The CRISPR/Cas9-System as used for biotech applications consists of only a few components. One is the protein Cas9 which always has to be present. It functions as a pair of scissors that can cut DNA. Additionally, there is the guide RNA, a variable component that directs the Cas9 protein to a specific sequence within a genome where it makes the cut. The guide RNA can be modified thereby reprogramming the Cas9 for a new target sequence. The recognition of target sequences via the guide RNA follows the simple rules also found in DNA: DNA consist of the components A, G, C and T. In a DNA double-strand, A-T or G-C are always positioned opposite to each other and form contacts. Thus, knowledge of a gene’s sequence is the only prerequisite for targeting it by CRISPR/Cas. Depending on the system used, the gene may be cut, or alternatively activated or deactivated without actually changing the genetic material.

After the DNA is cut, it is repaired within the cell. Two competing mechanisms exist: in the most straightforward case, the free ends are reconnected. This often results in small errors that can destroy a gene. Alternatively, a repair template, in other words a DNA fragment with the same or similar sequence, can be employed to repair the double-strand break. This mechanism can be utilised to insert “new” genetic material at a specific site, or to correct a defective gene.

In principle, producing a plant specifically modified by CRISPR/Cas is relatively easy. First, the adapted CRISPR/Cas system has to be delivered into the plant. This is commonly done using Agrobacterium, which is naturally able to transfer parts of its genetic material to plant cells. This genetic material is replaced by the CRISPR/Cas system. Every cell of a plant is totipotent. In other words, a complete organism can be regenerated from every cell. Thus, I can cut pieces of leaf or stem from a plant, treat them with my bacteria, and then attempt to breed plants, complete with shoot and roots, from these small pieces. The CRISPR/Cas components are then produced by the plant cells themselves, thus modifying the target sequence in the genetic material. Finally, individuals carrying the desired modification are selected from the regenerated plants.