and autonomous processes, as proposed in the model for this embryo set forth in 1989. Recent experimental results have greatly illuminated the mechanisms of specification in some early embryonic territories, though others remain obscure. We review the progressive process of specification within given lineage elements, and with reference to the early axial organization of the embryo. Evidence for the conditional specification of the veg2 lineage subelement of the endoderm and other potential interblastomere signaling interactions in the cleavage-stage embryo are summarized. Definitive boundaries between mesoderm and endoderm territories of the vegetal plate, and between endoderm and overlying ectoderm, are not established until later in development. These processes have been clarified by numerous observations on spatial expression of various genes, and by elegant lineage labeling studies. The early specification events depend on regional mobilization of maternal regulatory factors resulting at once in the zygotic expression of genes encoding transcription factors, as well as downstream genes encoding proteins characteristic of the cell types that will much later arise from the progeny of the specified blastomeres. This embryo displays a maximal form of indirect development. The gene regulatory network underlying the embryonic development reflects the relative simplicity of the completed larva and of the processes required for its formation. The requirements for postembryonic adult body plan formation in the larval rudiment include engagement of a new level of genetic regulatory apparatus, exemplified by the Hox gene complex.

scholarly journals Mit Einzelzell-Genomik die Entscheidungen von Zellen verfolgen

BIOspektrum ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 25-27
Author(s):  
Bo Hu ◽  
Jan Philipp Junker
Keyword(s):  
Developmental Biology ◽  
Embryonic Development ◽  
Cell Fate ◽  
Cell Types ◽  
Regulatory Mechanisms ◽  
Major Question ◽  
Cell Fate Decisions ◽  
New Methods ◽  
Gene Regulatory ◽  
Different Cell Types

AbstractDuring embryonic development, cells need to take a series of successive fate decisions in order to reach their final differentiated stage. Understanding the processes that give rise to the multitude of different cell types in an organism is a major question in developmental biology. New methods in single cell genomics enable researchers to decipher the transcriptional programs and gene regulatory mechanisms that underlie cell fate decisions during embryonic development.

scholarly journals Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

2016 ◽  
Vol 113 (3) ◽  
pp. E291-E299 ◽  
Author(s):  
James E. Dixon ◽  
Gizem Osman ◽  
Gavin E. Morris ◽  
Hareklea Markides ◽  
Michael Rotherham ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Disease Modeling ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Cell Penetrating Peptides ◽  
Cell Labeling ◽  
Human Escs ◽  
Native Proteins

Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.

scholarly journals Absolute quantification of transcription factors reveals principles of gene regulation in erythropoiesis

2019 ◽  
Author(s):  
Mark A. Gillespie ◽  
Carmen G. Palii ◽  
Daniel Sanchez-Taltavull ◽  
Paul Shannon ◽  
William J.R. Longabaugh ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Transcription Factors ◽  
Cell Fate ◽  
Regulatory Network ◽  
Copy Number ◽  
Absolute Quantification ◽  
Cellular Processes ◽  
Gene Regulatory ◽  
Surprising Discovery

SummaryDynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). Yet, despite years of studies we still do not know the protein copy number of TFs in the nucleus. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first Gene Regulatory Network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that in the nucleus, corepressors are dramatically more abundant than coactivators at the protein, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

scholarly journals ANANSE: An enhancer network-based computational approach for predicting key transcription factors in cell fate determination

2020 ◽  
Author(s):  
Quan Xu ◽  
Georgios Georgiou ◽  
Gert Jan C. Veenstra ◽  
Huiqing Zhou ◽  
Simon J. van Heeringen
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Cell Types ◽  
Cell Fate Determination ◽  
Cell Type ◽  
Fate Determination ◽  
Cell Type Specific ◽  
Gene Regulatory

AbstractProper cell fate determination is largely orchestrated by complex gene regulatory networks centered around transcription factors. However, experimental elucidation of key transcription factors that drive cellular identity is currently often intractable. Here, we present ANANSE (ANalysis Algorithm for Networks Specified by Enhancers), a network-based method that exploits enhancer-encoded regulatory information to identify the key transcription factors in cell fate determination. As cell type-specific transcription factors predominantly bind to enhancers, we use regulatory networks based on enhancer properties to prioritize transcription factors. First, we predict genome-wide binding profiles of transcription factors in various cell types using enhancer activity and transcription factor binding motifs. Subsequently, applying these inferred binding profiles, we construct cell type-specific gene regulatory networks, and then predict key transcription factors controlling cell fate conversions using differential gene networks between cell types. This method outperforms existing approaches in correctly predicting major transcription factors previously identified to be sufficient for trans-differentiation. Finally, we apply ANANSE to define an atlas of key transcription factors in 18 normal human tissues. In conclusion, we present a ready-to-implement computational tool for efficient prediction of transcription factors in cell fate determination and to study transcription factor-mediated regulatory mechanisms. ANANSE is freely available at https://github.com/vanheeringen-lab/ANANSE.

scholarly journals Two transcription factors, Pou4f2 and Isl1, are sufficient to specify the retinal ganglion cell fate

2015 ◽  
Vol 112 (13) ◽  
pp. E1559-E1568 ◽  
Author(s):  
Fuguo Wu ◽  
Tadeusz J. Kaczynski ◽  
Santhosh Sethuramanujam ◽  
Renzhong Li ◽  
Varsha Jain ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Ganglion Cells ◽  
Ectopic Expression ◽  
Cell Types ◽  
Retinal Cell ◽  
Retinal Ganglion ◽  
Pou Domain ◽  
Gene Enhancer ◽  
Gene Regulatory

As with other retinal cell types, retinal ganglion cells (RGCs) arise from multipotent retinal progenitor cells (RPCs), and their formation is regulated by a hierarchical gene-regulatory network (GRN). Within this GRN, three transcription factors—atonal homolog 7 (Atoh7), POU domain, class 4, transcription factor 2 (Pou4f2), and insulin gene enhancer protein 1 (Isl1)—occupy key node positions at two different stages of RGC development. Atoh7 is upstream and is required for RPCs to gain competence for an RGC fate, whereas Pou4f2 and Isl1 are downstream and regulate RGC differentiation. However, the genetic and molecular basis for the specification of the RGC fate, a key step in RGC development, remains unclear. Here we report that ectopic expression of Pou4f2 and Isl1 in the Atoh7-null retina using a binary knockin-transgenic system is sufficient for the specification of the RGC fate. The RGCs thus formed are largely normal in gene expression, survive to postnatal stages, and are physiologically functional. Our results indicate that Pou4f2 and Isl1 compose a minimally sufficient regulatory core for the RGC fate. We further conclude that during development a core group of limited transcription factors, including Pou4f2 and Isl1, function downstream of Atoh7 to determine the RGC fate and initiate RGC differentiation.

scholarly journals Mechanisms of miRNA-Mediated Gene Regulation from Common Downregulation to mRNA-Specific Upregulation

2014 ◽  
Vol 2014 ◽  
pp. 1-15 ◽  
Author(s):  
Ayla Valinezhad Orang ◽  
Reza Safaralizadeh ◽  
Mina Kazemzadeh-Bavili
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Regulatory Gene ◽  
Gene Families ◽  
Protein Translation ◽  
Cell Types ◽  
Posttranscriptional Gene Regulation ◽  
Gene Regulatory ◽  
To Come ◽  
Different Cell Types

Discovered in 1993, micoRNAs (miRNAs) are now recognized as one of the major regulatory gene families in eukaryotes. To date, 24521 microRNAs have been discovered and there are certainly more to come. It was primarily acknowledged that miRNAs result in gene expression repression at both the level of mRNA stability by conducting mRNA degradation and the level of translation (at initiation and after initiation) by inhibiting protein translation or degrading the polypeptides through binding complementarily to 3′UTR of the target mRNAs. Nevertheless, some studies revealed that miRNAs have the capability of activating gene expression directly or indirectly in respond to different cell types and conditions and in the presence of distinct cofactors. This reversibility in their posttranslational gene regulatory natures enables the bearing cells to rapidly response to different cell conditions and consequently block unnecessary energy wastage or maintain the cell state. This paper provides an overview of the current understandings of the miRNA characteristics including their genes and biogenesis, as well as their mediated downregulation. We also review up-to-date knowledge of miRNA-mediated gene upregulation through highlighting some notable examples and discuss the emerging concepts of their associations with other posttranscriptional gene regulation processes.

scholarly journals Chromatin Interaction Changes during the iPSC-NPC Model to Facilitate the Study of Biologically Significant Genes Involved in Differentiation

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1176
Author(s):  
Won-Young Choi ◽  
Ji-Hyun Hwang ◽  
Jin-Young Lee ◽  
Ann-Na Cho ◽  
Andrew J Lee ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Fate ◽  
Spatial Organization ◽  
Spinal Cord Injuries ◽  
Cell Types ◽  
Regulatory Elements ◽  
Chromatin Interaction ◽  
Patient Specific ◽  
Differentiation Process ◽  
Chromosome Conformation

Given the difficulties of obtaining diseased cells, differentiation of neurons from patient-specific human induced pluripotent stem cells (iPSCs) with neural progenitor cells (NPCs) as intermediate precursors is of great interest. While cellular and transcriptomic changes during the differentiation process have been tracked, little attention has been given to examining spatial re-organization, which has been revealed to control gene regulation in various cells. To address the regulatory mechanism by 3D chromatin structure during neuronal differentiation, we examined the changes that take place during differentiation process using two cell types that are highly valued in the study of neurodegenerative disease - iPSCs and NPCs. In our study, we used Hi-C, a derivative of chromosome conformation capture that enables unbiased, genome-wide analysis of interaction frequencies in chromatin. We showed that while topologically associated domains remained mostly the same during differentiation, the presence of differential interacting regions in both cell types suggested that spatial organization affects gene regulation of both pluripotency maintenance and neuroectodermal differentiation. Moreover, closer analysis of promoter–promoter pairs suggested that cell fate specification is under the control of cis-regulatory elements. Our results are thus a resourceful addition in benchmarking differentiation protocols and also provide a greater appreciation of NPCs, the common precursors from which required neurons for applications in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, schizophrenia and spinal cord injuries are utilized.

Regulation of Cell Fate Decisions in Early Mammalian Embryos

2020 ◽  
Vol 8 (1) ◽  
pp. 377-393
Author(s):  
Ramiro Alberio
Keyword(s):  
Cell Fate ◽  
Regulatory Networks ◽  
New Technologies ◽  
Cell Types ◽  
Cell Fate Decisions ◽  
Evolution And Development ◽  
Mammalian Embryos ◽  
Adult Organism ◽  
Gene Regulatory ◽  
Comparative Embryology

Early embryogenesis is characterized by the segregation of cell lineages that fulfill critical roles in the establishment of pregnancy and development of the fetus. The formation of the blastocyst marks the emergence of extraembryonic precursors, needed for implantation, and of pluripotent cells, which differentiate toward the major lineages of the adult organism. The coordinated emergence of these cell types shows that these processes are broadly conserved in mammals. However, developmental heterochrony and changes in gene regulatory networks highlight unique evolutionary adaptations that may explain the diversity in placentation and in the mechanisms controlling pluripotency in mammals. The incorporation of new technologies, including single-cell omics, imaging, and gene editing, is instrumental for comparative embryology. Broadening the knowledge of mammalian embryology will provide new insights into the mechanisms driving evolution and development. This knowledge can be readily translated into biomedical and biotechnological applications in humans and livestock, respectively.

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