Protocol: CRISPR-Cas9 knockout protocol (Part 2): Cell Transfection

Among the CRISPR/Cas9 protocols, the most commonly used and efficient method for CRISPR-Cas9 knockout cell line generation is to use CRISPR gRNA vectors to transfect into Cas9 stable cell lines, and obtain gene knockout cell clones through monoclonal isolation and screening.

Here is the part 2 of the CRISPR-Cas9 knockout protocol--Cell Transfection. The following procedure is for cas9 expressing cells transfection during KO cell line generation.

▍  Preliminary experiment

1. Exploring the concentration for antibiotic screening (Kill curve)
1) Digest the cas9 stable cells in the logarithmic growth phase into a single-cell suspension, and inoculate into a 12-well plate. The total volume of the culture medium is 1ml. Incubate the cells in 5% CO2 incubator at 37℃ for 24 hours.
2) When the confluence level reaches about 50%, change the medium to a antibiotic screening medium containing different concentrations of Puromycin, the concentration gradient is: 0, 1, 1.5, 2, 3, 4 μg/ml. 
3) After 2-3 days, observe the cells under a microscope, and select the lowest concentration of the cells that are all dead as the drug screening concentration for subsequent experiments.

2. Exploring the single-cell clonal formation ability
1) Digest the cas9 stable cells in the logarithmic growth phase into a single-cell suspension, and do the cell counting.
2) Take a certain amount of cell suspension, dilute and inoculate into a 96-well plate, giving the total volume of each well of the culture medium is 100 μl. Incubate the cells in 5% CO2 incubator at 37℃ for static culture.
3) After 7-10 days, observe under the microscope whether the cells have a proliferation trend and form cell clusters, and count the number of single-cell clones in each group.

3. Exploring the transfection method
1) Digest the cas9 stable cells in the logarithmic growth phase into a single-cell suspension, set up several different electroporation procedures for electrotransfection, observe the transfection efficiency 24 hours after the transfection and take pictures as record. Use the electroporation parameters with better transfection rate and cell viability as the electroporation conditions for the formal experiment.

4. Target site sequencing
1) Use blood/cell/tissue genomic DNA extraction kit to extract genomic DNA from cells.
2) Amplify the target region via PCR, and the PCR ingredients are as follows:

3) Evaluate the amplified DNA (PCR product) by agarose gel electrophoresis. If the band size of the PCR product is consistent with the theoretical size, then sequence the PCR product.

▍  Formal experiment

1. Electrotransfection of target cas9 expressing stable cells
1) Digest the cells in the logarithmic growth phase into a single-cell suspension, take some cells for cell counting and to investigate the cell viability. 
2) Transfer 3×10^6 cells into a sterile centrifuge tube and centrifuge at 300×g for 4 minutes. 
3) Discard the supernatant, resuspend the cell pellet in transfection buffer, add 30μg endotoxin-free CRISPR gRNA plasmid and mix well. (The amount of gRNA plasmid we recommend here is base on Neon transfection system, please adjust accordingly if the transfection system is different.)
4) Add 3ml Buffer E2 to the electroporation cuvette, and then put it into the insertion site of the electroporator.
5) Use a 100μl electroporation pipette tip to aspirate the cells and the above mixture, insert it into the electroporation cuvette, set electroporation conditions, and press start.
6) After the electroporation is completed, inoculate the cells in a 6-well plate with pre-warmed culture medium and continue to culture.

2. CRISPR-cas9 knockout cell pool screening
1) 24-48 hours after the transfection, observe the effect of electrotransfection under a microscope, and add antibiotic to screen the cells.
2) After 2-3 days of screening, a KO cell pool is ready for single-cell clone isolation.