Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells

Fabian Geyer, Maximilian Geyer, Sarah Klapproth, Klaus Dietrich Wolff, Markus Nieberler

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines.

Original languageEnglish
Article number102366
JournalSTAR Protocols
Volume4
Issue number3
DOIs
StatePublished - 15 Sep 2023
Externally publishedYes

Keywords

  • CRISPR
  • Cancer
  • Cell Biology
  • Molecular Biology
  • Sequencing
  • Single Cell

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