Researchers are beginning see the genome not as static and unchanging but as re-writable. Editing the genome of a living cell is not quite as simple as ctrl+C ctrl+V though. Researchers have had to find and alter some unique proteins to escape the issues of toxicity and mutation that editing can cause. Historically, genome editing has only been possible in mice, and it wasn’t very efficient (Hall et al., 2009). Homologous recombination by the cell is used to insert DNA into an induced break, and this has only been favourable enough in mice.
The biggest issue in developing editing systems in other organisms has been specificity. If DNA is going to be added or removed from the genome, you want to be very sure it targets only the region you want. This has proved difficult, and is still not completely solved, but some novel discoveries in protein engineering have made real headway. Zinc Finger Nuclease, TALEN, CRISPR/Cas9 systems are proving to be useful editors, each with their own advantages.
Genome Editing Techniques
Zinc Finger Nucleases (ZFNs): The oldest, and best studied mechanism of genome modificaion. Zinc fingers (ZF) are proteins composed of conjugated Cys2His2 motifs that each recognize a specific nucleotide triplet based on the residues in their α helix.
Transcription Activator-like Effector Nucleases (TALENs): Operate on almost the same principle as ZFNs, and are fusions of transcription activator-like (TAL) proteins and a FokI nuclease that have a strong recognition for specific nucleotides.
CRISPR/Cas9: This system uses the clustered regulatory interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) bacterial endonuclease and a short guide RNA (gRNA) to target the Cas9 protein to a genomic region of interest.
Genome Editing Additional Reading
Gaj, T., Gersbach, C.A., and Barbas, C.F.,3rd. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397-405.
This review covers the ZFNs, TALENs, and CRISPR a great detail. Historical perspective, domain structure, mechanism of binding, papers that use each, therapeutic applications, and future directions are discussed.
- Hall, B., Limaye, A., and Kulkarni, A.B. (2009). Overview: generation of gene knockout mice. Curr. Protoc. Cell. Biol. Chapter 19, Unit 19.12 19.12.1-17.