Targeted Genome Editing

Phil Simmons and Derek Douglas
Product Management, Biotechnology Division, Sigma-Aldrich
The determination of complete genome sequences for a wide variety of experimental organisms established a first step in the search for an in-depth understanding of the complex genetic functions that define living entities. Extensive genetic, biochemical, cytological and physiological analyses are now required to correlate genome sequence with the next level of understanding of genetic function. As such, technologies that allow researchers to routinely and efficiently edit the genomes of virtually any species, by directing mutations in a truly targeted fashion, would greatly enhance the understanding of basic biology, and potentially lead to novel ways of treating human disease.

Early Approaches to Targeted Genome Editing

While early approaches to genetic manipulation used random and/or non-targeted methods such as ionizing radiation and chemical-induced mutagenesis to make changes to the genome, more recent methods have employed targeted methods of genomic editing. The most well studied methods have relied on the process of homologous recombination (HR), a naturally occurring DNA-repair mechanism found in most cells that uses the second copy of a chromosome as a template to repair damage to a gene.
Starting with Nobel Prize winner Mario Capecchi’s earliest work, it was discovered that the HR process could be harnessed and directed in mouse embryonic stem cells (MESCs) by introducing exogenous DNA as a repair template to induce precise point mutations, gene deletions, and gene insertions. This approach to gene targeting led to the development of knockout mice as a research tool, still considered the gold standard for many functional genomics studies. However, HR was found to be woefully inefficient in many other relevant model systems, including mammalian somatic cells, in which the rate of HR is much lower than the rate of HR in MESCs. For this reason HR never found its footing as a robust means of mutatgenesis in cell types commonly used in functional genomics, target validation, and protein production settings.

The Zinc Finger as a DNA Targeting Tool

Zinc Finger Nucleases: Highly Specific 'Genomic Scissors'

ZFN-mediated Genome Editing

Targeted Gene Knockout

Targeted Gene Integration

Targeted Gene Correction

Sigma-Aldrich: Making ZFN Technology Accessible