CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells

Begin this procedure by adding 9 milliliters of reduced serum medium to the 15-milliliter tube containing the cell suspension, and centrifuge at 400G for 3 minutes at room temperature. Remove all the supernatant, and resuspend the pellet in an electroporation solution. Add a total amount of 10 micrograms DNA to two new tubes. Then, add electroporation solution to a final volume of 100 microliters and keep the cell-DNA mixture on ice.

Transfer the cell-DNA mixture to the electroporation cuvette. Place the cuvette in the electroporator chamber. Measure the impedance by pushing the appropriate button on the electroporator, and ensure that it is 0.030 to 0.055 ohm. Perform electroporation according to the settings indicated in the text protocol. Add 400 microliters of electroporation buffer plus ROCK inhibitor to the cuvette, and then transfer all of its contents to a 1.5-milliliter tube.

Incubate at room temperature for 30 minutes to allow the cells to recover. After 30 minutes, spin at 400G for 3 minutes at room temperature. Remove the supernatant and resuspend the pellet in 20 microliters per well of basement matrix. Seed approximately 1 x 10⁴ to 1 x 10⁵ cells per well in a 48-well plate, and add EGF plus Noggin GSK-3 inhibitor, ROCK inhibitor, and 1.25% dimethyl sulfoxide medium. Incubate at 37 degrees Celsius.