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.