Related Experiment Video
Updated: Jul 24, 2025
09:29
Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation
Published on: February 28, 2025
1.1K
Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells
Fabian Geyer1, Maximilian Geyer1, Sarah Klapproth2
1Department of Oral and Maxillofacial Surgery, Klinikum rechts der Isar der Technischen Universität München, 81675 Munich, Germany.
STAR Protocols
|July 8, 2023
Summary
This study details a CRISPR-Cas9 protocol for creating knockout cell lines. It covers guide design, ribonucleoprotein complex delivery, and monoclonal cell line verification in HNSCC cells.
Area of Science:
- Molecular Biology
- Genomics
- Cell Biology
Background:
- CRISPR-Cas9 technology offers precise genome editing capabilities.
- Generating stable knockout cell lines is crucial for functional genomics research.
Purpose of the Study:
- To present a comprehensive protocol for generating monoclonal knockout cell lines.
- To optimize CRISPR-Cas9, ribonucleoprotein complexes, and lipofection for HNSCC cells.
Main Methods:
- Guide and primer design for CRISPR-Cas9.
- Preparation and lipofection of ribonucleoprotein complexes.
- Single-cell cloning, PCR, DNA purification, and cell line verification.
Main Results:
- A complete workflow for generating monoclonal knockout cell lines was established.
- The protocol is optimized for adherent HNSCC cells.
Conclusions:
- This protocol provides a robust method for creating verified monoclonal knockout cell lines.
- It facilitates functional studies in head and neck squamous cell carcinoma research.
