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 language | English |
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Article number | 102366 |
Journal | STAR Protocols |
Volume | 4 |
Issue number | 3 |
DOIs | |
State | Published - 15 Sep 2023 |
Externally published | Yes |
Keywords
- CRISPR
- Cancer
- Cell Biology
- Molecular Biology
- Sequencing
- Single Cell