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Senger, Kate; Akhmetzyanova, Ilseyar; Haley, Benjamin; Rutz, Sascha; Oh, Soyoung A.

Plasmid‐Based Donor Templates for Nonviral CRISPR/Cas9‐Mediated Gene Knock‐In in Human T Cells

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  • Medientyp: E-Artikel
  • Titel: Plasmid‐Based Donor Templates for Nonviral CRISPR/Cas9‐Mediated Gene Knock‐In in Human T Cells
  • Beteiligte: Senger, Kate; Akhmetzyanova, Ilseyar; Haley, Benjamin; Rutz, Sascha; Oh, Soyoung A.
  • Erschienen: Wiley, 2022
  • Erschienen in: Current Protocols
  • Sprache: Englisch
  • DOI: 10.1002/cpz1.538
  • ISSN: 2691-1299
  • Schlagwörter: Medical Laboratory Technology ; Health Informatics ; General Pharmacology, Toxicology and Pharmaceutics ; General Immunology and Microbiology ; General Biochemistry, Genetics and Molecular Biology ; General Neuroscience
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  • Beschreibung: <jats:title>Abstract</jats:title><jats:p>Effective and precise gene editing of T lymphocytes is critical for advancing the understanding of T cell biology and the development of next‐generation cellular therapies. Although methods for effective CRISPR/Cas9‐mediated gene knock‐out in primary human T cells have been developed, complementary techniques for nonviral gene knock‐in can be cumbersome and inefficient. Here, we report a simple and efficient method for nonviral CRISPR/Cas9‐based gene knock‐in utilizing plasmid‐based donor DNA templates. © 2022 Wiley Periodicals LLC.</jats:p><jats:p><jats:bold>Basic Protocol 1</jats:bold>: Purification of human CD4<jats:sup>+</jats:sup> or CD8<jats:sup>+</jats:sup> T cells from blood</jats:p><jats:p><jats:bold>Basic Protocol 2</jats:bold>: Activation of purified CD4<jats:sup>+</jats:sup> or CD8<jats:sup>+</jats:sup> T cells using TransAct CD3/CD28 agonist‐conjugated nanomatrix</jats:p><jats:p><jats:bold>Basic Protocol 3</jats:bold>: Preparation of Cas9/sgRNA RNPs</jats:p><jats:p><jats:bold>Basic Protocol 4</jats:bold>: Transfection of CAS9‐RNP and knock‐in template into human T cells</jats:p><jats:p><jats:bold>Support Protocol 1</jats:bold>: Purity check following magnetic T cell isolation</jats:p><jats:p><jats:bold>Support Protocol 2</jats:bold>: Dextramer staining of TCR‐edited T cells</jats:p><jats:p><jats:bold>Support Protocol 3</jats:bold>: Functional characterization of TCR knock‐in T cells</jats:p><jats:p><jats:bold>Support Protocol 4</jats:bold>: Detection of knock‐in reporter activity in CRISPR/CAS9‐edited T cells</jats:p>