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…
Declarações
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