For decades, mammalian genomes have remained resistant to targeted modification and researchers have generally lacked robust tools for efficient and routine mutagenesis. This is not surprising as mammalian genomes are littered with large amounts of repetitive elements, some of which constitute ~21% of the entire genome.
If the human genome were more permissive to homologous recombination, the stability of the genome would be largely compromised. Thus, in light of this and other protective mechanisms mammalian cells have established for genome stability, the genetic engineering of mammalian cells has remained difficult.
In cell types where homologous recombination is naturally suppressed, methods have been developed to induce hyper-recombinant phenotypes. For example, phage proteins have been used to globally increase the rate of homologous recombination in Escherichia coli and Mycobacterium tuberculosis.
This technological advancement facilitated the rapid generation of a complete, genome-wide knockout library of E. coli strains, each lacking a specific gene. However, application of this or similar technologies is impractical in mammalian cells since a global increase in recombination would destabilize the genome through random recombination of repetitive elements. Indeed, such genomic instability is known to be associated with many human diseases.
The recent development of engineered zinc finger nucleases (ZFNs) provides researchers with a technological solution to mammalian genome editing. ZFNs can be designed to generate site-specific double-strand breaks (DSBs), which are repaired by either nonhomologous end joining (NHEJ), leading to gene knockout or homologous recombination (HR), achieving precise gene replacement, insertion, or deletion (Figure 1).
In this way, gene editing becomes possible in a localized area without compromising the stability of the entire genome. Consequently, in the area surrounding a ZFN-mediated DSB, the rate of DNA repair can be elevated by several orders of magnitude, often increasing mutagenic frequencies from 10-6–10-5 up to 10-2 without global changes in genomic stability.
Such a large increase in efficiency has enabled the direct screening of clonal cells for mutations in convenient 96-well formats, without relying on antibiotic selection markers. The freedom from using antibiotic selection enables a new level of “surgical” genome modification such as the mutation of codons and the modification of single nucleotide polymorphisms (SNPs).