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

To simultaneously knock out multiple genes using the CRISPR-concatemer-Cas9 system, begin with a tube containing the mouse intestinal cell suspension. Supplement the tube with CRISPR-concatemer vectors containing the expression cassette for multiple guide RNAs, or gRNAs, integrated adjacent to each other.

Each gRNA cassette is custom-designed to individually knock out the intended gene. Add expression vectors encoding Cas9 endonuclease to the same tube. Electroporate the cell-plasmid mixture - a technique that uses electric current to facilitate the entry of plasmids into the cell. Inside the cell, the co-expression of the CRISPR-concatemer and the Cas9 vector forms gRNA sequences and Cas9 nucleases, respectively.

Each gRNA sequence binds to the corresponding Cas9 enzyme, forming multiple Cas9-gRNA complexes that attach to the target sites in the host genome. This binding activates the Cas9 enzyme, which produces a nick in both DNA strands upstream to the protospacer adjacent motif, or PAM, site. This results in a double-strand break, or DSB, in the target DNA.

In the absence of any homologous sequence to the target gene, the cell's endogenous repair mechanism, called the non-homologous end joining, is triggered, allowing the repair proteins and kinase molecules to bind at the break site. Later, the ligase enzyme repairs the DSB, resulting in modifications of the target gene sequence. These modifications disrupt the function of the genes, resulting in multiple genes knockout.

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.