All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System

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

1. Prepare cell cultures for electroporation
   1. 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 for post-transfection incubation before cell sorting.
   2. Perform subculturing of RAW264.7 and Jurkat T cells according to the supplier's instruction....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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