zinc finger nuclease-based gene editing
Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells: this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair.
Such ’genome editing’ is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.
Zinc finger motifs occur in several transcription factors. The zinc ion, found in 8% of all human proteins, plays an important role in the organization of their three-dimensional structure.
In transcription factors, it is most often located at the protein-DNA interaction sites, where it stabilizes the motif. The C-terminal part of each finger is responsible for the specific recognition of the DNA sequence.
The recognized sequences are short, made up of around 3 base pairs, but by combining 6 to 8 zinc fingers whose recognition sites have been characterized, it is possible to obtain specific proteins for sequences of around 20 base pairs. It is therefore possible to control the expression of a specific gene.
It has been demonstrated that this strategy can be used to promote a process of angiogenesis in animals.
It is also possible to fuse a protein constructed in this way with the catalytic domain of an endonuclease in order to induce a targeted DNA break, and therefore to use these proteins as genome engineering tools.
The method generally adopted for this involves associating two proteins – each containing 3 to 6 specifically chosen zinc fingers – with the catalytic domain of the FokI endonuclease.
The two proteins recognize two DNA sequences that are a few nucleotides apart.
Linking the two zinc finger proteins to their respective sequences brings the two endonucleases associated with them closer together. This means that they can be dimerized and then cut the DNA molecule.
Several approaches are used to design specific zinc finger nucleases for the chosen sequences.
The most widespread involves combining zinc-finger units with known specificities (modular assembly).
Various selection techniques, using bacteria, yeast or mammal cells have been developed to identify the combinations that offer the best specificity and the best cell tolerance.
Although the direct genome-wide characterization of zinc finger nuclease activity has not been reported, an assay that measures the total number of double-strand DNA breaks in cells found that only one to two such breaks occur above background in cells treated with zinc finger nucleases with a 24 bp composite recognition site and obligate heterodimer FokI nuclease domains.
Zinc finger nucleases are research and development tools that have already been used to modify a range of genomes, in particular by the laboratories in the Zinc Finger Consortium.
The US company Sangamo Biosciences uses zinc finger nucleases to carry out research into the genetic engineering of stem cells and the modification of immune cells for therapeutic purposes.
Modified T lymphocytes are currently undergoing phase I clinical trials to treat a type of brain tumor (glioblastoma) and in the fight against AIDS.
References
Genome editing with engineered zinc finger nucleases. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Nat Rev Genet. 2010 Sep;11(9):636-46. PMID: #20717154#