Gene targeting

Gene targeting is a biotechnological tool used to change the DNA sequence of an organism (hence it is a form of Genome Editing).

It is also common practice to increase GT rates by causing a double-strand-break (DSB) in the targeted DNA region.

To target genes in mice, the DNA is inserted into mouse embryonic stem cells in culture.

This method has been applied to species including Drosophila melanogaster,[5] tobacco,[7][8] corn,[9] human cells,[10] mice[11] and rats.

[12] Genetic modification usually describes the insertion of a transgene (foreign DNA, i.e. a gene from another species) into a random location within the genome.

[15][16] In such cases the edits caused by gene-targeting would, in some jurisdictions, be considered as equivalent to Genetic Modification as insertion of foreign DNA has occurred.

[17][18] This site-specific or ‘targeted’ nature of genome editing is typically what makes genome-editing different to traditional ‘genetic modification’ which inserts a transgene at a non-specific location in the organisms' genome, as well as gene-editing making small edits to the DNA already present in the organisms, verses genetic modification insertion 'foreign' DNA from another species.

[19][20] Because gene editing makes smaller changes to endogenous DNA, many mutations created through genome-editing could in theory occur through natural mutagenesis or, in the context of plants, through mutation breeding which is part of conventional breeding (in contrast the insertion of a transgene to create a Genetically Modified Organism (GMO) could not occur naturally).

A summary of gene-targeting through HDR (also called Homologous Recombination) and targeted mutagenesis through NHEJ is shown in the figure below.

[22][23] Hence GT remains the primary method of targeted (location-specific) insertion of long DNA sequences for genome engineering.

However the low efficiency of delivery of the gene-targeting machinery into cells has hindered this, with research conducted into viral vectors for gene targeting to try and address these challenges.

[33] Gene targeting is relatively high efficiency in yeast, bacterial and moss (but is rare in higher eukaryotes).

The most significant improvement to gene targeting frequencies in plants was the induction of double-strand-breaks through site specific nucleases such as CRISPR, as described above.

[49] As explained above, Gene Targeting is technically capable of creating a range of sizes of genetic changes; from single base-pair mutations through to insertion of longer sequences, including potentially transgenes.

This means that products of gene targeting can be indistinguishable from natural mutation, or can be equivalent to GMOs due to their insertion of a transgene (see Venn diagram above).

Historically the European Union (EU) has broadly been opposed to Genetic Modification technology, on grounds of its precautionary principle.

A chimeric mouse gene targeted for the agouti coat color gene , with its offspring
Wild-type Physcomitrella and knockout mosses : Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores . For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B–E, Different mutants. [ 1 ]
A Venn Diagram to show the relationship between three types of 'Genetic engineering'; Genetic Modification, Gene Targeting and Genome Editing.
Possible DNA repair outcomes after cutting by CRISPR , leading to gene editing. Both strands of DNA are cut by CRISPR-Cas (or other site-specific nuclease) to create a double-strand-break (DSB). The DSB is then repaired through two alternative DNA repair pathways ( NHEJ or HR ) to lead to random mutations at the cut site ("targeted mutagenesis") or specific mutations if a repair template is supplied that contains those specific edits ("gene targeting").