Cell Biology

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    Protocols in Current Issue
    Optimized CRISPR-Cas9-based Strategy for Complex Gene Targeting in Murine Embryonic Stem Cells for Germline Transmission
    Authors:  Thomas J. O'Neill, Daniel Krappmann and Andreas Gewies, date: 05/20/2022, view: 792, Q&A: 0
    [Abstract]

    Although CRISPR-Cas9 genome editing can be performed directly in single-cell mouse zygotes, the targeting efficiency for more complex modifications such as the insertion of two loxP sites, multiple mutations in cis, or the precise insertion or deletion of longer DNA sequences often remains low (Cohen, 2016). Thus, targeting and validation of

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    Preparation of Cas9 Ribonucleoproteins for Genome Editing
    Authors:  Sheng-Wei Lin, Viet Quoc Nguyen and Steven Lin, date: 05/20/2022, view: 1283, Q&A: 0
    [Abstract]

    Genome editing by the delivery of pre-assembled Cas9 ribonucleoproteins (Cas9 RNP) is an increasingly popular approach for cell types that are difficult to manipulate genetically by the conventional plasmid and viral methods. Cas9 RNP editing is robust, precise, capable of multiplexing, and free of genetic materials. Its transient presence in

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    CRISPR/Cas9 Gene Editing of HeLa Cells to Tag Proteins with mNeonGreen
    Authors:  Sachin Surve and Alexander Sorkin, date: 05/20/2022, view: 1002, Q&A: 0
    [Abstract]

    Subcellular localization dynamics of proteins involved in signal transduction processes is crucial in determining the signaling outcome. However, there is very limited information about the localization of endogenous signaling proteins in living cells. For example, biochemical mechanisms underlying the signaling pathway from epidermal growth

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    RNA-mediated in vivo Directed Evolution in Yeast
    Authors:  Emil D. Jensen and Michael K. Jensen, date: 03/05/2022, view: 1488, Q&A: 0
    [Abstract]

    Directed evolution is a powerful approach to obtain genetically-encoded sought-for traits. Compared to the prolonged adaptation regimes to mutations occurring under natural selection, directed evolution unlocks rapid screening and selection of mutants with improved traits from vast mutated sequence spaces. Many systems have been developed to

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    Labeling Endogenous Proteins Using CRISPR-mediated Insertion of Exon (CRISPIE)
    Authors:  Evan A. Wilson, Tianyi Mao and Haining Zhong, date: 03/05/2022, view: 2796, Q&A: 0
    [Abstract]

    The CRISPR/Cas9 technology has transformed our ability to edit eukaryotic genomes. Despite this breakthrough, it remains challenging to precisely knock-in large DNA sequences, such as those encoding a fluorescent protein, for labeling or modifying a target protein in post-mitotic cells. Previous efforts focusing on sequence insertion to the

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    Conditional Gene Editing in Presynaptic Extinction-ensemble Cells via the CRISPR-SaCas9 System
    Authors:  Haojie Sun, Ming Yi and You Wan, date: 12/05/2021, view: 1048, Q&A: 0
    [Abstract]

    The CRISPR-Cas9 enables efficient gene editing in various cell types, including post-mitotic neurons. However, neuronal ensembles in the same brain region can still be functionally or anatomically different, and such heterogeneity requires gene editing in specific neuronal populations. We recently developed a CRISPR-SaCas9 system-based technique.

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    APEX-mediated Proximity Labeling of Proteins in Cells Targeted by Extracellular Vesicles
    Authors:  Lu Song, Jun Chen, Angela Sun and Randy Schekman, date: 11/05/2021, view: 1982, Q&A: 0
    [Abstract]

    Extracellular vesicles (EVs) are thought to mediate intercellular communication through the delivery of cargo proteins and RNA to target cells. The uptake of EVs is often followed visually using lipophilic-dyes or fluorescently-tagged proteins to label membrane constituents that are then internalized into recipient cells (Christianson et al.,

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    Cytoduction and Plasmiduction in Yeast
    Authors:  Jane E. Dorweiler and Anita L. Manogaran, date: 09/05/2021, view: 1688, Q&A: 0
    [Abstract]

    Cytoduction, and a related technique referred to as plasmiduction, have facilitated substantial advancements in the field of yeast prion biology by providing a streamlined method of transferring prions from one yeast strain to another. Prions are cytoplasmic elements consisting of aggregated misfolded proteins, and as such, they exhibit

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    CRISPR/Cas9-mediated Precise SNP Editing in Human iPSC Lines
    Authors:  Hanwen Zhang and Siwei Zhang, date: 06/20/2021, view: 2931, Q&A: 0
    [Abstract]

    Human induced pluripotent stem cells (hiPSCs) have been extensively used in the fields of developmental biology and disease modeling. CRISPR/Cas9 gene editing in iPSC lines often has a low frequency, which hampers its application in precise allele editing of disease-associated single nucleotide polymorphisms (SNPs), especially those in the

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    Production of Phenotypically Uniform Human Cerebral Organoids from Pluripotent Stem Cells
    Authors:  Adam Sivitilli, Parisa Ghiasi and Liliana Attisano, date: 04/20/2021, view: 3281, Q&A: 0
    [Abstract]

    Recent advances in stem cell technology have allowed researchers to generate 3D cerebral organoids (COs) from human pluripotent stem cells (hPSCs). Indeed, COs have provided an unprecedented opportunity to model the developing human brain in a 3D context, and in turn, are suitable for addressing complex neurological questions by leveraging

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