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NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in .
He S, Cuentas-Condori A, Miller DM
(2019) Genetics 212: 387-395
MeSH Terms: Animals, CRISPR-Cas Systems, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Fluorescence, Gene Editing, Green Fluorescent Proteins, Membrane Proteins, Nerve Tissue Proteins
Show Abstract · Added March 3, 2020
GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand ( ) at the genomic locus of each target protein. The resultant knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment () in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the nervous system.
Copyright © 2019 by the Genetics Society of America.
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MeSH Terms
Genome Editing and Induced Pluripotent Stem Cell Technologies for Personalized Study of Cardiovascular Diseases.
Chun YW, Durbin MD, Hong CC
(2018) Curr Cardiol Rep 20: 38
MeSH Terms: Cardiovascular Diseases, Cell Differentiation, Gene Editing, Humans, Induced Pluripotent Stem Cells, Models, Biological, Precision Medicine
Show Abstract · Added May 1, 2018
PURPOSE OF REVIEW - The goal of this review is to highlight the potential of induced pluripotent stem cell (iPSC)-based modeling as a tool for studying human cardiovascular diseases. We present some of the current cardiovascular disease models utilizing genome editing and patient-derived iPSCs.
RECENT FINDINGS - The incorporation of genome-editing and iPSC technologies provides an innovative research platform, providing novel insight into human cardiovascular disease at molecular, cellular, and functional level. In addition, genome editing in diseased iPSC lines holds potential for personalized regenerative therapies. The study of human cardiovascular disease has been revolutionized by cellular reprogramming and genome editing discoveries. These exceptional technologies provide an opportunity to generate human cell cardiovascular disease models and enable therapeutic strategy development in a dish. We anticipate these technologies to improve our understanding of cardiovascular disease pathophysiology leading to optimal treatment for heart diseases in the future.
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7 MeSH Terms
Gene-edited MLE-15 Cells as a Model for the Hermansky-Pudlak Syndromes.
Kook S, Qi A, Wang P, Meng S, Gulleman P, Young LR, Guttentag SH
(2018) Am J Respir Cell Mol Biol 58: 566-574
MeSH Terms: Alveolar Epithelial Cells, Animals, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, Disease Models, Animal, Gene Editing, Genetic Markers, Genetic Predisposition to Disease, Hermanski-Pudlak Syndrome, Humans, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Phenotype
Show Abstract · Added April 1, 2019
Defining the mechanisms of cellular pathogenesis in rare lung diseases such as Hermansky-Pudlak syndrome (HPS) is often complicated by loss of the differentiated phenotype of cultured primary alveolar type 2 (AT2) cells, as well as by a lack of durable cell lines that are faithful to both AT2-cell and rare disease phenotypes. We used CRISPR/Cas9 gene editing to generate a series of HPS-specific mutations in the MLE-15 cell line. The resulting MLE-15/HPS cell lines exhibit preservation of AT2 cellular functions, including formation of lamellar body-like organelles, complete processing of surfactant protein B, and known features of HPS specific to each trafficking complex, including loss of protein targeting to lamellar bodies. MLE-15/HPS1 and MLE-15/HPS2 (with a mutation in Ap3β1) express increased macrophage chemotactic protein-1, a well-described mediator of alveolitis in patients with HPS and in mouse models. We show that MLE-15/HPS9 and pallid AT2 cells (with a mutation in Bloc1s6) also express increased macrophage chemotactic protein-1, suggesting that mice and humans with BLOC-1 mutations may also be susceptible to alveolitis. In addition to providing a flexible platform to examine the role of HPS-specific mutations in trafficking AT2 cells, MLE-15/HPS cell lines provide a durable resource for high-throughput screening and studies of cellular pathophysiology that are likely to accelerate progress toward developing novel therapies for this rare lung disease.
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16 MeSH Terms
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR
(2017) Nature 551: 464-471
MeSH Terms: Adenosine Deaminase, Base Pairing, CRISPR-Associated Proteins, Cell Line, Tumor, DNA, DNA Cleavage, Gene Editing, Genome, Human, HEK293 Cells, Humans, Models, Molecular, Polymorphism, Single Nucleotide
Show Abstract · Added March 13, 2018
The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
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12 MeSH Terms
Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development.
Kim YK, Refaeli I, Brooks CR, Jing P, Gulieva RE, Hughes MR, Cruz NM, Liu Y, Churchill AJ, Wang Y, Fu H, Pippin JW, Lin LY, Shankland SJ, Vogl AW, McNagny KM, Freedman BS
(2017) Stem Cells 35: 2366-2378
MeSH Terms: Animals, Cell Adhesion, Cell Differentiation, Gene Editing, Humans, Kidney, Kidney Glomerulus, Mice, Organoids, Pluripotent Stem Cells, Podocytes, Sialoglycoproteins
Show Abstract · Added March 14, 2019
A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin podocin ZO-1 ) and microvillus-rich apical membranes (podocalyxin ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.
© 2017 AlphaMed Press.
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Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.
Ando K, Shah AK, Sachdev V, Kleinstiver BP, Taylor-Parker J, Welch MM, Hu Y, Salgia R, White FM, Parvin JD, Ozonoff A, Rameh LE, Joung JK, Bharti AK
(2017) Oncotarget 8: 43733-43751
MeSH Terms: BRCA1 Protein, Camptothecin, Cell Line, Tumor, DNA Topoisomerases, Type I, DNA-Binding Proteins, Drug Resistance, Neoplasm, Gene Editing, Humans, Ku Autoantigen, Multiprotein Complexes, PTEN Phosphohydrolase, Phosphorylation, Proteasome Endopeptidase Complex, Protein Binding, Protein Kinase C, Proteolysis, RNA Interference, Topoisomerase I Inhibitors, Ubiquitin
Show Abstract · Added November 26, 2018
Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.
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Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers.
Gu M, Shao NY, Sa S, Li D, Termglinchan V, Ameen M, Karakikes I, Sosa G, Grubert F, Lee J, Cao A, Taylor S, Ma Y, Zhao Z, Chappell J, Hamid R, Austin ED, Gold JD, Wu JC, Snyder MP, Rabinovitch M
(2017) Cell Stem Cell 20: 490-504.e5
MeSH Terms: Base Sequence, Bone Morphogenetic Protein 4, Bone Morphogenetic Protein Receptors, Type II, Cell Adhesion, Cell Movement, Cell Shape, Cell Survival, Endothelial Cells, Gene Editing, Gene Expression Regulation, Heterozygote, Humans, Hypertension, Pulmonary, Induced Pluripotent Stem Cells, Mutation, Neovascularization, Physiologic, Phosphorylation, Sequence Analysis, RNA, Signal Transduction, Smad Proteins, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added February 21, 2017
In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease. Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.
Copyright © 2017 Elsevier Inc. All rights reserved.
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21 MeSH Terms
Translational Advances in the Field of Pulmonary Hypertension Molecular Medicine of Pulmonary Arterial Hypertension. From Population Genetics to Precision Medicine and Gene Editing.
Austin ED, West J, Loyd JE, Hemnes AR
(2017) Am J Respir Crit Care Med 195: 23-31
MeSH Terms: Forecasting, Gene Editing, Genetic Therapy, Genetics, Population, Humans, Hypertension, Pulmonary, Male, Precision Medicine, Translational Medical Research
Added February 21, 2017
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9 MeSH Terms
Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.
Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK
(2014) Nat Biotechnol 32: 569-76
MeSH Terms: Bacterial Proteins, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Deoxyribonucleases, Type II Site-Specific, Endonucleases, Gene Editing, Humans, Protein Multimerization, RNA, Guide, Recombinant Fusion Proteins
Show Abstract · Added January 26, 2015
Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.
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10 MeSH Terms