Genome engineering refers to the strategies and techniques developed in recent years for the targeted, specific modification of the genetic information – or genome – of living organisms.
It represents a very active field of research because of the wide range of possible applications, particularly in the areas of human health - the correction of a gene carrying a harmful mutation, the production of therapeutic proteins, the elimination of persistent viral sequences - agricultural biotechnology - the development of new generations of genetically modified plants - and for the development of research tools - for example, to explore the function of a gene.
Various modifications can be performed:
How does it differ from previous genome modification technologies?
Early technologies developed to insert a gene into a living cell, such as transgenesis, are limited by the random nature of the insertion of the new sequence into the genome. The new gene is positioned blindly, and may inactivate or disturb the functioning of other genes or even cause severe unwanted effects; it may trigger a process of cancerization, for example. Furthermore, these technologies offer no degree of reproducibility, as there is no guarantee that the new sequence will be inserted at the same place in two different cells.
The major advantage of genome engineering, which uses more recent knowledge and technology, is that it enables a specific area of the DNA to be modified, thereby increasing the precision of the correction or insertion, preventing any cell toxicity and offering perfect reproducibility.
Genome engineering and synthetic genomics (designing artificial genomes) are currently among the most promising technologies in terms of applied biological research and industrial innovation.
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Early approaches to genome engineering involved modifying genetic sequences using only homologous recombination. Using a homologous sequence located on another strand as a model can lead this natural DNA maintenance mechanism to repair a DNA strand. It is possible to induce homologous recombinations between a cellular DNA strand and an exogenous DNA strand inserted in the cell by researchers, using a vector such as the modified genome of a retrovirus. The recombination phenomenon is flexible enough for a certain level of change (addition, suppression or modification of a DNA portion) to be introduced to the targeted homologous area.
In the 1980s, Mario R. Capecchi and Oliver Smithies worked on the homologous recombination of DNA as a “gene targeting” tool; in other words, as an instrument for the inactivation or modification of specific genes. Working with Martin J. Evans, they developed a process for the modification of the mouse genome by modifying the DNA of mouse embryonic stem cells in culture and injecting these modified stem cells into mouse embryos. Genetically modified mice generated using this method make useful laboratory models to study human diseases. This tool is now commonly used in medical research. The three researchers were awarded the 2007 Nobel Prize in Medicine for their work.[1]
Modifying genomes using only homologous recombination remained a long and random process, until additional developments were made that could increase the rate of homologous recombination in somatic cell types. These developments fall under to two mechanistically distinct methods of triggering the cells inherent DNA repair mechanisms which are required to insert a foreign gene sequence into a live cell. The first method is by site directed endonucleases (restriction enzymes), which include specific technologies such as zinc finger nucleases (ZFNs) and meganucleases. Site directed endonucleases achieve gene modification through causing double stranded DNA (dsDNA) breaks which triggers the cells natural DNA repair mechanism, predominantly non homologous end joining (NHEJ) as well as a low frequency of homologous recombination (HR). The second method is recombinant adeno-associated virus (rAAV) mediated genome engineering which induces high frequencies of homologous recombination alone, thus forgoing the need to perform dsDNA breaks.
Researchers wishing to efficiently eliminate a gene to study the resulting loss of its function are increasingly opting for a “molecular scissors” approach. These are enzymes with specific properties which enable them to cut the long double DNA strand along the sequence to be modified, thereby triggering the NHEJ and HR process at the required location. Restriction enzymes commonly used in molecular biology to cut DNA interact with sequences of 1 to 10 nucleotides. These sequences, which are very short and generally palindromic, often occur at several sites in the genome (the human genome comprises 6.4 billion bases). Restriction enzymes are therefore likely to cut the DNA molecule several times. In their efforts to find a genome surgery approach offering a higher degree of accuracy and security, scientists therefore turned to more precise tools. More targeted genome engineering can be performed by using enzymes which are able to recognize and interact with DNA sequences that are sufficiently long so as to occur only once, with high probability, in any given genome. The DNA modification therefore takes place precisely at the site of the target sequence. With recognition sites of over 12 base pairs, meganucleases and zinc finger nucleases offer this degree of precision. Once the DNA has been cut, natural DNA repair mechanisms and homologous recombination enable the incorporation of a modified sequence or a new gene. The success of these different stages (recognition, cleavage and recombination) depends on various factors, including the efficacy of the vector that introduces the enzyme into the cell, the enzyme cleavage activity, the cell’s capacity for homologous recombination and probably the state of the chromatin at the given locus.
Meganuclease-based Engineering
Meganucleases, discovered in the late 1980s, are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 12 to 40 base pairs).[2] The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence.
These enzymes were identified in the 1990s as promising tools for genome engineering. However, even though they occur in nature and each one exhibits slight variations in its DNA recognition site, there is virtually no chance of finding the exact meganuclease required to act on a specific DNA sequence. Each new genome engineering target therefore requires an initial protein engineering stage to produce a custom meganuclease.
There are two possible methods for creating custom meganucleases:
These two approaches can be combined. Scientists from the French biotechnology company Cellectis have identified in the structure of several meganucleases the areas responsible for DNA cleavage and the areas that interact with specific DNA sites. By acting on these recognition sites, they have been able to generate variants that interact with different DNA sequences from those of the initial meganucleases, while retaining their ability to cut the DNA and their high degree of specificity.
A large bank containing several tens of thousands of protein units has been created. These units can be combined to obtain chimeric meganucleases that recognize the target site, thereby providing research and development tools that meet a wide range of needs (fundamental research, health, agriculture, industry, energy, etc.).
This technique has enabled the development of several meganucleases specific for sequences in the genomes of viruses, plants, etc., and the industrial-scale production of two meganucleases able to cleave the human XPC gene; mutations in this gene result in Xeroderma pigmentosum, a severe monogenic disorder that predisposes the patients to skin cancer and burns whenever their skin is exposed to UV rays.[5]
Another approach involves using computer models to try to predict as accurately as possible the activity of the modified meganucleases and the specificity of the recognized nucleic sequence.[6] The Northwest Genome Engineering Consortium, a US consortium funded by the National Institutes of Health, has adopted this approach with the aim of treating leukemia by modifying hematopoietic stem cells. The model’s prediction has been verified and guided by means of directed mutagenesis and in vitro biochemical analysis.
A third approach has been taken by the American biotechnology company Precision Biosciences, Inc. The company, funded by the National Institutes of Health and the National Institute of Standards and Technology, has developed a fully rational design process called the Directed Nuclease Editor (DNE) which is capable of creating highly specific engineered meganucleases that successfully target and modify a user-defined location in a genome.[7]
Zinc finger nuclease-based Engineering
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.[8] 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.[9]
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.[10]
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.[11][12] Modified T lymphocytes are currently undergoing phase I clinical trials to treat a type of brain tumor (glioblastoma) and in the fight against AIDS.[10]
rAAV-based engineering
rAAV mediated genome engineering builds on Capecchi and Smithies’ Nobel Prize winning discovery that homologous recombination, a natural DNA repair mechanism, can be harnessed to perform precise genome alterations in mice. rAAV improves the efficiency of this approach to permit gene editing in any pre-established and differentiated human cell line,[13] which in contrast to mouse ES cells, have low rates of homologous recombination.[14] Genome editing in human and other mammalian somatic cell types using homologous recombination is now achievable using recombinant adeno-associated virus (rAAV) vectors. These single-stranded DNA viral vectors have high transduction rates and have a unique property of stimulating endogenous HR without causing double strand DNA breaks in the genome, which are typical of other site-directed endonuclease mediated genome editing methods.
Users can design a rAAV vector to any target genomic locus and perform either gross or subtle endogenous gene alterations in mammalian somatic cell types. These include gene knock-outs for functional genomics, or the ‘knock-in’ of protein tag insertions to track translocation events at physiological levels in live cells. Most importantly, rAAV targets a single allele at a time and does not result in any off-target genomic alterations. Because of this, it is able to routinely and accurately model genetic diseases caused by subtle SNPs or point mutations that are increasingly the targets of novel drug discovery programs.
The use of rAAV based engineering has been documented in over 160 peer-reviewed publications. Researchers have employed rAAV based genome editing to engineer human cell lines for use as disease models.[15] These isogenic human disease models are precisely matched pairs of cell lines, where one harbours a cancer-associated mutation in an endogenous gene, just as it occurs in real patients, while the other is a genetically identical cell line carrying a normal version of that gene. These isogenic disease models provide a definitive means to understand disease biology.[16]
Derived from this disease model panel, the company is also launching a range of genetically-defined genomic DNA diagnostic control materials. Further work is underway by Horizon to apply rAAV gene-editing in other translational areas, such as enhancing the yield of therapeutic biologic agents from engineered ‘bioproducer’ mammalian cell lines.
Another emerging application of rAAV based engineering is for gene therapy in patients, due to the accuracy and lack of off-target recombination events afforded by this approach. There are a number of clinical trials currently in progress.