Gene Correction
An Overview
Gene correction is one of the mechanisms of gene editing and gives researchers a powerful tool for both repairing and mutating DNA, for discovering gene functions and for engineering new genetic variants. Approaches for gene correction are diverse and mainly relied on homology recombination. Zinc finger nucleases (ZFNs), CRISPR/Cas9 system, and transcription activator-like effector nucleases (TALENS) are three common and powerful gene editing tools for gene correction.
Figure 1. Gene correction. (Gaj, 2016)
Principles of Gene Correction
As opposed to the unpredictable mutations resulting from non-homologous end joining (NHEJ), targeted double-strand DNA breaks (DSBs) can induce precise gene editing by stimulating homology-directed repair (HDR) with an exogenously supplied donor template. Be active mainly during the S and G2 phases of the cell cycle, HDR naturally utilizes the sister chromatid as a template for DNA repair. However, an exogenously supplied donor sequence may also be used as a repair template. Therefore, the codelivery of targeted nucleases along with a targeting vector containing DNA homologous guide to the break site enables high-efficiency HDR-based gene editing. Any sequence differences present in the donor template can thus be incorporated into the endogenous locus to correct disease-causing mutations, which has been demonstrated in many proof-of-concept studies. Although plasmids have traditionally been the most commonly used source of donor DNA, recent studies have indicated that single-stranded oligonucleotides (ssODNs), with as little as 80 base pairs of homologies, can serve as efficient donor templates for HDR.
Figure 2. Principle mechanism of homologous recombination (HR). (Akcay, 2015)
Tools in Gene Correction
CRISPR/Cas systems are currently the simplest, most versatile and precise method of genetic manipulation and their fundamentals lie in prokaryotes in the provision of adaptive immunity against viruses and plasmids. It is a special tool that makes geneticists and medical researchers edit parts of the genome by removing, adding or altering sections of the DNA sequence.
Figure 3. CRISPR/Cas9. (Akcay, 2015)
TALE nucleases are principally derived from the Xanthomonas bacteria, which are plant pathogens. TALENs are targeted to a specific DNA sequence with the help of their repeat-variable di-residues (RVDs). Highly conserved 33-35-amino-acid TALE repeats each bind a single base pair of DNA with specificity dictated by two hypervariable residues.
Figure 4. TALEN. (Maeder, 2016)
Approaches for Gene Correction
- RecTEPsy-Mediated Recombineering in Pseudomonas syringae
- Genome Manipulations with Bacterial Recombineering and Site-Specific Integration in Drosophila
- Multiple Genetic Manipulations of DT40 Cell Line
- Gene Targeting of Human Pluripotent Stem Cells by Homologous Recombination
References
- Gaj, T.; et al. (2016). Genome-editing technologies: principles and applications. Cold Spring Harbor perspectives in biology. 8(12): a023754.
- Akçay, D. (2015). Fare Miyoblast Hücre Hattinda (C2c12 Hücre Hatti) Klf5 Geni Üzerinde Hedefli İnsersiyon Gerçekleştirilmesi.
- Maeder, M. L.; Gersbach, C. A. (2016). Genome-editing technologies for gene and cell therapy. Molecular Therapy. 24(3): 430-446.
