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: 1057, 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: 1652, 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: 1293, 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: 1656, 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: 3733, 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: 1138, 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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    CRISPR/Cas9-mediated Precise SNP Editing in Human iPSC Lines
    Authors:  Hanwen Zhang and Siwei Zhang, date: 06/20/2021, view: 3119, 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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    Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies
    [Abstract] CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and ...
    CRISPR-Cas9 Genome Editing of Plasmodium knowlesi
    Authors:  Franziska Mohring, Melissa N. Hart, Avnish Patel, David A. Baker and Robert W. Moon, date: 02/20/2020, view: 4712, Q&A: 1
    [Abstract] Plasmodium knowlesi is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After P. falciparum, P. knowlesi is the only other malaria parasite capable of being maintained in ...
    CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice
    [Abstract] Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos’ genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation ...



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