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

Overview

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 instructions (See Table of Materials). When subculturing RAW264.7 cells, use trypsin-EDTA solution (0.25%) to detach cells. Use FBS supplemented with 10% (v/v) DMSO as a cryopreservation medium for RAW264.7 and Jurkat cells.
   3. Collect cells at 250 × g for 5 min for RAW264.7 macrophages and 90 × g for 8 min for Jurkat T cells. Then wash with 5-10 mL of 1× DPBS (without Ca²⁺ or Mg²⁺ ions). Remove the DPBS.
   4. Resuspend the cell pellet using 2 mL of 1× DPBS. Use 10 µL of cells and mix with an equal volume of 0.2% trypan blue to estimate the cell count and viability.
      NOTE: Ensure that the cell culture has >90% viability on the day of transfection.
   5. For a single knock-in experiment, perform electroporation with 10 µL nucleofection tips with five repetitions. Calculate the volume needed for 2.0 × 106 cells and pellet the cells by centrifugation. Wash the cell pellet again with 1× DPBS as described in step 1.1.2.
      NOTE: When using 10 µL nucleofection tips, 4.0 × 10⁵ cells are needed per electroporation. Accordingly, prepare at least 2.0 × 10⁶ cells for one knock-in experiment.
   6. Prepare a 24-well plate with 0.5 mL of complete growth medium (prepared in step 1.1.1) per well without Pen/Strep and prewarm in a 37 °C incubator.

2. Electroporation of CRISPR/Cas9 components and the targeting vector

1. Turn on the electroporation system. Use electroporation parameters optimized as follows: 1,400 V/20 ms/2 pulses for RAW264.7 macrophages and 1350 V/20 ms/2 pulses for Jurkat T cells.
2. Accounting for sample loss due to pipetting, prepare a 55 µL electroporation mixture in a sterile 1.5 mL microcentrifuge tube containing 2.5 µg of each CRISPR/Cas9 vector (pDsR-mR26-sg1 and pDsR-mR26-sg2 for mRosa26 locus knock-in, and pDsR-hR26-sg1 and pDsR-hR26-sg2 for the hROSA26 locus), 2.4 µg of the linearized targeting vector (pKR26-POI-iBFP or pKhR26-POI-iBFP for mRosa26 and hROSA26 knock-in, linearized by EcoRI or BamHI), and the Resuspension Buffer R.
   NOTE: To save time, the electroporation mixture can be prepared during centrifugation (step 1.1.5).
3. Resuspend 2.0 × 10⁶ cells (prepared in step 1.1.5) in the 55 µL electroporation mixture from step 1.2.2.
4. Aspirate the cell/electroporation mixture from step 1.2.3 using a 10 µL nucleofection tip with a pipette.
   NOTE: During pipetting, avoid introducing air bubbles, which may cause electroporation failure.
5. Add the sample to a tube filled with 3 mL of Buffer E from the electroporation kit.
6. Apply the electroporation parameters for the two cell types as described in step 1.2.1.
7. Transfer the sample into one well of the 24-well plate with prewarmed medium from step 1.1.6.
8. Repeat steps 1.2.4-1.2.7 for the other four repetitions as well as for the targeting vector only and CRISPR expression vector only controls.
   NOTE: Change the nucleofection tip and tube when switching to a different cell type/plasmid DNA.
9. Culture the transfected cells for 48-72 h to allow for recovery after electroporation and expression of CRISPR/Cas9 components prior to flow cytometry analysis or fluorescence-activated cell sorting (FACS).

Materials

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