Artigos de revistas: "Cas9-tagging"

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

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Wang, Qiang, e Jeffrey J. Coleman. "CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum". Fungal Genetics and Biology 126 (maio de 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 e Peter Kaiser. "Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion". Journal of Biological Chemistry 294, n.º 28 (28 de maio de 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 e Richard Wheeler. "Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase". Wellcome Open Research 7 (5 de dezembro de 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 e Richard J. Wheeler. "Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase". Wellcome Open Research 7 (23 de fevereiro de 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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Harazi, A., L. Yakovlev e S. Mitrani-Rosenbaum. "P.248CRISPR-Cas9 tagging allows the detection of endogenous gne in mice". Neuromuscular Disorders 29 (outubro de 2019): S139. http://dx.doi.org/10.1016/j.nmd.2019.06.362.

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Li, Pan, Lijun Zhang, Zhifang Li, Chunlong Xu, Xuguang Du e Sen Wu. "Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations". Cellular and Molecular Life Sciences 77, n.º 19 (17 de dezembro de 2019): 3875–84. http://dx.doi.org/10.1007/s00018-019-03396-8.

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AbstractEfficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.

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Calverley, Ben C., Karl E. Kadler e Adam Pickard. "Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging". Cells 9, n.º 9 (10 de setembro de 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 e David Horn. "CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes". Molecular and Biochemical Parasitology 249 (maio de 2022): 111476. http://dx.doi.org/10.1016/j.molbiopara.2022.111476.

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Blaeser, Anthony R., Pei Lu e Qi Long Lu. "347. Tagging FKRP and LARGE by CRISPR/Cas9 for Monitoring Expression and Localization". Molecular Therapy 23 (maio de 2015): S138. http://dx.doi.org/10.1016/s1525-0016(16)33956-9.

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Nitika e Andrew W. Truman. "Endogenous epitope tagging of heat shock protein 70 isoform Hsc70 using CRISPR/Cas9". Cell Stress and Chaperones 23, n.º 3 (24 de setembro de 2017): 347–55. http://dx.doi.org/10.1007/s12192-017-0845-2.

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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 e Robin C. Allshire. "SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast". Wellcome Open Research 5 (24 de novembro de 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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Matsuda, Takahiko, e Izumi Oinuma. "Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9". Molecular Biology of the Cell 30, n.º 22 (15 de outubro de 2019): 2838–55. http://dx.doi.org/10.1091/mbc.e19-04-0223.

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Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.

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Roberts, Brock, Amanda Haupt, Andrew Tucker, Tanya Grancharova, Joy Arakaki, Margaret A. Fuqua, Angelique Nelson et al. "Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization". Molecular Biology of the Cell 28, n.º 21 (15 de outubro de 2017): 2854–74. http://dx.doi.org/10.1091/mbc.e17-03-0209.

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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.

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Morrow, Christopher S., Tiaira J. Porter e Darcie L. Moore. "Fluorescent tagging of endogenous proteins with CRISPR/Cas9 in primary mouse neural stem cells". STAR Protocols 2, n.º 3 (setembro de 2021): 100744. http://dx.doi.org/10.1016/j.xpro.2021.100744.

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Lyu, Qing, Vidhi Dhagia, Yu Han, Bing Guo, Mary E. Wines-Samuelson, Christine K. Christie, Qiangzong Yin et al. "CRISPR-Cas9–Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin—Brief Report". Arteriosclerosis, Thrombosis, and Vascular Biology 38, n.º 9 (setembro de 2018): 2184–90. http://dx.doi.org/10.1161/atvbaha.118.311171.

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Cheng, Tian-Lin, e Zilong Qiu. "Long non-coding RNA tagging and expression manipulation via CRISPR/Cas9-mediated targeted insertion". Protein & Cell 9, n.º 9 (5 de setembro de 2017): 820–25. http://dx.doi.org/10.1007/s13238-017-0464-9.

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Aoto, Kazushi, Shuji Takabayashi, Hiroki Mutoh e Hirotomo Saitsu. "Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i-GONAD Enables Detection of Endogenous CaMKIIα and β Proteins". International Journal of Molecular Sciences 23, n.º 19 (7 de outubro de 2022): 11915. http://dx.doi.org/10.3390/ijms231911915.

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Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and β proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i-GONAD is a useful and convenient choice, especially if specific antibodies are unavailable.

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Li, Weicheng, Yaoyao Zhang, Katy Moffat, Venugopal Nair e Yongxiu Yao. "V5 and GFP Tagging of Viral Gene pp38 of Marek’s Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing". Viruses 14, n.º 2 (21 de fevereiro de 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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Leonetti, Manuel D., Sayaka Sekine, Daichi Kamiyama, Jonathan S. Weissman e Bo Huang. "A scalable strategy for high-throughput GFP tagging of endogenous human proteins". Proceedings of the National Academy of Sciences 113, n.º 25 (6 de junho de 2016): E3501—E3508. http://dx.doi.org/10.1073/pnas.1606731113.

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A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

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Wege, Sarah-Maria, Katharina Gejer, Fabienne Becker, Michael Bölker, Johannes Freitag e Björn Sandrock. "Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis". Journal of Fungi 7, n.º 2 (18 de fevereiro de 2021): 149. http://dx.doi.org/10.3390/jof7020149.

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The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

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Papasavva, Panayiota L., Petros Patsali, Constantinos C. Loucari, Ryo Kurita, Yukio Nakamura, Marina Kleanthous e Carsten W. Lederer. "CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation". International Journal of Molecular Sciences 23, n.º 3 (19 de janeiro de 2022): 1082. http://dx.doi.org/10.3390/ijms23031082.

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Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor BCL11A in erythroid cells by tagging the 3′ untranslated region (UTR) of BCL11A with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.

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Li, Qingyun, Scott Barish, Sumie Okuwa e Pelin C. Volkan. "Examination of Endogenous Rotund Expression and Function in DevelopingDrosophilaOlfactory System Using CRISPR-Cas9–Mediated Protein Tagging". G3: Genes|Genomes|Genetics 5, n.º 12 (23 de outubro de 2015): 2809–16. http://dx.doi.org/10.1534/g3.115.021857.

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Willems, Jelmer, Arthur P. H. de Jong, Nicky Scheefhals, Eline Mertens, Lisa A. E. Catsburg, Rogier B. Poorthuis, Fred de Winter, Joost Verhaagen, Frank J. Meye e Harold D. MacGillavry. "ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons". PLOS Biology 18, n.º 4 (10 de abril de 2020): e3000665. http://dx.doi.org/10.1371/journal.pbio.3000665.

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Beneke, Tom, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter e Eva Gluenz. "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids". Royal Society Open Science 4, n.º 5 (maio de 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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Han, Jeong Pil, Yoo Jin Chang, Dong Woo Song, Beom Seok Choi, Ok Jae Koo, Seung Youn Yi, Tae Sub Park e Su Cheong Yeom. "High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal". International Journal of Molecular Sciences 21, n.º 11 (26 de maio de 2020): 3747. http://dx.doi.org/10.3390/ijms21113747.

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In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

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Hou, Yuqing, Xi Cheng e George B. Witman. "Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis". PLOS ONE 17, n.º 12 (9 de dezembro de 2022): e0278972. http://dx.doi.org/10.1371/journal.pone.0278972.

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Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the strategy on two genes: LF5/CDKL5, lack of which causes a long-flagella phenotype, and Cre09.g416350/NAP1L1, which has not been studied previously in C. reinhardtii. We successfully tagged the C-terminus of wild-type LF5 with the hemagglutinin (HA) tag with an efficiency of 7.4%. Sequencing confirmed that these strains are correctly edited. Western blotting confirmed the expression of HA-tagged LF5, and immunofluorescence microscopy showed that LF5-HA is localized normally. These strains have normal length flagella and appear wild type. We successfully tagged the N-terminus of Cre09.g416350 with mNeonGreen-3xFLAG with an efficiency of 9%. Sequencing showed that the tag region in these strains is as expected. Western blotting confirmed the expression of tagged protein of the expected size in these strains, which appeared to have normal cell size, growth rate, and swimming speed. This is the first time that C. reinhardtii endogenous genes have been edited in situ to express a wild-type tagged protein. This effective, efficient, and convenient TIM-tagging strategy promises to be a useful tool for the study of nuclear genes, including essential genes, in C. reinhardtii.

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Mészár, Zoltán, Éva Kókai, Rita Varga, László Ducza, Tamás Papp, Monika Béresová, Marianna Nagy, Péter Szücs e Angelika Varga. "CRISPR/Cas9-Based Mutagenesis of Histone H3.1 in Spinal Dynorphinergic Neurons Attenuates Thermal Sensitivity in Mice". International Journal of Molecular Sciences 23, n.º 6 (15 de março de 2022): 3178. http://dx.doi.org/10.3390/ijms23063178.

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Burn injury is a trauma resulting in tissue degradation and severe pain, which is processed first by neuronal circuits in the spinal dorsal horn. We have recently shown that in mice, excitatory dynorphinergic (Pdyn) neurons play a pivotal role in the response to burn-injury-associated tissue damage via histone H3.1 phosphorylation-dependent signaling. As Pdyn neurons were mostly associated with mechanical allodynia, their involvement in thermonociception had to be further elucidated. Using a custom-made AAV9_mutH3.1 virus combined with the CRISPR/cas9 system, here we provide evidence that blocking histone H3.1 phosphorylation at position serine 10 (S10) in spinal Pdyn neurons significantly increases the thermal nociceptive threshold in mice. In contrast, neither mechanosensation nor acute chemonociception was affected by the transgenic manipulation of histone H3.1. These results suggest that blocking rapid epigenetic tagging of S10H3 in spinal Pdyn neurons alters acute thermosensation and thus explains the involvement of Pdyn cells in the immediate response to burn-injury-associated tissue damage.

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Lander, Noelia, Miguel A. Chiurillo, Melissa Storey, Anibal E. Vercesi e Roberto Docampo. "CRISPR/Cas9-mediated endogenous C-terminal Tagging ofTrypanosoma cruziGenes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor". Journal of Biological Chemistry 291, n.º 49 (28 de outubro de 2016): 25505–15. http://dx.doi.org/10.1074/jbc.m116.749655.

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Kuri, Paola, Nicole L. Schieber, Thomas Thumberger, Joachim Wittbrodt, Yannick Schwab e Maria Leptin. "Dynamics of in vivo ASC speck formation". Journal of Cell Biology 216, n.º 9 (12 de julho de 2017): 2891–909. http://dx.doi.org/10.1083/jcb.201703103.

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Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

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Thakare, Swapnil S., Navita Bansal, S. Vanchinathan, G. Rama Prashat, Veda Krishnan, Archana Sachdev, Shelly Praveen e T. Vinutha. "GFP tagging based method to analyze the genome editing efficiency of CRISPR/Cas9-gRNAs through transient expression in N. benthamiana". Journal of Plant Biochemistry and Biotechnology 29, n.º 2 (15 de novembro de 2019): 183–92. http://dx.doi.org/10.1007/s13562-019-00540-0.

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Leong, Shwee Khuan, Jye-Chian Hsiao e Jiun-Jie Shie. "A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model". International Journal of Molecular Sciences 23, n.º 15 (6 de agosto de 2022): 8754. http://dx.doi.org/10.3390/ijms23158754.

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Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.

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Naeimi Kararoudi, Meisam, Shibi Likhite, Ezgi Elmas, Maura Schwartz, Kinnari Sorathia, Kenta Yamamoto, Nitin Chakravarti, Branden S. Moriarity, Kathrin Meyer e Dean Anthony Lee. "CD33 Targeting Primary CAR-NK Cells Generated By CRISPR Mediated Gene Insertion Show Enhanced Anti-AML Activity". Blood 136, Supplement 1 (5 de novembro de 2020): 3. http://dx.doi.org/10.1182/blood-2020-142494.

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Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein complexes (PMID: 29985369 and 32603414). Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. We demonstrated that expansion of NK cells on feeder cells (CSTX002) expressing membrane-bound IL21 increases expression of HDR-related genes and provides optimum biological condition for targeted gene insertion. For proof-of-concept, we successfully generated stable mCherry-expressing primary NK cells (up to 89% mCherry+) and determined that sc vectors with 300bp homology arms were optimal. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ), which continued to show robust expansion on CSTX002 and stably maintained their CAR expression. This resulted in CAR-NK-cells of high number and purity (mean 68% CAR+) that demonstrated enhanced antileukemic activity against acute myeloid leukemia (AML) cell lines. This efficient method for site-directed insertion of genetic materials into primary NK cells has broad potential for fundamental discovery and therapeutic applications. Keywords: CRISPR, NK, Cas9/RNP, AAV6, CRISPaint, HR, CD33CAR-NK Figure Disclosures Naeimi Kararoudi: Kiadis Pharma Netherlans B.V: Patents & Royalties. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Nie, Zheng-Wen, Ying-Jie Niu, Wenjun Zhou, Dong-Jie Zhou, Ju-Yeon Kim e Xiang-Shun Cui. "AGS3-dependent trans-Golgi network membrane trafficking is essential for compaction in mouse embryos". Journal of Cell Science 133, n.º 23 (4 de novembro de 2020): jcs243238. http://dx.doi.org/10.1242/jcs.243238.

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ABSTRACTActivator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.

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Donlin-Asp, Paul G., Claudio Polisseni, Robin Klimek, Alexander Heckel e Erin M. Schuman. "Differential regulation of local mRNA dynamics and translation following long-term potentiation and depression". Proceedings of the National Academy of Sciences 118, n.º 13 (26 de março de 2021): e2017578118. http://dx.doi.org/10.1073/pnas.2017578118.

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Decades of work have demonstrated that messenger RNAs (mRNAs) are localized and translated within neuronal dendrites and axons to provide proteins for remodeling and maintaining growth cones or synapses. It remains unknown, however, whether specific forms of plasticity differentially regulate the dynamics and translation of individual mRNA species. To address this, we targeted three individual synaptically localized mRNAs, CamkIIa, β-actin, Psd95, and used molecular beacons to track endogenous mRNA movements. We used reporters and CRISPR/Cas9 gene editing to track mRNA translation in cultured neurons. We found alterations in mRNA dynamic properties occurred during two forms of synaptic plasticity, long-term potentiation (cLTP) and depression (mGluR-LTD). Changes in mRNA dynamics following either form of plasticity resulted in an enrichment of mRNA in the vicinity of dendritic spines. Both the reporters and tagging of endogenous proteins revealed the transcript-specific stimulation of protein synthesis following cLTP or mGluR-LTD. As such, the plasticity-induced enrichment of mRNA near synapses could be uncoupled from its translational status. The enrichment of mRNA in the proximity of spines allows for localized signaling pathways to decode plasticity milieus and stimulate a specific translational profile, resulting in a customized remodeling of the synaptic proteome.

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Wąchalska, Magda, Małgorzata Graul, Patrique Praest, Rutger D. Luteijn, Aleksandra W. Babnis, Emmanuel J. H. J. Wiertz, Krystyna Bieńkowska-Szewczyk e Andrea D. Lipińska. "Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter". Cells 8, n.º 12 (7 de dezembro de 2019): 1590. http://dx.doi.org/10.3390/cells8121590.

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Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

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Wall, Richard J., Eva Rico, Iva Lukac, Fabio Zuccotto, Sara Elg, Ian H. Gilbert, Yvonne Freund et al. "Clinical and veterinary trypanocidal benzoxaboroles target CPSF3". Proceedings of the National Academy of Sciences 115, n.º 38 (5 de setembro de 2018): 9616–21. http://dx.doi.org/10.1073/pnas.1807915115.

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African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization’s target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei. Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.

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Köhler, Simone, Michal Wojcik, Ke Xu e Abby F. Dernburg. "Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue". Proceedings of the National Academy of Sciences 114, n.º 24 (30 de maio de 2017): E4734—E4743. http://dx.doi.org/10.1073/pnas.1702312114.

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When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair.

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Raghuram, Viswanathan, Karim Salhadar, Kavee Limbutara, Euijung Park, Chin-Rang Yang e Mark A. Knepper. "Protein kinase A catalytic-α and catalytic-β proteins have nonredundant regulatory functions". American Journal of Physiology-Renal Physiology 319, n.º 5 (1 de novembro de 2020): F848—F862. http://dx.doi.org/10.1152/ajprenal.00383.2020.

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Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic β (PKA-Cβ). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cβ was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-Cα deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, whereas PKA-Cβ deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cβ serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding.

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Graustein, Andrew, Elizabeth A. Misch, Munyaradzi Musvosvi, Muki Shey, Javeed Shah, Rick Wells, Willem Hanekom, Mark Hatherill, Thomas Scriba e Thomas Hawn. "HSP90B1 Regulates TLR-dependent Monocyte Signaling and its Common Variants are Associated with BCG-specific T-cell Responses and Protection from Pediatric TB Disease". Journal of Immunology 196, n.º 1_Supplement (1 de maio de 2016): 200.18. http://dx.doi.org/10.4049/jimmunol.196.supp.200.18.

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Abstract Introduction HSP90B1 is a chaperone for multiple Toll-like receptors (TLRs), regulates innate immune signaling in murine myeloid cells and is expressed in T-cells. We hypothesized human HSP90B1 regulates monocyte responses to Mycobacterium tuberculosis (Mtb) and that its variants are associated with T-cell responses to the BCG vaccine and susceptibility to TB disease. Methods & Results We screened 17 haplotype-tagging HSP90B1 gene region SNPs for association with BCG-induced immune responses in South African infants vaccinated with BCG at birth. We stimulated peripheral blood drawn at 10 weeks of age with BCG in a whole blood cytokine assay. Three HSP90B1 SNPs (rs10507172, 10507173, and 2164747) were associated with increased IL-2 secretion (P<0.05, generalized linear model). SNPrs10507173 was associated with increased IL-2 production in the CD4+ T-cell subset (p=0.028, ANOVA) based on intracellular cytokine staining and flow cytometry. The same three alleles were associated with protection from pediatric TB disease in a gene association study of 226 children who contracted tuberculosis compared with 626 controls who remained healthy (dominant model, unadjusted p<0.05, OR 0.58–0.72; after adjusting for ethnicity, p<0.05 for 2/3 SNPs). We used CRISPR/Cas9 gene editing to reduce HSP90B1 expression in U937 monocyte cells. Relative to controls, HSP90B1-deficient cells secreted less TNF when exposed to TLR1/2/6 (p=0.02) and TLR4 (p=0.01) ligands and Mtb whole cell lysate (p=0.001). Conclusion These data suggest HSP90B1 deficiency is associated with an impaired monocyte response to Mtb ligands. In infants, HSP90B1 variants are associated with increased BCG-specific IL-2 responses T-cells and protection from TB disease.

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Alok, Anshu, Hanny Chauhan, Santosh Kumar Upadhyay, Ashutosh Pandey, Jitendra Kumar e Kashmir Singh. "Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages". Life 11, n.º 10 (28 de setembro de 2021): 1021. http://dx.doi.org/10.3390/life11101021.

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CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.

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Avagyan, Serine, Jonathan E. Henninger, William P. Mannherz, Meeta Mistry, Song P. Yang, Margaret Weber, Jessica Moore e Leonard I. Zon. "Mosaic Mutagenesis In Vivo Reveals Mutant Blood Stem Cells Intrinsically Resistant to Inflammatory Mediators in Clonal Hematopoiesis". Blood 136, Supplement 1 (5 de novembro de 2020): 27. http://dx.doi.org/10.1182/blood-2020-140903.

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Clonal hematopoiesis (CH) is a state of clonal dominance of a mutant hematopoietic stem and progenitor cell (HSPC) promoted by unknown mechanisms. To study the competitive behavior and the mechanism of such expansion in a native environment, we used a combinatorial approach of HSPCs labeling and mutagenesis in vivo in a technique we called Tissue editing With Inducible Stem cell Tagging via Recombination (TWISTR). TWISTR utilizes Zebrabow zebrafish to fluorescently label endogenous HSPCs, and allows following of the clonal activity and sorting of labeled HSPCs within the hematopoietic system. CRISPR/Cas9 mutagenesis was used to simultaneously induce mosaic insertions/deletions (indels) in zebrafish orthologs of 12 human CH genes during development, and zebrafish were followed for 8 months. As such, mutant stem cells were directly competed against endogenous wildtype stem cells. We achieved a high degree of mosaic mutagenesis with heterozygous edits, with a median of 5 targeted genes per zebrafish in total marrow cells at variant allele frequency (VAF) of 5% or greater. Serial sampling and sequencing of peripheral blood cells showed selective expansion of clones with frameshift indels in exon 12 of asxl1, while clones with other CH mutations did not show a significant change over the 4 months period evaluated. No changes were noted in control gene indels or in the CH gene indels in non-hematopoietic tissue in the same time period. We identified significant enrichment of frameshift indels in asxl1 in sorted dominant clones compared to sorted smaller clones in the same zebrafish (p<0.01) at 8 months, while indels in DNMT3A ortholog dnmt8 were present in clones of various competitive nature. Introduction of asxl1 mutations singly in a mosaic fashion also resulted in clonally dominant states. To identify gene expression signatures associated with the clonal expansion, we used single cell RNA sequencing of marrow cells in zebrafish with a dominant clone with unique sets of mutations. We identified increased expression of inflammatory signaling genes in these zebrafish compared to controls, consistent with recent findings in other organisms. Within sorted dominant clones, mutant neutrophils and macrophages exhibited more than 4-fold increased expression of cytokines, such as il1b and tnfb. In mutant HSPCs and myeloid-biased progenitor cells, we observed elevated levels of genes involved in suppressing responses to cytokines and inflammatory lipids, like socs3a, nr4a1, atf3 and ier2a. This supports a model in which the mutant HSPCs of the dominant clone express anti-inflammatory genes that dampen their response to the inflammatory mediators produced by mature cells of the same clone, limiting the inflammation-induced HSPC exhaustion and providing a selective mechanism for the mutant stem cells to expand over time. We successfully used the Zebrabow color labeling system and prospective endogenous mosaic mutagenesis to show that mutant asxl1 is a potent inducer of CH, and that CH gene mutations lead to clonal dominance by modifying the response of stem cells to inflammatory cues. Disclosures Zon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.

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Pantier, Raphaël, Tülin Tatar, Douglas Colby e Ian Chambers. "Endogenous epitope-tagging of Tet1, Tet2 and Tet3 identifies TET2 as a naïve pluripotency marker". Life Science Alliance 2, n.º 5 (outubro de 2019): e201900516. http://dx.doi.org/10.26508/lsa.201900516.

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Tet1, Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.

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Avagyan, Serine, Jonathan E. Henninger, William P. Mannherz, Meeta Mistry, Song Yang, Margaret C. Weber, Jessica Moore e Leonard I. Zon. "Loss of nr4a1 abrogates Fitness of asxl1-mutant Hematopoietic Clones". Blood 138, Supplement 1 (5 de novembro de 2021): 3272. http://dx.doi.org/10.1182/blood-2021-149731.

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Abstract Clonal fitness of mutant hematopoietic stem and progenitor cells (HSPCs) underlies clonal hematopoiesis (CH), a state of clonal expansion associated with increased risk of blood malignancies and cardiovascular disease. Mechanisms by which acquired mutations lead to clonal fitness are not known. We used a zebrafish model to study the effect of acquired asxl1 mutations on HSPC clonality with TWISTR (Tissue editing With Inducible Stem cell Tagging via Recombination) that combined mosaic CRISPR-Cas9 mutagenesis with color labeling of HSPC clones. TWISTR asxl1 mutants showed clonal dominant states with the expansion of single-colored clones contributing to over 30% of myelopoiesis. These zebrafish had normal hematopoietic output and no major lineage skewing. Single-cell RNA sequencing analysis of TWISTR mutant marrow cells harboring asxl1 mutations showed >10-fold upregulation of inflammatory cytokines in mutant mature myeloid cells and >30-fold upregulation of anti-inflammatory modulators in immature progenitors. Increased inflammation has been widely documented in persons with CH with acquired mutations in DNMT3A, TET2, ASXL1 and other genes. Moreover, chronic inflammation due to infection was shown to promote relative clonal fitness in Dnmt3a mutant mice. Based on our results, we proposed the hypothesis that upregulation of the anti-inflammatory genes, including nr4a1, served as a mechanism of resistance to chronic inflammation created by the mutant HSPC progeny, resulting in a self-perpetuating cycle of clonal fitness in that environment. To test this, we used TWISTR to generate mosaic mutants of asxl1 and nr4a1 by injecting zebrafish with guide RNAs targeting exon 12 of asxl1 and exon 3 of nr4a1 together. Our model would predict that abrogation of nr4a1 expression in asxl1-mutant clones would weaken their fitness relative to clones that maintained nr4a1 expression. We sorted over 300 clones of various sizes from this cohort of zebrafish and sequenced the two targeted genes. We found that clones with frameshift mutations in asxl1 with either no nr4a1 mutations or heterozygous nr4a1 mutations had a clone size of 20%±14% or 19.7%±15% in myeloid cells, respectively. Asxl1-mutant clones with biallelic frameshift mutations in nr4a1 were significantly smaller with a clone size of 13.8±11.5% (p < 0.015). Clones with intact asxl1 did not differ in their clone size independent of nr4a1 genetic status (11.9% wildtype nr4a1 vs 11.5% homozygous nr4a1 mutant). Chemical inhibition of nr4a1 over 3 months resulted in reduced change of edited clones in asxl1-mutant zebrafish compared to vehicle-treated zebrafish, with median change in allelic fraction of 3%±4.8% vs 5.3%±7.5%, respectively. This suggested that upregulation of nr4a1 in asxl1-mutant clones maintains their fitness in inflammatory conditions, potentially by limiting HSPC exhaustion. We successfully used TWISTR to study asxl1 induced CH in zebrafish and identified nr4a1 upregulation as a critical pathway engaged for establishing clonal fitness. Disclosures Zon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Amagma Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Branch Biosciences: Current holder of individual stocks in a privately-held company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.

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Snijders, Kirsten E., Anita Fehér, Zsuzsanna Táncos, István Bock, Annamária Téglási, Linda van den Berk, Marije Niemeijer et al. "Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages". Archives of Toxicology 95, n.º 10 (4 de setembro de 2021): 3285–302. http://dx.doi.org/10.1007/s00204-021-03127-8.

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AbstractTagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62–1000 nM) or diethyl maleate (5.62–1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

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Kesavan, Gokul, Anja Machate e Michael Brand. "CRISPR/Cas9-Based Split Fluorescent Protein Tagging". Zebrafish, 7 de setembro de 2021. http://dx.doi.org/10.1089/zeb.2021.0031.

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47

Dewari, Pooran Singh, Benjamin Southgate, Katrina Mccarten, German Monogarov, Eoghan O'Duibhir, Niall Quinn, Ashley Tyrer et al. "An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein". eLife 7 (11 de abril de 2018). http://dx.doi.org/10.7554/elife.35069.

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CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

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Baker, Oliver, Ashish Gupta, Mandy Obst, Youming Zhang, Konstantinos Anastassiadis, Jun Fu e A. Francis Stewart. "RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses". Scientific Reports 6, n.º 1 (24 de maio de 2016). http://dx.doi.org/10.1038/srep25529.

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Lackner, Daniel H., Alexia Carré, Paloma M. Guzzardo, Carina Banning, Ramu Mangena, Tom Henley, Sarah Oberndorfer et al. "A generic strategy for CRISPR-Cas9-mediated gene tagging". Nature Communications 6, n.º 1 (dezembro de 2015). http://dx.doi.org/10.1038/ncomms10237.

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Gutierrez-Triana, Jose Arturo, Tinatini Tavhelidse, Thomas Thumberger, Isabelle Thomas, Beate Wittbrodt, Tanja Kellner, Kerim Anlas, Erika Tsingos e Joachim Wittbrodt. "Efficient single-copy HDR by 5’ modified long dsDNA donors". eLife 7 (29 de agosto de 2018). http://dx.doi.org/10.7554/elife.39468.

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CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

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