For decades geneticists have theorized that targeted gene editing would provide an eloquent method to repair a defective gene, treat or prevent disease or allow selection of genetic traits in offspring. Regardless, successful efforts to incorporate targeted genetic material into higher organisms remain risky at best. Several approaches are currently being tested including the swapping of an abnormal gene for a good one, repairing a segment know to contain the deleterious error, or altering control of gene expression via editing of its transcriptional regulatory elements. All of the above approaches currently remain in the experimental mode.
One such example of gene therapy involves a mutated form of the gene RPE65 linked to an inherited disorder causing vision loss. In this case a harmless virus carrying the healthy gene was engineered as a vector to deliver the good gene1. While these studies have shown some success more importantly they point the way to future work.
Gene editing evolved in the early 2000s with the discovery of zinc finger nucleases and more highly evolved synthetic nucleases called TALENs. More recently a new technology is being adopted with high expectation of revolutionizing genome editing: CRISPR-Cas9 based genome editing. Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins were first discovered in bacteria and other prokaryotic organisms forming an immune system providing a form of acquired immunity to foreign genetic elements such as plasmids and phages. The CRISPR/Cas system has been adapted to gene editing in a wide variety of species by delivering the Cas9 protein and appropriate guide RNAs into the target cell. This approach allows the host genome to be cut at any desired target sequence location.
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA: http://dx.doi.org/10.1016/j.cell.2014.02.001Currently in press, researchers are working to functionally correct Factor VIII gene chromosomal inversions in hemophilia A patients by using the CRISPR-Cas9 system and induced pluripotent stem cells (iPSCs). Proof of concept has shown rescue of factor VIII deficiency in a lethal mouse model2. The hope is this technology will be transferrable to human disease targets in the near future.
1. Jacobson SG et al. “Improvement and decline in vision with gene therapy in childhood blindness.” New Engl J Med. DOI:10.1056/NEJMbr1412965
2. Park CY et al. “Functional Correction of Large Factor VIII Gene Chromosomal Inverstions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9.” Cell Stem Cell. http://dx.doi.org/10.1016/j.stem.2015.07.001
By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA