scholarly journals Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

2017 ◽  
Author(s):  
Brock Roberts ◽  
Amanda Haupt ◽  
Andrew Tucker ◽  
Tanya Grancharova ◽  
Joy Arakaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quality Control ◽  
Genome Editing ◽  
Cell Biology ◽  
Cellular Structures ◽  
Human Stem Cells ◽  
Alpha Tubulin ◽  
Junction Protein ◽  
Endogenous Proteins ◽  
Clonal Lines

AbstractWe present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ∼25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

scholarly journals Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

2017 ◽  
Vol 28 (21) ◽  
pp. 2854-2874 ◽  
Author(s):  
Brock Roberts ◽  
Amanda Haupt ◽  
Andrew Tucker ◽  
Tanya Grancharova ◽  
Joy Arakaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Fluorescent Protein ◽  
Genomic Stability ◽  
Gene Tagging ◽  
Cellular Structures ◽  
Generation Process ◽  
Differentiation Potential ◽  
Junction Protein ◽  
Clonal Lines

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1–4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line–generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community.

scholarly journals New frontiers in human cell biology and medicine: Can pluripotent stem cells deliver?

2012 ◽  
Vol 199 (4) ◽  
pp. 577-581 ◽  
Author(s):  
Lawrence S.B. Goldstein
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cell Biology ◽  
Model System ◽  
Human Model ◽  
Human Stem Cells ◽  
Policy Issues ◽  
The Many ◽  
Medical Policy ◽  
Lateral Sclerosis

Human pluripotent stem cells provide enormous opportunities to treat disease using cell therapy. But human stem cells can also drive biomedical and cell biological discoveries in a human model system, which can be directly linked to understanding disease or developing new therapies. Finally, rigorous scientific studies of these cells can and should inform the many science and medical policy issues that confront the translation of these technologies to medicine. In this paper, I discuss these issues using amyotrophic lateral sclerosis as an example.

scholarly journals ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

2019 ◽  
Author(s):  
Jelmer Willems ◽  
Arthur P.H. de Jong ◽  
Nicky Scheefhals ◽  
Harold D. MacGillavry
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Protein Complexes ◽  
Neuronal Cell ◽  
Super Resolution ◽  
Protein Distribution ◽  
Neuronal Processes ◽  
Endogenous Proteins ◽  
Fluorescent Tags ◽  
Super Resolution Microscopy

ABSTRACTThe correct subcellular distribution of protein complexes establishes the complex morphology of neurons and is fundamental to their functioning. Thus, determining the dynamic distribution of proteins is essential to understand neuronal processes. Fluorescence imaging, in particular super-resolution microscopy, has become invaluable to investigate subcellular protein distribution. However, these approaches suffer from the limited ability to efficiently and reliably label endogenous proteins. We developed ORANGE: an Open Resource for the Application of Neuronal Genome Editing, that mediates targeted genomic integration of fluorescent tags in neurons. This toolbox includes a knock-in library for in-depth investigation of endogenous protein distribution, and a detailed protocol explaining how knock-in can be developed for novel targets. In combination with super-resolution microscopy, ORANGE revealed the dynamic nanoscale organization of endogenous neuronal signaling molecules, synaptic scaffolding proteins, and neurotransmitter receptors. Thus, ORANGE enables quantitation of expression and distribution for virtually any protein in neurons at high resolution and will significantly further our understanding of neuronal cell biology.

Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells

2015 ◽  
Vol 10 (11) ◽  
pp. 1842-1859 ◽  
Author(s):  
Jia Liu ◽  
Thomas Gaj ◽  
Yifeng Yang ◽  
Nan Wang ◽  
Sailan Shui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Primary Cells ◽  
Human Stem Cells ◽  
Efficient Delivery

Genome editing: a robust technology for human stem cells

2017 ◽  
Vol 74 (18) ◽  
pp. 3335-3346 ◽  
Author(s):  
Arun Pandian Chandrasekaran ◽  
Minjung Song ◽  
Suresh Ramakrishna
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Human Stem Cells

scholarly journals NADPH Oxidases: Redox Regulators of Stem Cell Fate and Function

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 973
Author(s):  
Tullia Maraldi ◽  
Cristina Angeloni ◽  
Cecilia Prata ◽  
Silvana Hrelia
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Cell Biology ◽  
Immune Modulation ◽  
Ex Vivo ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Stem Cells ◽  
Stem Cell Fate

One of the major sources of reactive oxygen species (ROS) generated within stem cells is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOXs), which are critical determinants of the redox state beside antioxidant defense mechanisms. This balance is involved in another one that regulates stem cell fate: indeed, self-renewal, proliferation, and differentiation are decisive steps for stem cells during embryo development, adult tissue renovation, and cell therapy application. Ex vivo culture-expanded stem cells are being investigated for tissue repair and immune modulation, but events such as aging, senescence, and oxidative stress reduce their ex vivo proliferation, which is crucial for their clinical applications. Here, we review the role of NOX-derived ROS in stem cell biology and functions, focusing on positive and negative effects triggered by the activity of different NOX isoforms. We report recent findings on downstream molecular targets of NOX-ROS signaling that can modulate stem cell homeostasis and lineage commitment and discuss the implications in ex vivo expansion and in vivo engraftment, function, and longevity. This review highlights the role of NOX as a pivotal regulator of several stem cell populations, and we conclude that these aspects have important implications in the clinical utility of stem cells, but further studies on the effects of pharmacological modulation of NOX in human stem cells are imperative.

scholarly journals Comparative RNAi Screens in Isogenic Human Stem Cells RevealSMARCA4as a Differential Regulator

2018 ◽  
Author(s):  
Ceren Güneş ◽  
Maciej Paszkowski-Rogacz ◽  
Susann Rahmig ◽  
Shahryar Khattak ◽  
Martin Wermke ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Large Scale ◽  
Model Organisms ◽  
Human Stem Cells ◽  
Cell Type ◽  
Hematopoietic Stem ◽  
Neural Lineage ◽  
Powerful Approach

SUMMARYLarge-scale RNAi screens are a powerful approach to identify functions of genes in a cell-type specific manner. For model organisms, genetically identical (isogenic) cells from different cell-types are readily available, making comparative studies meaningful. For humans, however, screening isogenic cells is not straightforward. Here, we show that RNAi screens are possible in genetically identical human stem cells, employing induced pluripotent stem cell as intermediates. The screens revealedSMARCA4(SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4) as a stemness regulator, while balancing differentiation distinctively for each cell type.SMARCA4knockdown in hematopoietic stem progenitor cells (HSPC) caused impaired self-renewalin-vitroandin-vivowith skewed myeloid differentiation; whereas in neural stem cells (NSC), it impaired selfrenewal while biasing differentiation towards neural lineage, through combinatorial SWI/SNF subunit assembly. Our findings pose a powerful approach for deciphering human stem cell biology and attribute distinct roles toSMARCA4in stem cell maintenance.

scholarly journals “Ancestralization” of human pluripotent stem cells by multiplexed precise genome editing

2018 ◽  
Author(s):  
Stephan Riesenberg ◽  
Tomislav Maricic ◽  
Svante Pääbo
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Nucleotide Substitution ◽  
Human Stem Cells ◽  
Animal Cells ◽  
Isolated Cells ◽  
Dependent Protein Kinase ◽  
Drastic Increase ◽  
Dependent Protein

We show that inactivation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) results in a drastic increase in efficiency of precise genome editing with CRISPR enzymes in human stem cells, allowing up to 79% of chromosomes to carry an intended nucleotide substitution when a single genomic site is targeted. When three different genes are simultaneously targeted, 12% of the isolated cells carry the targeted amino acid-changing substitutions in homozygous forms. These substitutions represent the first step towards resurrecting the proteome ancestral to Neandertals and modern humans. DNA-PKcs inactivation will greatly facilitate multiplexed precise genome editing in animal cells.

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