Creating a genetically modified organism (GMO) is a multi-step process. Genetic engineers must isolate the gene they wish to insert into the host organism. This gene can be taken from a cell or artificially synthesized. If the chosen gene or the donor organism's genome has been well studied it may already be accessible from a genetic library. The gene is then combined with other genetic elements, including a promoter and terminator region and a selectable marker.
A number of techniques are available for inserting the isolated gene into the host genome. Bacteria can be induced to take up foreign DNA, usually by exposed heat shock or electroporation. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus, or through the use of viral vectors. In plants the DNA is often inserted using Agrobacterium'-mediated recombination, biolistics or electroporation.
As only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through tissue culture. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene.
Traditionally the new genetic material was inserted randomly within the host genome. Gene targeting techniques, which creates double-stranded breaks and takes advantage on the cells natural homologous recombination repair systems, have been developed to target insertion to exact locations. Genome editing uses artificially engineered nucleases that create breaks at specific points. There are four families of engineered nucleases: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the Cas9-guideRNA system (adapted from CRISPR). TALEN and CRISPR are the two most commonly used and each has its own advantages. TALENs have greater target specificity, while CRISPR is easier to design and more efficient.