Genome editing
Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly insert genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain, double-strand breaks in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination or non-homologous end joining.
The development of CRISPR gene editing in 2015 improved the efficiency, specificity, and practicality of large-scale genome editing.
Since 2015, genome editing has been experimentally investigated on non-viable human embryos. In 2019, the first humans were born from genome-edited embryos using the CRISPR technique, as a result of the controversial He Jiankui affair.
History
Genome editing was pioneered in the 1990s, before the advent of the common current nuclease-based gene-editing platforms, but its use was limited by low efficiencies of editing. Genome editing with engineered nucleases, i.e. all three major classes of these enzymes—zinc finger nucleases, transcription activator-like effector nucleases and engineered meganucleases—were selected by Nature Methods as the 2011 Method of the Year. The CRISPR-Cas system was selected by Science as 2015 Breakthrough of the Year., four families of engineered nucleases were used: meganucleases, zinc finger nucleases, transcription activator-like effector-based nucleases, and the clustered regularly interspaced short palindromic repeats system. Nine genome editors were available as of 2017.
In 2018, the common methods for such editing used engineered nucleases, or "molecular scissors". These nucleases create site-specific double-strand breaks at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining or homologous recombination, resulting in targeted mutations.
In May 2019, lawyers in China reported, in light of the purported creation by Chinese scientist He Jiankui of the first gene-edited humans, the drafting of regulations that anyone manipulating the human genome by gene-editing techniques, like CRISPR, would be held responsible for any related adverse consequences. A cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies has been recently discussed, focusing on the stochastic nature of cellular control processes.
The University of Edinburgh Roslin Institute engineered pigs resistant to a virus that causes porcine reproductive and respiratory syndrome, which costs US and European pig farmers $2.6 billion annually.
In February 2020, a US trial safely showed CRISPR gene editing on 3 cancer patients. In 2020 Sicilian Rouge High GABA, a tomato that makes more of an amino acid said to promote relaxation, was approved for sale in Japan.
In 2021, England planned to remove restrictions on gene-edited plants and animals, moving from European Union-compliant regulation to rules closer to those of the US and some other countries. An April 2021 European Commission report found "strong indications" that the current regulatory regime was not appropriate for gene editing. Later in 2021, researchers announced a CRISPR alternative, labelled obligate mobile element–guided activity proteins including IscB, IsrB and TnpB as endonucleases found in transposons, and guided by small ωRNAs.
Background
, as method of introducing new genetic elements into organisms, has been around since the 1970s. One drawback of this technology has been the random nature with which the DNA is inserted into the host's genome, which can impair or alter other genes within the organism. However, several methods have been discovered that target the inserted genes to specific sites within an organism's genome. It has also enabled the editing of specific sequences within a genome, as well as reduced off-target effects. This could be used for research purposes, by targeting mutations to specific genes, and in gene therapy. By inserting a functional gene into an organism, and targeting it to replace the defective one, it could be possible to cure certain genetic diseases.Gene targeting
Homologous recombination
Early methods to target genes to certain sites within a genome of an organism relied on homologous recombination. By creating DNA constructs that contain a template that matches the targeted genome sequence, it is possible that the HR processes within the cell will insert the construct at the desired location. Using this method on embryonic stem cells led to the development of transgenic mice with targeted genes knocked out. It has also been possible to knock in genes or alter gene expression patterns. In recognition of their discovery of how homologous recombination can be used to introduce genetic modifications in mice through embryonic stem cells, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine.Conditional targeting
If a vital gene is knocked out, it can prove lethal to the organism. In order to study the function of these genes, site specific recombinases were used. The two most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites. The Flip-FRT system operates in a similar way, with the Flip recombinase recognising FRT sequences. By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that express the SSR under control of tissue specific promoters, it is possible to knock out or switch on genes only in certain cells. These techniques were also used to remove marker genes from transgenic animals. Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired times or stages of development.Process
Double strand break repair
A common form of genome editing relies on the concept of DNA double stranded break repair mechanics. There are two major pathways that repair DSB; non-homologous end joining and homology directed repair. NHEJ uses a variety of enzymes to directly join the DNA ends, while the more accurate HDR uses a homologous sequence as a template for regeneration of missing DNA sequences at the break point. This can be exploited by creating a vector with the desired genetic elements within a sequence that is homologous to the flanking sequences of a DSB. This will result in the desired change being inserted at the site of the DSB. While HDR based gene editing is similar to the homologous recombination based gene targeting, the rate of recombination is increased by at least three orders of magnitude.Engineered nucleases
The key to genome editing is creating a DSB at a specific point within the genome. Commonly used restriction enzymes are effective at cutting DNA, but generally recognize and cut at multiple sites. To overcome this challenge and create site-specific DSB, three distinct classes of nucleases have been discovered and bioengineered to date. These are the Zinc finger nucleases, transcription-activator like effector nucleases, meganucleases and the clustered regularly interspaced short palindromic repeats system.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. The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence.Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences thus making them naturally very specific. However, there is virtually no chance of finding the exact meganuclease required to act on a chosen specific DNA sequence. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Others have been able to fuse various meganucleases and create hybrid enzymes that recognize a new sequence. Yet others have attempted to alter the DNA interacting aminoacids of the meganuclease to design sequence specific meganucleases in a method named rationally designed meganuclease. 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.
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 These include 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.
Meganucleases have the benefit of causing less toxicity in cells than methods such as Zinc finger nuclease, likely because of more stringent DNA sequence recognition; however, the construction of sequence-specific enzymes for all possible sequences is costly and time-consuming, as one is not benefiting from combinatorial possibilities that methods such as ZFNs and TALEN-based fusions utilize.
Zinc finger nucleases
As opposed to meganucleases, the concept behind ZFNs and TALEN technology is based on a non-specific DNA cutting catalytic domain, which can then be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors. The first step to this was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not the most common among restriction enzymes. Once this enzyme was found, its cleaving portion could be separated which would be very non-specific as it would have no recognition ability. This portion could then be linked to sequence recognizing peptides that could lead to very high specificity.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 DNA binding proteins – each containing 3 to 6 specifically chosen zinc fingers – with the catalytic domain of the FokI endonuclease which need to dimerize to cleave the double-strand DNA. 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 FokI domains closer together. FokI requires dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner would recognize a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered that can only function as heterodimers.
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. 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.
The heterodimer functioning nucleases would avoid the possibility of unwanted homodimer activity and thus increase specificity of the DSB. Although the nuclease portions of both ZFNs and TALEN constructs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2-His2 zinc fingers and TALEN constructs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically happen in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins such as transcription factors. Each finger of the Zinc finger domain is completely independent and the binding capacity of one finger is impacted by its neighbor. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Zinc fingers have been more established in these terms and approaches such as modular assembly, OPEN, and bacterial one-hybrid screening of zinc finger libraries among other methods have been used to make site specific nucleases.
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 and in the fight against AIDS.