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

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).