All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System
Published: April 30, 2023
Abstract
Source: Zhang, L. et al, Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines. J. Vis. Exp. (2021).
In this video, we demonstrate all-in-one CRISPR-Cas9 based genome editing in cultured cells where Cas9 and sgRNA are provided as a single plasmid construct to the cells. The CRISPR-Cas9 system and desired gene to be inserted was introduced in cells through electroporation technique to facilitate successful gene editing.
Protocol
1. Electroporation of Macrophage and T Cell Lines Prepare cell cultures for electroporation Prepare Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) as complete growth medium for Jurkat T cells. Prepare Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS for culturing RAW264.7 macrophages. Supplement all complete growth media with 100 U/mL penicillin and 100 µg/mL streptomycin (Pen/Strep) except for media used fo…
Disclosures
The authors have nothing to disclose.
Materials
| Ampicillin, sodium salt | MP Biomedicals | 194526 | |
| Cellometer Mini Automated Cell Counter | Nexcelom Bioscience | ||
| DPBS (10X), no calcium, no magnesium | ThermoFisher Scientific | 14200075 | |
| Dulbecco's Modified Eagle Medium (DMEM) with high glucose | HyClone | SH30022.01 | |
| Falcon 5 ml polystyrene round bottom test tube | BD Biosciences | 352003 | |
| Fetal bovine serum (FBS) | ThermoFisher Scientific | 10099141 | |
| Jurkat | ATCC | TIB-152 | https://www.atcc.org/ |
| Kanamycin sulfate | MP Biomedicals | 194531 | |
| Multi-channel Pipette (30-300 μL) | Eppendorf, or similar | ||
| Neon Transfection System | ThermoFisher Scientific | MPK5000 | |
| Neon Transfection System, 10 μL kit | ThermoFisher Scientific | MPK1096 | |
| Nunc 15 mL Conical Sterile Centrifuge Tubes | ThermoFisher Scientific | 339651 | |
| Pipette tip 0.1-20µl | Eppendorf, or similar | 0030 075.005 | |
| Pipette tip 2-200µl | Eppendorf, or similar | 0030 075.021 | |
| Pipette tip 50-1000µl | Eppendorf, or similar | 0030 075.064 | |
| pX458-DsRed2 | Addgene | 112219 | |
| RAW264.7 | ATCC | TIB-71 | https://www.atcc.org/ |
| RPMI 1640 Medium | HyClone | SH30027.01 | |
| Penicillin-Streptomycin | ThermoFisher Scientific | 15140122 | |
| Trypan Blue Solution, 0.4% | ThermoFisher Scientific | 15250061 | |
| 1.5 mL microtubes, PCR-clean | Eppendorf, or similar | 0030 125.215 | |
| 24-well Clear TC-treated Multiple Well Plates | Corning | 3524 |
Tags
Cite This Article
All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System. J. Vis. Exp. (Pending Publication), e20971, doi: (2023).