To create a gene-targeted organism, DNA must be introduced into its cells. This DNA must contain all of the parts necessary to complete the gene targeting. At a minimum this is the homology repair template, containing the desired edit flanked by regions of DNA homologous (identical in sequence to) the targeted region (these homologous regions are called “homology arms” ). Often a reporter gene and/or a selectable marker is also required, to help identify and select for cells (or “events”) where GT has actually occurred. It is also common practice to increase GT rates by causing a double-strand-break (DSB) in the targeted DNA region. Hence the genes encoding for the site-specific-nuclease of interest may also be transformed along with the repair template. These genetic elements required for GT may be assembled through conventional molecular cloning in bacteria.
Gene targeting methods are established for several model organisms and may vary depending on the species used. To target genes in mice, the DNA is inserted into mouse embryonic stem cells in culture. Cells with the insertion can contribute to a mouse's tissue via embryo injection. Finally, chimeric mice where the modified cells make up the reproductive organs are bred. After this step the entire body of the mouse is based on the selected embryonic stem cell.
To target genes in moss, the DNA is incubated together with freshly isolated protoplasts and with polyethylene glycol. As mosses are haploid organisms, moss filaments (protonema) can be directly screened for the target, either by treatment with antibiotics or with PCR. Unique among plants, this procedure for reverse genetics is as efficient as in yeast. Gene targeting has been successfully applied to cattle, sheep, swine and many fungi.
The frequency of gene targeting can be significantly enhanced through the use of site-specific endonucleases such as zinc finger nucleases, engineered homing endonucleases, TALENS, or most commonly the CRISPR-Cas system.  This method has been applied to species including Drosophila melanogaster, tobacco, corn, human cells, mice and rats.