Genetically modified animal

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Genetically_modified_animal 

 

Genetically modified animals are animals that have been genetically modified for a variety of purposes including producing drugs, enhancing yields, increase resistance to disease, etc. The vast majority of genetically modified animals are at the research stage with the number close to entering the market remains small.

Production

The process of genetically engineering mammals is a slow, tedious, and expensive process. As with other genetically modified organisms (GMOs), first genetic engineers must isolate the gene they wish to insert into the host organism. This can be taken from a cell containing the gene or artificially synthesised. If the chosen gene or the donor organism's genome has been well studied it may already be accessible from a genetic library. The gene is then combined with other genetic elements, including a promoter and terminator region and usually a selectable marker.

A number of techniques are available for inserting the isolated gene into the host genome. With animals DNA is generally inserted into using microinjection, where it can be injected through the cell's nuclear envelope directly into the nucleus, or through the use of viral vectors. The first transgenic animals were produced by injecting viral DNA into embryos and then implanting the embryos in females. It is necessary to ensure that the inserted DNA is present in the embryonic stem cells. The embryo would develop and it would be hoped that some of the genetic material would be incorporated into the reproductive cells. Then researchers would have to wait until the animal reached breeding age and then offspring would be screened for presence of the gene in every cell, using PCR, Southern hybridization, and DNA sequencing.

New technologies are making genetic modifications easier and more precise. Gene targeting techniques, which creates double-stranded breaks and takes advantage on the cells natural homologous recombination repair systems, have been developed to target insertion to exact locations. Genome editing uses artificially engineered nucleases that create breaks at specific points. There are four families of engineered nucleases: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the Cas9-guideRNA system (adapted from CRISPR). TALEN and CRISPR are the two most commonly used and each has its own advantages. TALENs have greater target specificity, while CRISPR is easier to design and more efficient. The development of the CRISPR-Cas9 gene editing system has effectively halved the amount of time needed to develop genetically modified animals.

History

In 1974 Rudolf Jaenisch created the first GM animal.

Humans have domesticated animals since around 12,000 BCE, using selective breeding or artificial selection (as contrasted with natural selection). The process of selective breeding, in which organisms with desired traits (and thus with the desired genes) are used to breed the next generation and organisms lacking the trait are not bred, is a precursor to the modern concept of genetic modification Various advancements in genetics allowed humans to directly alter the DNA and therefore genes of organisms. In 1972 Paul Berg created the first recombinant DNA molecule when he combined DNA from a monkey virus with that of the lambda virus.

In 1974 Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world's first transgenic animal. However it took another eight years before transgenic mice were developed that passed the transgene to their offspring. Genetically modified mice were created in 1984 that carried cloned oncogenes, predisposing them to developing cancer. Mice with genes knocked out (knockout mouse) were created in 1989. The first transgenic livestock were produced in 1985 and the first animal to synthesise transgenic proteins in their milk were mice, engineered to produce human tissue plasminogen activator in 1987.

The first genetically modified animal to be commercialised was the GloFish, a Zebra fish with a fluorescent gene added that allows it to glow in the dark under ultraviolet light. It was released to the US market in 2003. The first genetically modified animal to be approved for food use was AquAdvantage salmon in 2015. The salmon were transformed with a growth hormone-regulating gene from a Pacific Chinook salmon and a promoter from an ocean pout enabling it to grow year-round instead of only during spring and summer.

Mammals

Some chimeras, like the blotched mouse shown, are created through genetic modification techniques like gene targeting.

 

GM mammals are created for research purposes, production of industrial or therapeutic products, agricultural uses or improving their health. There is also a market for creating genetically modified pets.

Medicine

Mammals are the best models for human disease, making genetic engineered ones vital to the discovery and development of cures and treatments for many serious diseases. Knocking out genes responsible for human genetic disorders allows researchers to study the mechanism of the disease and to test possible cures. Genetically modified mice have been the most common mammals used in biomedical research, as they are cheap and easy to manipulate. Pigs are also a good target as they have a similar body size and anatomical features, physiology, pathophysiological response and diet. Nonhuman primates are the most similar model organisms to humans, but there is less public acceptance towards using them as research animals. In 2009, scientists announced that they had successfully transferred a gene into a primate species (marmosets) and produced a stable line of breeding transgenic primates for the first time. Their first research target for these marmosets was Parkinson's disease, but they were also considering amyotrophic lateral sclerosis and Huntington's disease.

Transgenic pig for cheese production

 

Human proteins expressed in mammals are more likely to be similar to their natural counterparts than those expressed in plants or microorganisms. Stable expression has been accomplished in sheep, pigs, rats and other animals. In 2009, the first human biological drug produced from such an animal, a goat., was approved. The drug, ATryn, is an anticoagulant which reduces the probability of blood clots during surgery or childbirth was extracted from the goat's milk. Human alpha-1-antitrypsin is another protein that is used in treating humans with this deficiency. Another area is in creating pigs with greater capacity for human organ transplants (xenotransplantation). Pigs have been genetically modified so that their organs can no longer carry retroviruses or have modifications to reduce the chance of rejection. Pig lungs from genetically modified pigs are being considered for transplantation into humans. There is even potential to create chimeric pigs that can carry human organs.

Livestock

Livestock are modified with the intention of improving economically important traits such as growth-rate, quality of meat, milk composition, disease resistance and survival. Animals have been engineered to grow faster, be healthier and resist diseases. Modifications have also improved the wool production of sheep and udder health of cows.

Goats have been genetically engineered to produce milk with strong spiderweb-like silk proteins in their milk. The goat gene sequence has been modified, using fresh umbilical cords taken from kids, in order to code for the human enzyme lysozyme. Researchers wanted to alter the milk produced by the goats, to contain lysozyme in order to fight off bacteria causing diarrhea in humans.

Enviropig was a genetically enhanced line of Yorkshire pigs in Canada created with the capability of digesting plant phosphorus more efficiently than conventional Yorkshire pigs. The A transgene construct consisting of a promoter expressed in the murine parotid gland and the Escherichia coli phytase gene was introduced into the pig embryo by pronuclear microinjection. This caused the pigs to produce the enzyme phytase, which breaks down the indigestible phosphorus, in their saliva. As a result, they excrete 30 to 70% less phosphorus in manure depending upon the age and diet. The lower concentrations of phosphorus in surface runoff reduces algal growth, because phosphorus is the limiting nutrient for algae. Because algae consume large amounts of oxygen, excessive growth can result in dead zones for fish. Funding for the Enviropig program ended in April 2012, and as no new partners were found the pigs were killed. However, the genetic material will be stored at the Canadian Agricultural Genetics Repository Program. In 2006, a pig was engineered to produce omega-3 fatty acids through the expression of a roundworm gene.

Herman the Bull on display in Naturalis Biodiversity Center

In 1990, the world's first transgenic bovine, Herman the Bull, was developed. Herman was genetically engineered by micro-injected embyonic cells with the human gene coding for lactoferrin. The Dutch Parliament changed the law in 1992 to allow Herman to reproduce. Eight calves were born in 1994 and all calves inherited the lactoferrin gene. With subsequent sirings, Herman fathered a total of 83 calves. Dutch law required Herman to be slaughtered at the conclusion of the experiment. However the Dutch Agriculture Minister at the time, Jozias van Aartsen, granted him a reprieve provided he did not have more offspring after public and scientists rallied to his defence. Together with cloned cows named Holly and Belle, he lived out his retirement at Naturalis, the National Museum of Natural History in Leiden. On 2 April 2004, Herman was euthanised by veterinarians from the University of Utrecht because he suffered from osteoarthritis. At the time of his death Herman was one of the oldest bulls in the Netherlands. Herman's hide has been preserved and mounted by taxidermists and is permanently on display in Naturalis. They say that he represents the start of a new era in the way man deals with nature, an icon of scientific progress, and the subsequent public discussion of these issues.

Researchers have developed GM dairy cattle to grow without horns (sometimes referred to as "polled") which can cause injuries to farmers and other animals. DNA was taken from the genome of Red Angus cattle, which is known to suppress horn growth, and inserted into cells taken from an elite Holstein bull called "Randy". Each of the progeny will be a clone of Randy, but without his horns, and their offspring should also be hornless. In 2011, Chinese scientists generated dairy cows genetically engineered with genes from human beings to produce milk that would be the same as human breast milk. This could potentially benefit mothers who cannot produce breast milk but want their children to have breast milk rather than formula. The researchers claim these transgenic cows to be identical to regular cows. Two months later, scientists from Argentina presented Rosita, a transgenic cow incorporating two human genes, to produce milk with similar properties as human breast milk. In 2012, researchers from New Zealand also developed a genetically engineered cow that produced allergy-free milk.

Research

Scientists have genetically engineered several organisms, including some mammals, to include green fluorescent protein (GFP), for research purposes. GFP and other similar reporting genes allow easy visualisation and localisation of the products of the genetic modification. Fluorescent pigs have been bred to study human organ transplants, regenerating ocular photoreceptor cells, and other topics. In 2011 green-fluorescent cats were created to find therapies for HIV/AIDS and other diseases as feline immunodeficiency virus (FIV) is related to HIV.

Conservation

Genetic modification of the myxoma virus has been proposed to conserve European wild rabbits in the Iberian peninsula and to help regulate them in Australia. To protect the Iberian species from viral diseases, the myxoma virus was genetically modified to immunize the rabbits, while in Australia the same myxoma virus was genetically modified to lower fertility in the Australian rabbit population. There have also been suggestions that genetic engineering could be used to bring animals back from extinction. It involves changing the genome of a close living relative to resemble the extinct one and is currently being attempted with the passenger pigeon. Genes associated with the woolly mammoth have been added to the genome of an African Elephant, although the lead researcher says he has no intention of using live elephants.

Humans

Gene therapy uses genetically modified viruses to deliver genes which can cure disease in humans. Although gene therapy is still relatively new, it has had some successes. It has been used to treat genetic disorders such as severe combined immunodeficiency, and Leber's congenital amaurosis. Treatments are also being developed for a range of other currently incurable diseases, such as cystic fibrosis, sickle cell anemia, Parkinson's disease, cancer, diabetes, heart disease and muscular dystrophy. These treatments only affect somatic cells, meaning any changes would not be inheritable. Germline gene therapy results in any change being inheritable, which has raised concerns within the scientific community. In 2015, CRISPR was used to edit the DNA of non-viable human embryos. In November 2018, He Jiankui announced that he had edited the genomes of two human embryos, to attempt to disable the CCR5 gene, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier and that they carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.

Fish

Genetically modified fish are used for scientific research, as pets and as a food source. Aquaculture is a growing industry, currently providing over half the consumed fish worldwide. Through genetic engineering It is possible to increase growth rates, reduce food intake, remove allergenic properties, increase cold tolerance and provide disease resistance.

Detecting pollution

Fish can also be used to detect aquatic pollution or function as bioreactors. Several groups have been developing zebrafish to detect pollution by attaching fluorescent proteins to genes activated by the presence of pollutants. The fish will then glow and can be used as environmental sensors.

Pets

The GloFish is a brand of genetically modified fluorescent zebrafish with bright red, green, and orange fluorescent color. It was originally developed by one of the groups to detect pollution, but is now part of the ornamental fish trade, becoming the first genetically modified animal to become publicly available as a pet when it was introduced for sale in 2003.

Research

GM fish are widely used in basic research in genetics and development. Two species of fish, zebrafish and medaka, are most commonly modified because they have optically clear chorions (membranes in the egg), rapidly develop, and the 1-cell embryo is easy to see and microinject with transgenic DNA. Zebrafish are model organisms for developmental processes, regeneration, genetics, behaviour, disease mechanisms and toxicity testing. Their transparency allows researchers to observe developmental stages, intestinal functions and tumour growth. The generation of transgenic protocols (whole organism, cell or tissue specific, tagged with reporter genes) has increased the level of information gained by studying these fish.

Growth

GM fish have been developed with promoters driving an over-production of "all fish" growth hormone for use in the aquaculture industry to increase the speed of development and potentially reduce fishing pressure on wild stocks. This has resulted in dramatic growth enhancement in several species, including salmon, trout and tilapia.

AquaBounty Technologies have produced a salmon that can mature in half the time as wild salmon. The fish is an Atlantic salmon with a Chinook salmon (Oncorhynchus tshawytscha) gene inserted. This allows the fish to produce growth hormones all year round compared to the wild-type fish that produces the hormone for only part of the year. The fish also has a second gene inserted from the eel-like ocean pout that acts like an "on" switch for the hormone. Pout also have antifreeze proteins in their blood, which allow the GM salmon to survive near-freezing waters and continue their development. The wild-type salmon takes 24 to 30 months to reach market size (4–6 kg) whereas the producers of the GM salmon say it requires only 18 months for the GM fish to achieve this. In November 2015, the FDA of the USA approved the AquAdvantage salmon for commercial production, sale and consumption, the first non-plant GMO food to be commerialised.

AquaBounty say that to prevent the genetically modified fish inadvertently breeding with wild salmon, all the fish will be female and reproductively sterile, although a small percentage of the females may remain fertile. Some opponents of the GM salmon have dubbed it the "Frankenfish".

Insects

Research

In biological research, transgenic fruit flies (Drosophila melanogaster) are model organisms used to study the effects of genetic changes on development. Fruit flies are often preferred over other animals due to their short life cycle and low maintenance requirements. It also has a relatively simple genome compared to many vertebrates, with typically only one copy of each gene, making phenotypic analysis easy. Drosophila have been used to study genetics and inheritance, embryonic development, learning, behavior, and aging. Transposons (particularly P elements) are well developed in Drosophila and provided an early method to add transgenes to their genome, although this has been taken over by more modern gene-editing techniques.

Population control

Due to their significance to human health, scientists are looking at ways to control mosquitoes through genetic engineering. Malaria-resistant mosquitoes have been developed in the laboratory by inserting a gene that reduces the development of the malaria parasite and then use homing endonucleases to rapidly spread that gene throughout the male population (known as a gene drive). This has been taken further by swapping it for a lethal gene. In trials the populations of Aedes aegypti mosquitoes, the single most important carrier of dengue fever and Zika virus, were reduced by between 80% and by 90%. Another approach is to use the sterile insect technique, whereby males genetically engineered to be sterile out compete viable males, to reduce population numbers.

Other insect pests that make attractive targets are moths. Diamondback moths cause US$4 to $5 billion of damage a year worldwide. The approach is similar to the mosquitoes, where males transformed with a gene that prevents females from reaching maturity will be released. They underwent field trials in 2017. Genetically modified moths have previously been released in field trials. A strain of pink bollworm that were sterilised with radiation were genetically engineered to express a red fluorescent protein making it easier for researchers to monitor them.

Industry

Silkworm, the larvae stage of Bombyx mori, is an economically important insect in sericulture. Scientists are developing strategies to enhance silk quality and quantity. There is also potential to use the silk producing machinery to make other valuable proteins. Proteins expressed by silkworms include; human serum albumin, human collagen α-chain, mouse monoclonal antibody and N-glycanase. Silkworms have been created that produce spider silk, a stronger but extremely difficult to harvest silk, and even novel silks.

Birds

Attempts to produce genetically modified birds began before 1980. Chickens have been genetically modified for a variety of purposes. This includes studying embryo development, preventing the transmission of bird flu and providing evolutionary insights using reverse engineering to recreate dinosaur-like phenotypes. A GM chicken that produces the drug Kanuma, an enzyme that treats a rare condition, in its egg passed regulatory approval in 2015.

Disease control

One potential use of GM birds could be to reduce the spread of avian disease. Researchers at Roslin Institute have produced a strain of GM chickens (Gallus gallus domesticus) that does not transmit avian flu to other birds; however, these birds are still susceptible to contracting it. The genetic modification is an RNA molecule that prevents the virus reproduction by mimicking the region of the flu virus genome that controls replication. It is referred to as a "decoy" because it diverts the flu virus enzyme, the polymerase, from functions that are required for virus replication.

Evolutionary insights

A team of geneticists led by University of Montana paleontologist Jack Horner is seeking to modify a chicken to express several features present in ancestral maniraptorans but absent in modern birds, such as teeth and a long tail, creating what has been dubbed a 'chickenosaurus'. Parallel projects have produced chicken embryos expressing dinosaur-like skull, leg, and foot anatomy.

Amphibians

Genetically modified frogs, in particular Xenopus laevis and Xenopus tropicalis, are used in development biology. GM frogs can also be used as pollution sensors, especially for endocrine disrupting chemicals. There are proposals to use genetic engineering to control cane toads in Australia.

Nematodes

The nemotode Caenorhabditis elegans is one of the major model organisms for researching molecular biology. RNA interference (RNAi) was discovered in C elegans and could be induced by simply feeding them bacteria modified to express double stranded RNA. It is also relatively easy to produce stable transgenic nemotodes and this along with RNAi are the major tools used in studying their genes. The most common use of transgenic nematodes has been studying gene expression and localisation by attaching reporter genes. Transgenes can also be combined with RNAi to rescue phenotypes, altered to study gene function, imaged in real time as the cells develop or used to control expression for different tissues or developmental stages. Transgenic nematodes have been used to study viruses, toxicology, and diseases and to detect environmental pollutants.

Other

Systems have been developed to create transgenic organisms in a wide variety of other animals. The gene responsible for Albinism in sea cucumbers has been found and used to engineer white sea cucumbers, a rare delicacy. The technology also opens the way to investigate the genes responsible for some of the cucumbers more unusual traits, including hibernating in summer, eviscerating their intestines, and dissolving their bodies upon death. Flatworms have the ability to regenerate themselves from a single cell. Until 2017 there was no effective way to transform them, which hampered research. By using microinjection and radiation scientist have now created the first genetically modified flatworms. The bristle worm, a marine annelid, has been modified. It is of interest due to its reproductive cycle being synchronised with lunar phases, regeneration capacity and slow evolution rate. Cnidaria such as Hydra and the sea anemone Nematostella vectensis are attractive model organisms to study the evolution of immunity and certain developmental processes. Other organisms that have been genetically modified include snails, geckos, turtles, crayfish, oysters, shrimp, clams, abalone and sponges.

Genome editing

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Genome_editing 

 

The different generations of nucleases used for genome editing

and the DNA repair pathways used to modify target DNA.

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 inserts genetic material into a host genome, genome editing targets the insertions to site specific locations.

History

Genome editing with engineered nucleases, i.e. all three major classes of these enzymes—zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) 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.

As of 2015 four families of engineered nucleases were used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) 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 (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('edits').

In May 2019, lawyers in China reported, in light of the purported creation by Chinese scientist He Jiankui of the first gene-edited humans (see Lulu and Nana controversy), 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. 

In February 2020, a US trial safely showed CRISPR gene editing on 3 cancer patients.

Background

Genetic engineering 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 hosts genome. This can impair or alter other genes within the organism. Methods were sought which targeted the inserted genes to specific sites within an organism genome. As well as reducing off-target effects it also enabled the editing of specific sequences within a genome. 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 (called gene targeting) relied on homologous recombination (HR). 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 (SSR) 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

Genome editing relies on the concept of DNA double stranded break (DSB) repair mechanics. There are two major pathways that repair DSB; non-homologous end joining (NHEJ) and homology directed repair (HDR). 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

Groups of engineered nucleases. Matching colors signify DNA recognition patterns

 

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 (ZFNs), transcription-activator like effector nucleases (TALEN), meganucleases and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.

Meganucleases

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 14 to 40 base pairs). 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 (>14bp) 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 meganucelases 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 (fundamental research, health, agriculture, industry, energy, etc.) 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 (ZFN), 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 (TALEs). 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 (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.

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 (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), 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 (glioblastoma) and in the fight against AIDS.

TALEN

General overview of the TALEN process

Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins that feature an array of 33 or 34-amino acid repeats. TALENs are artificial restriction enzymes designed by fusing the DNA cutting domain of a nuclease to TALE domains, which can be tailored to specifically recognize a unique DNA sequence. These fusion proteins serve as readily targetable "DNA scissors" for gene editing applications that enable to perform targeted genome modifications such as sequence insertion, deletion, repair and replacement in living cells. The DNA binding domains, which can be designed to bind any desired DNA sequence, comes from TAL effectors, DNA-binding proteins excreted by plant pathogenic Xanthomanos app. TAL effectors consists of repeated domains, each of which contains a highly conserved sequence of 34 amino acids, and recognize a single DNA nucleotide within the target site. The nuclease can create double strand breaks at the target site that can be repaired by error-prone non-homologous end-joining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions. Each repeat is conserved, with the exception of the so-called repeat variable di-residues (RVDs) at amino acid positions 12 and 13. The RVDs determine the DNA sequence to which the TALE will bind. This simple one-to-one correspondence between the TALE repeats and the corresponding DNA sequence makes the process of assembling repeat arrays to recognize novel DNA sequences straightforward. These TALEs can be fused to the catalytic domain from a DNA nuclease, FokI, to generate a transcription activator-like effector nuclease (TALEN). The resultant TALEN constructs combine specificity and activity, effectively generating engineered sequence-specific nucleases that bind and cleave DNA sequences only at pre-selected sites. The TALEN target recognition system is based on an easy-to-predict code. TAL nucleases are specific to their target due in part to the length of their 30+ base pairs binding site. TALEN can be performed within a 6 base pairs range of any single nucleotide in the entire genome.

TALEN constructs are used in a similar way to designed zinc finger nucleases, and have three advantages in targeted mutagenesis: (1) DNA binding specificity is higher, (2) off-target effects are lower, and (3) construction of DNA-binding domains is easier.

CRISPR

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as a kind of acquired immunity to protect against viruses. They consist of short sequences that originate from viral genomes and have been incorporated into the bacterial genome. Cas (CRISPR associated proteins) process these sequences and cut matching viral DNA sequences. By introducing plasmids containing Cas genes and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired position. Several companies, including Cellectis and Editas, have been working to monetize the CRISPR method while developing gene-specific therapies.

Precision and efficiency of engineered nucleases

Meganucleases method of gene editing is the least efficient of the methods mentioned above. Due to the nature of its DNA-binding element and the cleaving element, it is limited to recognizing one potential target every 1,000 nucleotides. ZFN was developed to overcome the limitations of meganuclease. The number of possible targets ZFN can recognized was increased to one in every 140 nucleotides. However, both methods are unpredictable due to the ability of their DNA-binding elements affecting each other. As a result, high degrees of expertise and lengthy and costly validations processes are required.

TALE nucleases being the most precise and specific method yields a higher efficiency than the previous two methods. It achieves such efficiency because the DNA-binding element consists of an array of TALE subunits, each of them having the capability of recognizing a specific DNA nucleotide chain independent from others, resulting in a higher number of target sites with high precision. New TALE nucleases take about one week and a few hundred dollars to create, with specific expertise in molecular biology and protein engineering.

CRISPR nucleases have a slightly lower precision when compared to the TALE nucleases. This is caused by the need of having a specific nucleotide at one end in order to produce the guide RNA that CRISPR uses to repair the double-strand break it induces. It has been shown to be the quickest and cheapest method, only costing less than two hundred dollars and a few days of time. CRISPR also requires the least amount of expertise in molecular biology as the design lays in the guide RNA instead of the proteins. One major advantage that CRISPR has over the ZFN and TALEN methods is that it can be directed to target different DNA sequences using its ~80nt CRISPR sgRNAs, while both ZFN and TALEN methods required construction and testing of the proteins created for targeting each DNA sequence.

Because off-target activity of an active nuclease would have potentially dangerous consequences at the genetic and organismal levels, the precision of meganucleases, ZFNs, CRISPR, and TALEN-based fusions has been an active area of research. While variable figures have been reported, ZFNs tend to have more cytotoxicity than TALEN methods or RNA-guided nucleases, while TALEN and RNA-guided approaches tend to have the greatest efficiency and fewer off-target effects. Based on the maximum theoretical distance between DNA binding and nuclease activity, TALEN approaches result in the greatest precision.

Multiplex Automated Genomic Engineering (MAGE)

Synthetic DNA is repeatedly introduced at multiple targeted areas of the chromosome and/or loci and then is replicated producing cells with/without mutations.

 

The methods for scientists and researchers wanting to study genomic diversity and all possible associated phenotypes were very slow, expensive, and inefficient. Prior to this new revolution, researchers would have to do single-gene manipulations and tweak the genome one little section at a time, observe the phenotype, and start the process over with a different single-gene manipulation. Therefore, researchers at the Wyss Institute at Harvard University designed the MAGE, a powerful technology that improves the process of in vivo genome editing. It allows for quick and efficient manipulations of a genome, all happening in a machine small enough to put on top of a small kitchen table. Those mutations combine with the variation that naturally occurs during cell mitosis creating billions of cellular mutations.

Chemically combined, synthetic single-stranded DNA (ssDNA) and a pool of oligionucleotides are introduced at targeted areas of the cell thereby creating genetic modifications. The cyclical process involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. Experiments targeting selective phenotypic markers are screened and identified by plating the cells on differential medias. Each cycle ultimately takes 2.5 hours to process, with additional time required to grow isogenic cultures and characterize mutations. By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population. There can be up to 50 genome edits, from single nucleotide base pairs to whole genome or gene networks simultaneously with results in a matter of days.

MAGE experiments can be divided into three classes, characterized by varying degrees of scale and complexity: (i) many target sites, single genetic mutations; (ii) single target site, many genetic mutations; and (iii) many target sites, many genetic mutations. An example of class three was reflected in 2009, where Church and colleagues were able to program Escherichia coli to produce five times the normal amount of lycopene, an antioxidant normally found in tomato seeds and linked to anti-cancer properties. They applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) metabolic pathway in Escherichia coli to overproduce isoprenoid lycopene. It took them about 3 days and just over $1,000 in materials. The ease, speed, and cost efficiency in which MAGE can alter genomes can transform how industries approach the manufacturing and production of important compounds in the bioengineering, bioenergy, biomedical engineering, synthetic biology, pharmaceutical, agricultural, and chemical industries.

Applications

Plants, animals and human genes that are successfully targeted using ZFN, which demonstrates the generality of this approach

As of 2012 efficient genome editing had been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest, and was becoming a standard experimental strategy in research labs. The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants and the improvements in TALEN-based approaches testify to the significance of the methods, and the list is expanding rapidly. Genome editing with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans. For instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease to add or remove genomic elements and therefore create complex systems. In addition, gene functions can be studied using stem cells with engineered nucleases.

Listed below are some specific tasks this method can carry out:

• Targeted gene mutation
• Gene therapy
• Creating chromosome rearrangement
• Study gene function with stem cells
• Transgenic animals
• Endogenous gene labeling
• Targeted transgene addition

Targeted gene modification in animals

The combination of recent discoveries in genetic engineering, particularly gene editing and the latest improvement in bovine reproduction technologies (e.g. in vitro embryo culture) allows for genome editing directly in fertilised oocytes using synthetic highly specific endonucleases. RNA-guided endonucleases:clustered regularly interspaced short palindromic repeats associated Cas9 (CRISPR/Cas9) are a new tool, further increasing the range of methods available. In particular CRISPR/Cas9 engineered endonucleases allows the use of multiple guide RNAs for simultaneous Knockouts (KO) in one step by cytoplasmic direct injection (CDI) on mammalian zygotes.

Thanks to the parallel development of single-cell transcriptomics, genome editing and new stem cell models we are now entering a scientifically exciting period where functional genetics is no longer restricted to animal models but can be performed directly in human samples. Single-cell gene expression analysis has resolved a transcriptional road-map of human development from which key candidate genes are being identified for functional studies. Using global transcriptomics data to guide experimentation, the CRISPR based genome editing tool has made it feasible to disrupt or remove key genes in order to elucidate function in a human setting.

Targeted gene modification in plants

Overview of GEEN workflow and editing possibilities

Genome editing using Meganuclease, ZFNs, and TALEN provides a new strategy for genetic manipulation in plants and are likely to assist in the engineering of desired plant traits by modifying endogenous genes. For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes. Progress in such cases have been recently reported in Arabidopsis thaliana and Zea mays. In Arabidopsis thaliana, using ZFN-assisted gene targeting, two herbicide-resistant genes (tobacco acetolactate synthase SuRA and SuRB) were introduced to SuR loci with as high as 2% transformed cells with mutations. In Zea mays, disruption of the target locus was achieved by ZFN-induced DSBs and the resulting NHEJ. ZFN was also used to drive herbicide-tolerance gene expression cassette (PAT) into the targeted endogenous locus IPK1 in this case. Such genome modification observed in the regenerated plants has been shown to be inheritable and was transmitted to the next generation. A potentially successful example of the application of genome editing techniques in crop improvement can be found in banana, where scientists used CRISPR/Cas9 editing to inactivate the endogenous banana streak virus in the B genome of banana (Musa spp.) to overcome a major challenge in banana breeding.

In addition, TALEN-based genome engineering has been extensively tested and optimized for use in plants. TALEN fusions have also been used by a U.S. food ingredient company, Calyxt, to improve the quality of soybean oil products and to increase the storage potential of potatoes.

Several optimizations need to be made in order to improve editing plant genomes using ZFN-mediated targeting. There is a need for reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting, the routes of induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases.

A common delivery method for CRISPR/Cas9 in plants is Agrobacterium-based transformation. T-DNA is introduced directly into the plant genome by a T4SS mechanism. Cas9 and gRNA-based expression cassettes are turned into Ti plasmids, which are transformed in Agrobacterium for plant application. To improve Cas9 delivery in live plants, viruses are being used more effective transgene delivery.

Research

Gene therapy

The ideal gene therapy practice is that which replaces the defective gene with a normal allele at its natural location. This is advantageous over a virally delivered gene as there is no need to include the full coding sequences and regulatory sequences when only a small proportions of the gene needs to be altered as is often the case. The expression of the partially replaced genes is also more consistent with normal cell biology than full genes that are carried by viral vectors.

The first clinical use of TALEN-based genome editing was in the treatment of CD19+ acute lymphoblastic leukemia in an 11-month old child in 2015. Modified donor T cells were engineered to attack the leukemia cells, to be resistant to Alemtuzumab, and to evade detection by the host immune system after introduction.

Extensive research has been done in cells and animals using CRISPR-Cas9 to attempt to correct genetic mutations which cause genetic diseases such as Down syndrome, spina bifida, anencephaly, and Turner and Klinefelter syndromes.

In February 2019, medical scientists working with Sangamo Therapeutics, headquartered in Richmond, California, announced the first ever "in body" human gene editing therapy to permanently alter DNA - in a patient with Hunter Syndrome. Clinical trials by Sangamo involving gene editing using Zinc Finger Nuclease (ZFN) are ongoing.

Eradicating diseases

Researchers have used CRISPR-Cas9 gene drives to modify genes associated with sterility in A. gambiae, the vector for malaria. This technique has further implications in eradicating other vector borne diseases such as yellow fever, dengue, and Zika.

The CRISPR-Cas9 system can be programmed to modulate the population of any bacterial species by targeting clinical genotypes or epidemiological isolates. It can selectively enable the beneficial bacterial species over the harmful ones by eliminating pathogen, which gives it an advantage over broad-spectrum antibiotics.

Antiviral applications for therapies targeting human viruses such as HIV, herpes, and hepatitis B virus are under research. CRISPR can be used to target the virus or the host to disrupt genes encoding the virus cell-surface receptor proteins. In November 2018, He Jiankui announced that he had edited two human embryos, to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.

In January 2019, scientists in China reported the creation of five identical cloned gene-edited monkeys, using the same cloning technique that was used with Zhong Zhong and Hua Hua – the first ever cloned monkeys - and Dolly the sheep, and the same gene-editing Crispr-Cas9 technique allegedly used by He Jiankui in creating the first ever gene-modified human babies Lulu and Nana. The monkey clones were made in order to study several medical diseases.

In the near future the new CRISPR system will also be able to eradicate diseases and conditions that humans are predisposed for. With this new technology scientists will be able to take the genes of a human sperm cell and egg, and replace the genes that activate cancer or other abnormal or unwanted defects. This will take the stress off of parents worrying about having a child and them not being able to live it like a normal child should. After just one generation of this process, the entire future of the human race would never have to worry about the problems of deformities or predisposed conditions.

Prospects and limitations

In the future, an important goal of research into genome editing with engineered nucleases must be the improvement of the safety and specificity of the nucleases. For example, improving the ability to detect off-target events can improve our ability to learn about ways of preventing them. In addition, zinc-fingers used in ZFNs are seldom completely specific, and some may cause a toxic reaction. However, the toxicity has been reported to be reduced by modifications done on the cleavage domain of the ZFN.

In addition, research by Dana Carroll into modifying the genome with engineered nucleases has shown the need for better understanding of the basic recombination and repair machinery of DNA. In the future, a possible method to identify secondary targets would be to capture broken ends from cells expressing the ZFNs and to sequence the flanking DNA using high-throughput sequencing.

Because of the ease of use and cost-efficiency of CRISPR, extensive research is currently being done on it. There are now more publications on CRISPR than ZFN and TALEN despite how recent the discovery of CRISPR is. Both CRISPR and TALEN are favored to be the choices to be implemented in large-scale productions due to their precision and efficiency.

Genome editing occurs also as a natural process without artificial genetic engineering. The agents that are competent to edit genetic codes are viruses or subviral RNA-agents.

Although GEEN has higher efficiency than many other methods in reverse genetics, it is still not highly efficient; in many cases less than half of the treated populations obtain the desired changes. For example, when one is planning to use the cell's NHEJ to create a mutation, the cell's HDR systems will also be at work correcting the DSB with lower mutational rates.

Traditionally, mice have been the most common choice for researchers as a host of a disease model. CRISPR can help bridge the gap between this model and human clinical trials by creating transgenic disease models in larger animals such as pigs, dogs, and non-human primates. Using the CRISPR-Cas9 system, the programmed Cas9 protein and the sgRNA can be directly introduced into fertilized zygotes to achieve the desired gene modifications when creating transgenic models in rodents. This allows bypassing of the usual cell targeting stage in generating transgenic lines, and as a result, it reduces generation time by 90%.

One potential that CRISPR brings with its effectiveness is the application of xenotransplantation. In previous research trials, CRISPR demonstrated the ability to target and eliminate endogenous retroviruses, which reduces the risk of transmitting diseases and reduces immune barriers. Eliminating these problems improves donor organ function, which brings this application closer to a reality. 

In plants, genome editing is seen as a viable solution to the conservation of biodiversity. Gene drive are a potential tool to alter the reproductive rate of invasive species, although there are significant associated risks.

Human enhancement

Many transhumanists see genome editing as a potential tool for human enhancement. Australian biologist and Professor of Genetics David Andrew Sinclair notes that "the new technologies with genome editing will allow it to be used on individuals (...) to have (...) healthier children" – designer babies. According to a September 2016 report by the Nuffield Council on Bioethics in the future it may be possible to enhance people with genes from other organisms or wholly synthetic genes to for example improve night vision and sense of smell.

The American National Academy of Sciences and National Academy of Medicine issued a report in February 2017 giving qualified support to human genome editing. They recommended that clinical trials for genome editing might one day be permitted once answers have been found to safety and efficiency problems "but only for serious conditions under stringent oversight."

Risks

In the 2016 Worldwide Threat Assessment of the US Intelligence Community statement United States Director of National Intelligence, James R. Clapper, named genome editing as a potential weapon of mass destruction, stating that genome editing conducted by countries with regulatory or ethical standards "different from Western countries" probably increases the risk of the creation of harmful biological agents or products. According to the statement the broad distribution, low cost, and accelerated pace of development of this technology, its deliberate or unintentional misuse might lead to far-reaching economic and national security implications. For instance technologies such as CRISPR could be used to make "killer mosquitoes" that cause plagues that wipe out staple crops.

According to a September 2016 report by the Nuffield Council on Bioethics, the simplicity and low cost of tools to edit the genetic code will allow amateurs – or "biohackers" – to perform their own experiments, posing a potential risk from the release of genetically modified bugs. The review also found that the risks and benefits of modifying a person's genome – and having those changes pass on to future generations – are so complex that they demand urgent ethical scrutiny. Such modifications might have unintended consequences which could harm not only the child, but also their future children, as the altered gene would be in their sperm or eggs. In 2001 Australian researchers Ronald Jackson and Ian Ramshaw were criticized for publishing a paper in the Journal of Virology that explored the potential control of mice, a major pest in Australia, by infecting them with an altered mousepox virus that would cause infertility as the provided sensitive information could lead to the manufacture of biological weapons by potential bioterrorists who might use the knowledge to create vaccine resistant strains of other pox viruses, such as smallpox, that could affect humans. Furthermore, there are additional concerns about the ecological risks of releasing gene drives into wild populations.