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Gotowe bibliografie tematyczne / CRISPR/Cas9-tagging

Gotowa bibliografia na temat „CRISPR/Cas9-tagging”

Autor: Grafiati

Data publikacji: 1 kwietnia 2023

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Artykuły w czasopismach na temat "CRISPR/Cas9-tagging":

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Thöne, Fabian M. B., Nina S. Kurrle, Harald von Melchner i Frank Schnütgen. "CRISPR/Cas9-mediated generic protein tagging in mammalian cells". Methods 164-165 (lipiec 2019): 59–66. http://dx.doi.org/10.1016/j.ymeth.2019.02.018.

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Wang, Qiang, i Jeffrey J. Coleman. "CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum". Fungal Genetics and Biology 126 (maj 2019): 17–24. http://dx.doi.org/10.1016/j.fgb.2019.02.002.

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Lin, Da-Wei, Benjamin P. Chung, Jia-Wei Huang, Xiaorong Wang, Lan Huang i Peter Kaiser. "Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion". Journal of Biological Chemistry 294, nr 28 (28.05.2019): 10877–85. http://dx.doi.org/10.1074/jbc.ra119.008422.

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Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His6FLAG, His6-Biotin-TEV-RGSHis6, mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.

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Beneke, Tom, Ulrich Dobramysl, Carolina Moura Costa Catta-Preta, Jeremy Charles Mottram, Eva Gluenz i Richard Wheeler. "Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase". Wellcome Open Research 7 (5.12.2022): 294. http://dx.doi.org/10.12688/wellcomeopenres.18575.1.

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We present the genome sequence of Leishmania mexicana MNYC/BZ/62/M379 modified to express Cas9 and T7 RNA-polymerase, revealing high similarity to the reference genome (MHOM/GT2001/U1103). Through RNAseq-based annotation of coding sequences and untranslated regions, we provide primer sequences for construct and sgRNA template generation for CRISPR-assisted gene deletion and endogenous tagging.

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Beneke, Tom, Ulrich Dobramysl, Carolina Moura Costa Catta-Preta, Jeremy Charles Mottram, Eva Gluenz i Richard J. Wheeler. "Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase". Wellcome Open Research 7 (23.02.2023): 294. http://dx.doi.org/10.12688/wellcomeopenres.18575.2.

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We present the genome sequence of Leishmania mexicana MNYC/BZ/62/M379 modified to express Cas9 and T7 RNA-polymerase, revealing high similarity to the reference genome (MHOM/GT2001/U1103). Through RNAseq-based annotation of coding sequences and untranslated regions, we provide primer sequences for construct and sgRNA template generation for CRISPR-assisted gene deletion and endogenous tagging.

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Li, Weicheng, Yaoyao Zhang, Katy Moffat, Venugopal Nair i Yongxiu Yao. "V5 and GFP Tagging of Viral Gene pp38 of Marek’s Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing". Viruses 14, nr 2 (21.02.2022): 436. http://dx.doi.org/10.3390/v14020436.

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Marek’s disease virus (MDV) is a member of alphaherpesviruses associated with Marek’s disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have focused on gene deletion. However, analysis of the expression and interactions of the viral proteins during virus replication in infected cells and tumor cells is also important for studying its role in MDV pathogenesis. The unavailability of antibodies against most of the MDV proteins has hindered the progress in such studies. This prompted us to develop pipelines to tag MDV genes as an alternative method for this purpose. Here we describe the application of CRISPR/Cas9 gene-editing approaches to tag the phosphoprotein 38 (pp38) gene of the MDV vaccine strain CVI988 with both V5 and green fluorescent protein (GFP). This rapid and efficient viral-gene-tagging technique can overcome the shortage of specific antibodies and speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

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Torres-Garcia, Sito, Lorenza Di Pompeo, Luke Eivers, Baptiste Gaborieau, Sharon A. White, Alison L. Pidoux, Paulina Kanigowska, Imtiyaz Yaseen, Yizhi Cai i Robin C. Allshire. "SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast". Wellcome Open Research 5 (24.11.2020): 274. http://dx.doi.org/10.12688/wellcomeopenres.16405.1.

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The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6+ or ura4+ genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.

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Beneke, Tom, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter i Eva Gluenz. "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids". Royal Society Open Science 4, nr 5 (maj 2017): 170095. http://dx.doi.org/10.1098/rsos.170095.

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Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

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Calverley, Ben C., Karl E. Kadler i Adam Pickard. "Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging". Cells 9, nr 9 (10.09.2020): 2070. http://dx.doi.org/10.3390/cells9092070.

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The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.

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Kovářová, Julie, Markéta Novotná, Joana Faria, Eva Rico, Catriona Wallace, Martin Zoltner, Mark C. Field i David Horn. "CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes". Molecular and Biochemical Parasitology 249 (maj 2022): 111476. http://dx.doi.org/10.1016/j.molbiopara.2022.111476.

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Więcej źródeł

Rozprawy doktorskie na temat "CRISPR/Cas9-tagging":

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Lindvall, Jenny. "Green and red fluorescent protein tagging of endogenous proteins in glioblastoma using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-314151.

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Glioblastoma multiforme is the most malignant primary brain tumor that affects adults, recognized by the World Health Organization as an aggressive grade IV astrocytoma. Patients diagnosed with this type of tumor are left with a poor prognosis even with the most advanced treatment available. The cancer is quite heterogeneous and is typically categorized into four different subtypes depending on genetic aberrations and patient characteristics. Furthermore, researchers have discovered a subpopulation of glioblastoma cells, known as cancer stem cells, which are thought to be resistant to current therapies and responsible for tumor reoccurrence and relapse. Previous studies, in addition to this one, have found that the differentiation of glioblastoma cells downregulate nestin protein expression, the selected stem cell marker, and upregulate glial fibrillary acid protein expression, the selected differentiation marker, using immunofluorescence. Thus, one alternative treatment option is to understand the mechanism underlying the differentiation of cancer stem cells. Four cell cultures representative of each glioblastoma subtype will be endogenously tagged using the genome editing system, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9). The representative stem cell marker, nestin, will be tagged with a green fluorescent protein, while the chosen differentiation marker, glial fibrillary acid protein, will be tagged with a red fluorescent protein. Several drugs were screened to analyze whether the drugs had a differentiation effect on the glioblastoma cells. As a result, strong evidence indicated that bone morphogenetic protein four upregulated glial fibrillary acid protein expression levels to the same extent as the differentiation control media using 5% fetal bovine serum. The goal of this study is to establish a method to directly monitor the differentiation process of glioblastoma cells as a novel molecular screening method. In this case, all glioblastoma cells, even the ones resistant to treatment, can be eliminated through an initial “pre-treatment” by forcing differentiation of cancer stem cells, making the cells more susceptible to the chemotherapy drugs. In the long run, glioblastoma patients would have a chance at a more positive prognosis; a longer life that is free of glioblastoma.
Master Thesis in Applied Biotechnology

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Ratz, Michael [Verfasser], Stefan [Akademischer Betreuer] [Gutachter] Jakobs i Peter [Gutachter] Rehling. "CRISPR-Cas9-mediated protein tagging in human cells for RESOLFT nanoscopy and the analysis of mitochondrial prohibitins / Michael Ratz ; Gutachter: Stefan Jakobs, Peter Rehling ; Betreuer: Stefan Jakobs". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/1121909892/34.

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Jones, Matthew Leslie. "The subnuclear localisation of Notch responsive genes". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274909.

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Title: The subnuclear localisation of Notch responsive genes. Candidate Name: Matthew Jones Notch signalling is a highly conserved cell-cell communication pathway with critical roles in metazoan development and mutations in Notch pathway components are implicated in many types of cancer. Notch is an excellent and well-studied model of biological signalling and gene regulation, with a single intracellular messenger, one receptor and two ligands in Drosophila. However, despite the limited number of chemical players involved, a striking number of different outcomes arise. Molecular studies have shown that Notch activates different targets in different cell types and it is well known that Notch is important for maintaining a stem cell fate in some situations and driving differentiation in others. Thus some of the factors affecting the regulation of Notch target genes are yet to be discovered. Previous studies in various organisms have found that the location of a gene within the nucleus is important for its regulation and genome reorganisation can occur following gene activation or during development. Therefore this project aimed to label individual Notch responsive loci and determine their subnuclear localisation. In order to tag loci of interest a CRISPR/Cas9 genome-editing method was established that enabled the insertion of locus tags at Notch targets, namely the well-characterized Enhancer of split locus and also dpn and Hey, two transcription factors involved in neural cell fate decisions. The ParB/Int system is a recently developed locus tagging system and is not well characterised in Drosophila. It has a number of advantages over the traditional LacO/LacI-GFP locus tagging system as it does not rely on binding site repeats for signal amplification and can label two loci simultaneously in different colours. This thesis characterised the ParB/Int system in the Drosophila salivary gland and larval L3 neuroblast. Using 3D image segmentation hundreds of nuclei were reconstructed and a volume based normalisation method was applied to determine the subnuclear localisation of several Notch targets with and without genetic manipulations of the Notch pathway.

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Reinoite, Filipa Barroqueiro. "CRISPR/Cas9-mediated tagging of stem cell specific genes to study regeneration in the flatworm Macrostomum lignano". Master's thesis, 2019. http://hdl.handle.net/10316/88127.

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Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
A regeneração de tecidos é um processo que está dependente da capacidade de proliferação e diferenciação de células estaminais. No entanto, os mecanismos moleculares envolvidos neste processo são ainda uma incógnita, tornando ainda mais fascinante este atributo tão raro em alguns animais. Macrostomum lignano é um platelminta marinho que apresenta um conjunto qualidades únicas que fazem com que se adeqúe perfeitamente ao estudo de regeneração e células estaminais. É de fácil manutenção, transparente, tem um curto tempo de vida, produz ovos individuais fertilizados o que possibilita a criação de organismos transgénicos via micro-injecção. Além de todas as características descritas tem capacidade de regeneração ao longo do corpo, apenas possível devido à população pluripotente de células estaminais denominada neoblastos. O estudo dos neoblastos possibilita a compreensão dos mecanismos envolvidos no processo de regeneração.Neste estudo foi possível desenvolver linhas transgénicas de M. lignano que marcam os neoblastos pela integração de uma proteína fluorescente pelo sistema CRISPR/Cas9 no locus de um gene específico dos neoblastos, H2AX. Esta linha foi um ponto de partida para a criação de linhas transgénicas duplas que marcam neoblastos assim como variados tipos celulares com proteínas fluorescentes distintas. A expressão fluorescente de uma linha dupla que marca neoblastos e células musculares após amputação permitiu identificar células progenitoras, neoblastos destinados a se diferenciarem em determinado tipo celular. A criação destas linhas transgénicas possibilitou o acompanhamento in vivo do comportamento dos neoblastos no processo de regeneração assim como a separação de diferentes populações de neoblastos nunca identificadas.Além de nos dar uma perspectiva mais ampla da função dos neoblastos, este projecto permitiu a melhor compreensão do processo de diferenciação destas células após amputação. O desenvolvimento da tecnologia CRISPR/Cas9 posiciona Macrostomum lignano como o platelminta de eleição para o estudo de comportamento celular in vivo e para o estudo da genómica funcional dos neoblastos tanto em homeostasia como em regeneração. Também alarga o seu estudo em diferentes áreas como cancro, manutenção genómica e envelhecimento.
Regeneration of lost or damaged tissues is dependent on proliferation and differentiation of stem cells. The understanding of this molecular process is one of the longstanding scientific problems that require a suitable model organism. The recently developed model Macrostomum lignano is a marine free‐living flatworm with a unique set of biological properties perfectly positioned for the study of stem cells and regeneration. It is easy to culture, transparent, has a short generation time and produces single-cell fertilized eggs, enabling transgenesis via microinjection. Whole-body regeneration in flatworms is fueled by a pluripotent population of stem cells called neoblasts. Studying the biology of neoblasts improves our understanding of the mechanisms involved in regeneration. In the current project we were able to develop by microinjection a M. lignano transgenic line labeling neoblasts by CRISPR/Cas9‐mediated knock‐in of a fluorescent protein into a neoblast‐specific gene, H2AX. Such line was a starting point for generating multiple double transgenic lines labeling both neoblasts and differentiated cell types tagged with distinct fluorescent proteins. Fluorescent protein expression in a double transgenic line with a muscle-specific marker allowed identification of progenitor cells upon regeneration, a neoblast population committed to differentiate into specific cell-types. These transgenic lines allow for in vivo tracking of neoblast behavior during regeneration and isolation of several never identified stem cell populations for further characterization.This project improves our understanding of the molecular mechanisms of neoblast differentiation and provides a broader multidimensional image of the function of stem cells in the process of regeneration. The enlargement of its experimental potential places M. lignano as the invertebrate model organism of choice for studying in vivo cell behavior and stem cell-specific gene function in tissue regeneration and homeostasis. As well as expand its value in other research fields as cancer, genome maintenance and ageing.

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Ratz, Michael. "CRISPR-Cas9-mediated protein tagging in human cells for RESOLFT nanoscopy and the analysis of mitochondrial prohibitins". Doctoral thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-002B-7CDA-C.

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Części książek na temat "CRISPR/Cas9-tagging":

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Xiang, Xi, Conghui Li, Xi Chen, Hongwei Dou, Yong Li, Xiuqing Zhang i Yonglun Luo. "CRISPR/Cas9-Mediated Gene Tagging: A Step-by-Step Protocol". W Methods in Molecular Biology, 255–69. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9170-9_16.

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Ghosh, Sanjay, i Ji-Long Liu. "Genomic Tagging of AGO1 Using CRISPR/Cas9-Mediated Homologous Recombination". W Methods in Molecular Biology, 217–35. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7339-2_15.

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Shvets, Elena, i Carolina Mendoza-Topaz. "Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology". W Methods in Molecular Biology, 149–66. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0732-9_14.

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Beneke, Tom, i Eva Gluenz. "LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9". W Methods in Molecular Biology, 189–210. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9210-2_9.

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Geny, Sylvain, Simon Pichard, Arnaud Poterszman i Jean-Paul Concordet. "Gene Tagging with the CRISPR-Cas9 System to Facilitate Macromolecular Complex Purification". W Methods in Molecular Biology, 153–74. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1406-8_8.

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Ehrke-Schulz, Eric, Maren Schiwon, Claudia Hagedorn i Anja Ehrhardt. "Establishment of the CRISPR/Cas9 System for Targeted Gene Disruption and Gene Tagging". W Methods in Molecular Biology, 165–76. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7231-9_11.

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Geny, Sylvain, Simon Pichard, Alice Brion, Jean-Baptiste Renaud, Sophie Jacquemin, Jean-Paul Concordet i Arnaud Poterszman. "Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors". W Methods in Molecular Biology, 39–57. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-1126-5_3.

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