Guide for generating single-cell–derived knockout clones in mammalian cell lines using the CRISPR/Cas9 system

Taeyeon Hong, Seung Min Bae, Gwonhwa Song, Whasun Lim

Research output: Contribution to journalLetterpeer-review

1 Citation (Scopus)

Abstract

Genome editing has developed rapidly in various research fields for targeted genome modifications in many organisms, including cells, plants, viruses, and animals. The clustered regularly interspaced short palindromic repeats-associated protein 9 system stands as a potent tool in gene editing for generating cells and animal models with high precision. The clinical potential of clustered regularly interspaced short palindromic repeats-associated protein 9 has been extensively reported, with applications in genetic disease correction, inhibition of viral replication, and personalized or targeted therapeutics for various cancers. In this study, we provide a guide on single-guide RNA design, cloning single-guide RNA into plasmid vectors, single-cell isolation via transfection, and identification of knockout clones using next-generation sequencing. In addition, by providing the results of insertion into mammalian cell lines through next-generation sequencing, we offer useful information to those conducting research on human and animal cell lines.

Original languageEnglish
Article number100087
JournalMolecules and cells
Volume47
Issue number7
DOIs
Publication statusPublished - 2024 Jul

Bibliographical note

Publisher Copyright:
© 2024 The Authors

Keywords

  • Clustered regularly interspaced short palindromic repeats-associated protein 9
  • Culture system
  • Genome editing
  • Single-guide RNA

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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