Gene Targeting by Homologous Recombination

Abstract

Gene targeting by homologous recombination involves the exchange of genetic information between genomic and exogenous deoxyribonucleic acid (DNA) molecules via crossover events. These exchanges are guided by homologous sequences acted on by enzymatic machinery of the cell. Homologous recombination provides a mechanism for targeting defined modifications into genes of interest, making gene‐targeting technologies valuable tools to explore gene function and to develop human, genetic disease models. Gene targeting, however, is inefficient, making the process challenging. Advances in technology now allow us to direct repair enzymes to the targeted site by inducing site‐specific DNA damage using zinc‐finger nucleases, transcription activator‐like effector nucleases, clustered regularly interspaced short palindromic repeats and triplex‐forming oligonucleotides. Further, antirecombinogenic pathways can now be transiently suppressed using ribonucleic acid (RNA) interference (RNAi) and other small molecule approaches. These and other techniques can greatly enhance gene‐targeting efficiency. The coincident development of human stem cell technology brings forth the potential of gene‐targeting strategies for therapeutic application.

Key Concepts:

  • Homology‐directed gene targeting utilises homologous recombination to introduce defined modifications into sequences of interest in mammalian genomes.

  • Gene‐targeted knockout and knock‐in models are instrumental in elucidating gene function and studying human genetic diseases.

  • Combining stem cell and gene‐targeting technologies opens potential avenues for gene therapy.

  • Recent technological advances have greatly enhanced gene‐targeting efficiencies.

  • Due to technological advances, most genes in a variety of mammalian species can now be manipulated by homology‐directed gene targeting.

Keywords: homologous recombination; gene targeting; gene therapy; random integration; site‐specific recombinase; models of human disease; gene‐targeting efficiency

Figure 1.

Schematic of strategies to improve gene‐targeting efficiency. Described is the integration of a basic replacement‐targeting vector for creating a conditional knockout locus. The vector contains positive and negative selectable markers (green boxes) to enrich for targeting events as well as LoxP sites (pink triangles) for subsequent removal of exon 2. The targeting vector can integrate via a homology‐directed targeting event at the target locus or by randomly integrating into other nontargeted sites in the genome. Targeted integration results from homologous recombination (HR) between the homologous arms of the vector and the genomic locus. Random integration can occur by ligation of the targeting vector into spontaneous breaks in the genome or off‐target HR and generally occurs >1000 times more frequently than targeted events. To stimulate gene targeting, ZFNs, TALENs, CRISPR or TFOs can be used to induce site‐specific damage to elicit an HR response at the target locus. Also, synchronising cells, overexpressing HR components or loading HR proteins onto the vector prior to transfection can enrich HR proteins to the target locus and targeting vector. Further stimulation can be acquired by using RNAi or small molecules to transiently inhibit regulatory proteins that impede HR processes, such as end resection, strand invasion and crossover choice. This approach can also be used to transiently inhibit proteins that lead to random integration, such as components of NHEJ pathways. Following integration, there are several strategies to enrich for targeted integration events. The first is the presence of a positive selectable marker that is used to select cells that have integrated the vector. In this vector, the positive marker has been set up as a polyadenylation trap whereby the marker must integrate upstream of a polyadenylation signal to be efficiently expressed. If the vector is integrated in a targeted manner, the marker gains the polyadenylation signal at the target locus. If the vector integrates randomly, the chances of gaining a polyadenylation signal would be quite low, thus most cells that randomly integrate the targeting vector will be lost during selection. This vector also contains a negative selectable marker, the herpes simplex‐thymidine kinase (HS‐ThK) gene, outside the homologous arms. Targeted integration would result in the loss of this gene due to recombination within the homologous sequences. The marker would be readily integrated when the vector undergoes random integration. HS‐ThK will poison cells when under ganciclovir selection. Ganciclovir selection would thus kill only cells with randomly integrated vectors enriching for homology‐directed integration. After selection and verification of targeted integration (usually by Southern blot or long‐range polymerase chain reaction (PCR)), expression of the CRE recombinase induces recombination between LoxP sites (pink triangles), resulting in the removal of a vital exon to knock out the targeted gene at that locus. CRE recombinase also removes the positive selectable marker from the genome so that the same targeting vector can be used in a second round of gene targeting to knock out the other allele.

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Reh, Wade A, and Vasquez, Karen M(Apr 2014) Gene Targeting by Homologous Recombination. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005988.pub2]