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-homol