Genome editing is used to make targeted genetic modifications in all cell types, thanks to specific molecular scissors. Available since the 1980s, these tools have become more efficient and specialized over time. In 2012, the advent of the CRISPR-Cas9 system, characterized by its great simplicity and modest cost, revolutionized this approach with genome editing having since spread to all domains of science and medicine.
It enables researchers to make the genetic modifications of their choice, in order to develop tailor made animal and cell models, to further knowledge of the development of living organisms, diseases, or test therapeutic compounds. Initial clinical trials based on this approach have commenced, with the objective of treating monogenic diseases, some cancers or even infectious diseases.
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Understanding genome editing
Modifying a DNA sequence in a targeted way
Genome editing consists of modifying a cell’s genome with great precision. It is possible to deactivate a gene, introduce a targeted mutation, correct a specific mutation or insert a new gene. This genetic engineering technique uses modified nucleases, known as molecular scissors.
These nucleases cut the DNA at a predefined location in the genome, depending on its sequence. A system of natural DNA repair, known as non-homologous end-joining (NHEJ), is then activated in order to "reattach" the two free extremities generated by the cut. But this system introduces errors, leading to the mutation of the gene targeted by the nuclease. In this case, the mutation introduced is therefore random.
It is also possible to modify the targeted sequence according to one’s requirements. This requires delivering to the cell, in addition to the nucleases, a strand of DNA which presents the desired sequence, flanked by ends which are homologous to those of the site of the cut. Another cellular repair system (homologous recombination) then comes into play, "incorporating" the DNA sequence supplied at the time of the repair, leading to its definitive insertion into the genome.
Base editing: genome editing without DNA cutting
Recently, Cas nucleases have been transformed so that they no longer cut the recognized genome site: the nuclease serves as an anchor point for the transport of other proteins capable of transforming one DNA base into another, thereby inducing a targeted mutation without cutting. This technique, called base editing, could prove to be particularly interesting in cells in which the natural repair processes of DNA breaks are inefficient, rendering traditional genome editing (with double-stranded cutting) ineffective.
These various techniques work in all cell types: human, animal, plant, bacterial, adult or embryonic.
Various types of molecular scissors available
The nucleases used in genome editing are all derived from natural bacterial systems. These so-called restriction enzymes are capable of cutting double-stranded DNA at specific locations. These enzymes are modified in the laboratory in order to recognize and cut the required sequences in the DNA.
These proteins are extremely specific restriction enzymes, capable of recognizing and cleaving a DNA sequence by assembling themselves in pairs of identical subunits (homodimers). Since their natural repertoire is limited, it is necessary to engineer new meganucleases in order to target a specific site within a genome. As a result, this approach is difficult and reserved for specialists of this system. Their use is very limited.
These artificial proteins are comprised of so-called zinc-finger peptides, which recognize a DNA sequence, and a nuclease (Fok1), which cuts the DNA. Each zinc-finger peptide recognizes a short sequence of three nucleotides and assembling several of these makes it possible to target longer sequences, in a more specific way. Furthermore, in order to cut, zinc-finger nucleases act in pairs on two sites close to each other. This enables the catalytic action of the Fok1 enzymes. Therefore, a genome modification requires two zinc-finger nucleases, whose construction and assembly are very complex. This restricts their use.
TALENs (transcription activator like-effectors) are also used in pairs, targeting two close DNA sequences. They comprise a DNA binding domain made up of a combination of four peptides, each of these peptides specifically recognizing one of the four bases of the DNA. By acting on the linkage of these peptides, it is possible to target a specific DNA sequence. This binding domain is associated with a Fok1 nuclease which ensures double-strand cutting.
As with zinc-finger nucleases, protein engineering is necessary to construct and assemble the TALENs intended for genome editing. Computer programs such as E-Talen facilitate this task and a library of TALENs capable of recognizing more than 18,700 genes is available. TALENs are easier to produce than zinc-finger nucleases and offer very good efficacy.
This time it is a guide RNA (CRISPR, which stands for clustered regularly interspaced short palindromic repeats) and not a protein which recognizes the target sequence to be cut. It is associated with a Cas nuclease, generally Cas9, which cuts the DNA at this precise location.
Available since 2012, the simplicity of the CRISPR-Cas9 system has revolutionized genome editing. Scientists now use it daily in all areas of research: medicine, agronomy, environment, etc. Producing guide RNA is infinitely easier than producing proteins. It is also a lot faster (requiring a few days as opposed to a few weeks or months for the production of zinc-finger nucleases or TALENs) and a lot less costly.
Barely three months after this tool was developed, several laboratories were already publishing results obtained with this technique, confirming its potential. Five years later, several thousands of research articles - concerning basic or applied research, conducted in countless species, targeting all sorts of applications - had been published.
Use in all domains of life and particularly in biomedical research
Genome editing is used in various domains: agri-food to produce improved species (for example, sheep and veal with increased muscle mass in South America), agronomy (for example, with the genetic modification of invasive plant species, to restrict their growth) and, of course, health. And this is taking place at all levels of research: fundamental, applied and clinical. However, this research is all still very much at the experimental stage.
To produce animal models
Genome editing enables the development of new animal models (sheep, cows, ferrets, rabbits, pigs, etc.) by modifying the genetic heritage of embryos using the CRISPR-Cas9 system before transferring them to the females. In this way, the researchers can have at their disposal unlimited and varied animal models suitable for the study of development, diseases or for use in therapeutic trials.
Two genetically modified macaque monkeys were, for example, born in 2014 following the introduction of mutations in two different genes, one involved in metabolism and the other in immunity. These births have proven that it is possible to obtain genetically modified non-human primates for the study of diseases. Up until then, this type of research was more or less only possible with mice, drosophila and zebrafish.
To produce cell models
In addition to animal models, it is possible to produce tailor made cell culture models. Up until then, the study of rare diseases was notably limited by the difficulty of having homozygous cells for a rare recessive mutation. Now it is possible to create these mutations from healthy cells or to deactivate one of the alleles in heterozygous individuals for this rare mutation.
To treat using gene therapy
By making it possible to introduce a healthy gene or to correct a mutation in the cells of a patient, genome editing paves the way for potential gene therapies. But it faces the same difficulties as the other gene therapy techniques, particularly in regard to the vectorization of the therapeutic DNA and the nucleases (the step which consists of incorporating this material into the cells to be treated).
Several possibilities are available for researchers for an ex vivo procedure (the cells to be treated are taken from the patient, modified in the laboratory and then re-administered). The Cas nuclease can be delivered in various forms (DNA, RNA or protein) with the guide RNA, and several delivery methods are possible, such as the application of an electrical field (electroporation) or the use of chemical vectors which increase the permeability of the cell membranes. Nevertheless, the viral vectors remain highly efficient, particularly the lentiviruses and adenoviruses for tests conducted in vivo.
Challenges facing research
The immense majority of genome editing concerns fundamental or preclinical research, for the study of diseases, normal or pathological development and the testing of therapeutic compounds. Nevertheless, several clinical trials have begun in humans in monogenic diseases, as well as in infectious diseases and oncology.
A trial is commencing in patients with hemophilia B. Zinc-finger nucleases will be delivered to their liver cells using a viral vector (AAV). The objective is to introduce a healthy copy of the gene coding for coagulation factor IX into an active region of the genome, enabling its continuous expression. Phase I trials are also starting in the treatment of lysosomal storage diseases caused by a deficiency in the production of the IDUA (alpha-L-iduronidase) enzyme: mucopolysaccharidoses. Here too, the strategy tested consists of using zinc-finger nucleases delivered to the hepatocytes of patients, to force the expression of the deficient enzyme.
A phase II trial is underway in the domain of infectious diseases, against HIV. It is based on the ex vivo use of zinc-finger nucleases in the uninfected hematopoietic stem cells of patients. The objective is to deactivate the CCR5 gene. Since the mutation of this gene is known to protect against infection with HIV, the researchers hope to render the modified cells resistant to the virus and to restore patient immunity. Trials are also ongoing with various types of nucleases in the treatment of cervical dysplasia. The idea is to eliminate the HPV 16 or 18 virus in the precancerous cells. Indeed, the persistence of this infection contributes to the development of cancer and to a poor prognosis. The treatment tested consists of deactivating viral proteins (E6 and E7) associated with this persistence.
In the domain of cancer, genome editing is also used to arm patients’ T-cells against their own tumor. The modification takes place ex vivo, following extraction of the blood cells, and consists of bringing about the expression of a synthetic receptor (or CAR, which stands for chimeric antigen receptor) which recognizes tumor antigens. Another approach consists of eliminating an obstacle to immune cell activation: it was used in B-cell lymphoma, with T-cells modified in order to be capable of targeting the CD19 tumor surface antigen. Several clinical trials are also being launched to test the deactivation of the PD-1 gene in order to stimulate the immune system against the advanced stages of esophageal, lung and nasopharyngeal cancer or lymphomas. Blood cells will be taken from patients, genetically modified using CRISPR-Cas9, multiplied and then re-injected.
CRISPR-Cas9 in the human embryo
Chinese and US teams have tested the CRISPR-Cas9 technique in the human embryo to correct a mutation conferring beta thalassemia or another mutation associated with a severe heart disease. It consists of basic research intended to evaluate the efficacy and safety of CRISPR-Cas9 on embryos which will then be destroyed. The effects obtained to date remain largely perfectible: the percentage of embryos modified is low and the risk of mosaicism (the risk of the cells of a same embryo not all possessing the same genetic heritage) is high.
Concerning genetic modifications which could be passed on to offspring, France has ratified the Oviedo Convention which forbids this type of procedure. For many scientific bodies and ethics committees, including that of Inserm, even if the Oviedo Convention were to be modified, it is at this stage inconceivable to carry out procedures on embryos which would be intended to achieve the birth of a child, for lack of sufficient guarantees of efficacy and safety.
The risk of off-target mutations and others
As with all medicines, a major risk of genome editing in therapy is that of side effects.
In the case of genome editing, there is the particular risk of creating off-target mutations outside the zone initially targeted. The nucleases target specific sequences of a length of 15-20 bases, but they can "mistakenly" cut very close sequences which can only be distinguished by a single base. These undesired mutations can modify the expression of genes which were not targeted, deactivate them, or even lead to the development of cancer. At present, whole-genome sequencing approaches of cells genetically modified ex vivo make it possible, in principle, to check for the absence of off-target mutations. The good representativity of these checks remains to be verified. This problem must be solved before conducting in vivo tests. Bioinformatic tools have been developed for this purpose in order to better predict the risk of off-target mutations and guarantee better nuclease specificity. Furthermore, the performance and specificity of the latter continue to be improved.
Other difficulties have been identified, such as mosaicism: during an experiment, not all the cells subject to the genome editing attempt are genetically modified in a strictly identical way at the end of it. This is explained by the fact that this technique uses natural processes of DNA repair and that these can occur in an unequal manner from one cell to another.
Finally, the absence of experience does not make it possible to rule on the long-term safety of a genetic modification created in a cell. The clinical trials being launched will provide valuable information on the tolerance and safety of this approach. They will enable us to know, in two or three years’ time, whether or not the off-target effects are controlled.
A new variant of genome editing, called epigenome editing has been proposed. It uses the CRISPR-Cas system, but the Cas nuclease does not cut the DNA: it enables the import of transcription-regulating molecules to block or, on the contrary, stimulate the expression of a targeted gene. The sequence of the gene is therefore not modified.
The proof of concept was provided at the end of 2017 in vivo in mice, with the forced activation of genes implicated in the control of diabetes, Duchenne muscular dystrophy and an acute renal disease.
This approach avoids the risk of off-target mutation, even if side effects of off-target binding can exist. Furthermore, it avoids the irreversible modification of a cell’s genetic heritage.
The all-out use of genome editing raises ethical questions, especially since the initial applications are taking shape before the technique has been fully mastered.
This is especially the case for gene drives. This strategy makes it possible to genetically modify (using CRISPR-Cas9) an animal population by forcing a modified gene to pass itself on. The aim is to render the population resistant to a disease or to sterilize it if the species is considered harmful. Gene drives can be used to control invasive plant species or to eliminate resistance to herbicides or pesticides. It is also envisaged for fighting vectors of disease transmission, such as the mosquitoes implicated in the transmission of malaria or dengue. A test study, conducted in Panama in 2015, appears to back up the efficacy of the technique: it is thought to have reduced the populations of the Aedes aegypti mosquito which transmits dengue.
These practices raise many questions, in addition to those already discussed on the off-target effects: what is the risk of contaminating species other than the target population? What is the impact on the environment and biodiversity of eradicating insects that pollinate and serve as food for fish larvae? What are the long-term risks for the species? How can the propagation of the gene be stopped efficiently if there is a loss of control of the technology? Evaluations must be performed over long periods, with the preparation of multiple scenarios by multidisciplinary teams combining molecular biology, ecology, social sciences, for the prudent evaluation of the long-term benefit/risk balance.
Other questions are raised with the genetic modification of species for commercial gain. As such, in Argentina and Uruguay, experimental farms are modifying the genomes of sheep and veal to increase the size of their muscles with the aim of doubling the quantities of meat produced. What are the impacts on animal quality of life and for consumers?
In a human embryo which would be used to achieve the birth of a child, this type of procedure is totally inconceivable at this stage, for lack of sufficient guarantees of efficacy and safety. But ultimately, should the technique become safe and reliable, it could be used in rare and very specific indications: for example, to avoid the transmission of a severe disease when both parents are affected and in the case of a 100% risk of giving birth to a sick child. It would then involve correcting the mutation in the embryo or even upstream at germ-cell level prior to fertilization. The French Academy of Medicine has ruled in favor of this possibility, should the technology achieve the necessary levels of efficacy and safety. However, the greatest vigilance is required to avoid any deviations in favor of genetic modifications "of convenience".