From Wikipedia, the free encyclopedia https://en.wikipedia.org/wiki/Genetic_engineering_techniques Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene
they wish to insert, modify, or delete. The gene must then be isolated
and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.
The ability to genetically engineer organisms is built on years
of research and discovery on gene function and manipulation. Important
advances included the discovery of restriction enzymes, DNA ligases, and the development of polymerase chain reaction and sequencing.
Added genes are often accompanied by promoter and terminator regions as well as a selectable marker gene. The added gene may itself be modified to make it express
more efficiently. This vector is then inserted into the host organism's
genome. For animals, the gene is typically inserted into embryonic stem cells, while in plants it can be inserted into any tissue that can be cultured into a fully developed plant.
Tests are carried out on the modified organism to ensure stable integration, inheritance and expression. First generation offspring are heterozygous,
requiring them to be inbred to create the homozygous pattern necessary
for stable inheritance. Homozygosity must be confirmed in second
generation specimens.
Many different discoveries and advancements led to the development of genetic engineering. Human-directed genetic manipulation began with the domestication of plants and animals through artificial selection in about 12,000 BC. Various techniques were developed to aid in breeding and selection. Hybridization
was one way rapid changes in an organism's genetic makeup could be
introduced. Crop hybridization most likely first occurred when humans
began growing genetically distinct individuals of related species in
close proximity.Some plants were able to be propagated by vegetative cloning.
After discovering the existence and properties of DNA, tools had to be developed that allowed it to be manipulated. In 1970 Hamilton Smiths lab discovered restriction enzymes, enabling scientists to isolate genes from an organism's genome. DNA ligases, which join broken DNA together, were discovered earlier in 1967. By combining the two enzymes it became possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952, became important tools for transferring information between cells and replicating DNA sequences. Polymerase chain reaction (PCR), developed by Kary Mullis in 1983, allowed small sections of DNA to be amplified (replicated) and aided identification and isolation of genetic material.
As well as manipulating DNA, techniques had to be developed for
its insertion into an organism's genome. Griffith's experiment had
already shown that some bacteria had the ability to naturally uptake and express foreign DNA. Artificial competence was induced in Escherichia coli in 1970 by treating them with calcium chloride solution (CaCl2). Transformation using electroporation was developed in the late 1980s, increasing the efficiency and bacterial range In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, had been discovered. In the early 1970s it was found that this bacteria inserted its DNA into plants using a Ti plasmid.
By removing the genes in the plasmid that caused the tumor and adding
in novel genes, researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants.
Choosing target genes
The
first step is to identify the target gene or genes to insert into the
host organism. This is driven by the goal for the resultant organism. In
some cases only one or two genes are affected. For more complex
objectives entire biosynthetic pathways
involving multiple genes may be involved. Once found genes and other
genetic information from a wide range of organisms can be inserted into
bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal,
multiply quickly, relatively easy to transform and can be stored at
-80 °C almost indefinitely. Once a gene is isolated it can be stored
inside the bacteria providing an unlimited supply for research.
Genetic screens
can be carried out to determine potential genes followed by other tests
that identify the best candidates. A simple screen involves randomly mutating
DNA with chemicals or radiation and then selecting those that display
the desired trait. For organisms where mutation is not practical,
scientists instead look for individuals among the population who present
the characteristic through naturally-occurring mutations. Processes
that look at a phenotype and then try and identify the gene responsible are called forward genetics. The gene then needs to be mapped by comparing the inheritance of the phenotype with known genetic markers. Genes that are close together are likely to be inherited together.
Another option is reverse genetics. This approach involves targeting a specific gene with a mutation and then observing what phenotype develops. The mutation can be designed to inactivate the gene or only allow it to become active under certain conditions. Conditional mutations are useful for identifying genes that are normally lethal if non-functional. As genes with similar functions share similar sequences (homologous) it is possible to predict the likely function of a gene by comparing its sequence to that of well-studied genes from model organisms. The development of microarrays, transcriptomes and genome sequencing has made it much easier to find desirable genes.
The bacteria Bacillus thuringiensis was first discovered in 1901 as the causative agent in the death of silkworms. Due to these insecticidal properties, the bacteria was used as a biological insecticide, developed commercially in 1938. The cry proteins
were discovered to provide the insecticidal activity in 1956, and by
the 1980s, scientists had successfully cloned the gene that encodes this
protein and expressed it in plants. The gene that provides resistance to the herbicide glyphosate was found after seven years of searching in bacteria living in the outflow pipe of a MonsantoRoundUp manufacturing facility. In animals, the majority of genes used are growth hormone genes.
Gene manipulation
All
genetic engineering processes involve the modification of DNA.
Traditionally DNA was isolated from the cells of organisms. Later, genes
came to be cloned from a DNA segment after the creation of a DNA library or artificially synthesised.
Once isolated, additional genetic elements are added to the gene to
allow it to be expressed in the host organism and to aid selection.
First the cell must be gently opened,
exposing the DNA without causing too much damage to it. The methods
used vary depending on the type of cell. Once it is open, the DNA must
be separated from the other cellular components. A ruptured cell
contains proteins and other cell debris. By mixing with phenol and/or chloroform, followed by centrifuging, the nucleic acids can be separated from this debris into an upper aqueous phase.
This aqueous phase can be removed and further purified if necessary by
repeating the phenol-chloroform steps. The nucleic acids can then be precipitated from the aqueous solution using ethanol or isopropanol. Any RNA can be removed by adding a ribonuclease that will degrade it. Many companies now sell kits that simplify the process.
Gene isolation
The
gene researchers are looking to modify (known as the gene of interest)
must be separated from the extracted DNA. If the sequence is not known
then a common method is to break the DNA up with a random digestion
method. This is usually accomplished using restriction enzymes (enzymes that cut DNA). A partial restriction digest
cuts only some of the restriction sites, resulting in overlapping DNA
fragment segments. The DNA fragments are put into individual plasmid vectors and grown inside bacteria. Once in the bacteria the plasmid is copied as the bacteria divides. To determine if a useful gene is present in a particular fragment, the DNA library is screened for the desired phenotype. If the phenotype is detected then it is possible that the bacteria contains the target gene.
If the gene does not have a detectable phenotype or a DNA library
does not contain the correct gene, other methods must be used to
isolate it. If the position of the gene can be determined using molecular markers then chromosome walking is one way to isolate the correct DNA fragment. If the gene expresses close homology
to a known gene in another species, then it could be isolated by
searching for genes in the library that closely match the known gene.
For known DNA sequences, restriction enzymes that cut the DNA on either side of the gene can be used. Gel electrophoresis then sorts the fragments according to length. Some gels can separate sequences that differ by a single base-pair. The DNA can be visualised by staining it with ethidium bromide and photographing under UV light. A marker
with fragments of known lengths can be laid alongside the DNA to
estimate the size of each band. The DNA band at the correct size should
contain the gene, where it can be excised from the gel.Another technique to isolate genes of known sequences involves polymerase chain reaction (PCR).
PCR is a powerful tool that can amplify a given sequence, which can
then be isolated through gel electrophoresis. Its effectiveness drops
with larger genes and it has the potential to introduce errors into the
sequence.
It is possible to artificially synthesise genes. Some synthetic sequences are available commercially, forgoing many of these early steps.
Modification
The
gene to be inserted must be combined with other genetic elements in
order for it to work properly. The gene can be modified at this stage
for better expression or effectiveness. As well as the gene to be
inserted most constructs contain a promoter and terminator region as well as a selectable marker gene. The promoter region initiates transcription
of the gene and can be used to control the location and level of gene
expression, while the terminator region ends transcription. A selectable
marker, which in most cases confers antibiotic resistance
to the organism it is expressed in, is used to determine which cells
are transformed with the new gene. The constructs are made using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning.
Once the gene is constructed it must be stably integrated into the genome of the target organism or exist as extrachromosomal DNA.
There are a number of techniques available for inserting the gene into
the host genome and they vary depending on the type of organism
targeted. In multicellular eukaryotes, if the transgene is incorporated into the host's germline cells, the resulting host cell can pass the transgene to its progeny. If the transgene is incorporated into somatic cells, the transgene can not be inherited.
Bacterial
transformation involves moving a gene from one bacteria to another. It
is integrated into the recipients plasmid. and can then be expressed by
the new host.
Transformation is the direct alteration of a cell's genetic components by passing the genetic material through the cell membrane. About 1% of bacteria are naturally able to take up foreign DNA, but this ability can be induced in other bacteria. Stressing the bacteria with a heat shock or electroporation can make the cell membrane
permeable to DNA that may then be incorporated into the genome or exist
as extrachromosomal DNA. Typically the cells are incubated in a
solution containing divalentcations (often calcium chloride)
under cold conditions, before being exposed to a heat pulse (heat
shock). Calcium chloride partially disrupts the cell membrane, which
allows the recombinant DNA to enter the host cell. It is suggested that
exposing the cells to divalent cations in cold condition may change or
weaken the cell surface structure, making it more permeable to DNA. The
heat-pulse is thought to create a thermal imbalance across the cell
membrane, which forces the DNA to enter the cells through either cell
pores or the damaged cell wall. Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm,
which is thought to create holes in the cell membrane through which the
plasmid DNA may enter. After the electric shock, the holes are rapidly
closed by the cell's membrane-repair mechanisms. Up-taken DNA can
either integrate with the bacterials genome or, more commonly, exist as extrachromosomal DNA.
A gene gun uses biolistics to insert DNA into plant tissue.A. tumefaciens attaching itself to a carrot cell
In plants the DNA is often inserted using Agrobacterium-mediated recombination, taking advantage of the Agrobacteriums T-DNA sequence that allows natural insertion of genetic material into plant cells. Plant tissue are cut into small pieces and soaked in a fluid containing suspended Agrobacterium. The bacteria will attach to many of the plant cells exposed by the cuts. The bacteria uses conjugation to transfer a DNA segment called T-DNA
from its plasmid into the plant. The transferred DNA is piloted to the
plant cell nucleus and integrated into the host plants genomic DNA.The
plasmid T-DNA is integrated semi-randomly into the genome of the host cell.
By modifying the plasmid to express the gene of interest,
researchers can insert their chosen gene stably into the plants genome.
The only essential parts of the T-DNA are its two small (25 base pair)
border repeats, at least one of which is needed for plant
transformation. The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the plasmid. An alternative method is agroinfiltration.
Another method used to transform plant cells is biolistics, where particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material enters the cells and transforms them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids.
Plants cells can also be transformed using electroporation, which uses
an electric shock to make the cell membrane permeable to plasmid DNA.
Due to the damage caused to the cells and DNA the transformation
efficiency of biolistics and electroporation is lower than agrobacterial
transformation.
Transformation has a different meaning
in relation to animals, indicating progression to a cancerous state, so
the process used to insert foreign DNA into animal cells is usually
called transfection. There are many ways to directly introduce DNA into animal cells in vitro. Often these cells are stem cells that are used for gene therapy. Chemical based methods uses natural or synthetic compounds to form particles that facilitate the transfer of genes into cells. These synthetic vectors have the ability to bind DNA and accommodate large genetic transfers. One of the simplest methods involves using calcium phosphate to bind the DNA and then exposing it to cultured cells. The solution, along with the DNA, is encapsulated by the cells. Liposomes and polymers
can be used as vectors to deliver DNA into cultured animal cells.
Positively charged liposomes bind with DNA, while polymers can designed
that interact with DNA.
They form lipoplexes and polyplexes respectively, which are then
up-taken by the cells. Other techniques include using electroporation
and biolistics. In some cases, transfected cells may stably integrate external DNA into their own genome, this process is known as stable transfection.
To create transgenic animals the DNA must be inserted into viable embryos or eggs. This is usually accomplished using microinjection, where DNA is injected through the cell's nuclear envelope directly into the nucleus. Superovulated fertilised eggs are collected at the single cell stage and cultured in vitro. When the pronuclei from the sperm head and egg are visible through the protoplasm the genetic material is injected into one of them. The oocyte is then implanted in the oviduct of a pseudopregnant animal. Another method is Embryonic Stem Cell-Mediated Gene Transfer. The gene is transfected into embryonic stem cells and then they are inserted into mouse blastocysts that are then implanted into foster mothers. The resulting offspring are chimeric, and further mating can produce mice fully transgenic with the gene of interest.
Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. Genetically modified viruses can be used as viral vectors to transfer target genes to another organism in gene therapy.
First the virulent genes are removed from the virus and the target
genes are inserted instead. The sequences that allow the virus to insert
the genes into the host organism must be left intact. Popular virus
vectors are developed from retroviruses or adenoviruses. Other viruses used as vectors include, lentiviruses, pox viruses and herpes viruses. The type of virus used will depend on the cells targeted and whether the DNA is to be altered permanently or temporarily.
Regeneration
As often only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through the use of tissue culture.[45][46]
Each plant species has different requirements for successful
regeneration. If successful, the technique produces an adult plant that
contains the transgene in every cell. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Offspring can be screened for the gene. All offspring from the first generation are heterozygous for the inserted gene and must be inbred to produce a homozygous specimen. Bacteria consist of a single cell and reproduce clonally so regeneration is not necessary. Selectable markers are used to easily differentiate transformed from untransformed cells.
Cells that have been successfully transformed with the DNA
contain the marker gene, while those not transformed will not. By
growing the cells in the presence of an antibiotic or chemical that selects
or marks the cells expressing that gene, it is possible to separate
modified from unmodified cells. Another screening method involves a DNA probe
that sticks only to the inserted gene. These markers are usually
present in the transgenic organism, although a number of strategies have
been developed that can remove the selectable marker from the mature
transgenic plant.
Confirmation
Finding
that a recombinant organism contains the inserted genes is not usually
sufficient to ensure that they will be appropriately expressed in the
intended tissues. Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene.
These tests can also confirm the chromosomal location and copy number
of the inserted gene. Once confirmed methods that look for and measure
the gene products (RNA and protein) are also used to assess gene
expression, transcription, RNA processing patterns and expression and
localization of protein product(s). These include northern hybridisation, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis. When appropriate, the organism's offspring are studied to confirm that the transgene and associated phenotype are stably inherited.
Traditional methods of genetic engineering generally insert the new
genetic material randomly within the host genome. This can impair or
alter other genes within the organism. Methods were developed that
inserted the new genetic material into specific sites within an organism genome. Early methods that targeted genes at certain sites within a genome 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 knocked out. It has also been possible to knock in genes or alter gene expression patterns.
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 recognizing FRT sequences. By
crossing an organism containing the recombinase sites flanking the gene
of interest with an organism that expresses the SSR under control of tissue specific promoters,
it is possible to knock out or switch on genes only in certain cells.
This has also been 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.
Genome editing uses artificially engineered nucleases that create specific double-stranded breaks at desired locations in the genome. The breaks are subject to cellular DNA repair
processes that can be exploited for targeted gene knock-out, correction
or insertion at high frequencies. If a donor DNA containing the
appropriate sequence (homologies) is present, then new genetic material
containing the transgene will be integrated at the targeted site with
high efficiency by homologous recombination. There are four families of engineered nucleases: meganucleases, ZFNs, transcription activator-like effector nucleases (TALEN), the CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPRassociated protein (e.g. CRISPR/Cas9). Among the four types, TALEN and CRISPR/Cas are the two most commonly used.
Recent advances have looked at combining multiple systems to exploit
the best features of both (e.g. megaTAL that are a fusion of a TALE DNA
binding domain and a meganuclease).
Recent research has also focused on developing strategies to create
gene knock-out or corrections without creating double stranded breaks
(base editors).
Meganucleases and Zinc finger nucleases
Meganucleases were first used in 1988 in mammalian cells. Meganucleases are endodeoxyribonucleases that function as restriction enzymes with long recognition sites, making them more specific to their target site than other restriction enzymes.
This increases their specificity and reduces their toxicity as they
will not target as many sites within a genome. The most studied
meganucleases are the LAGLIDADG family.
While meganucleases are still quite susceptible to off-target binding,
which makes them less attractive than other gene editing tools, their
smaller size still makes them attractive particularly for viral
vectorization perspectives.
Zinc-finger nucleases (ZFNs), used for the first time in 1996,
are typically created through the fusion of Zinc-finger domains and the FokI nuclease domain. ZFNs have thus the ability to cleave DNA at target sites.
By engineering the zinc finger domain to target a specific site within
the genome, it is possible to edit the genomic sequence at the desired location. ZFNs have a greater specificity, but still hold the potential to bind
to non-specific sequences.. While a certain amount of off-target
cleavage is acceptable for creating transgenic model organisms, they
might not be optimal for all human gene therapy treatments.
TALEN and CRISPR
Access
to the code governing the DNA recognition by transcription
activator-like effectors (TALE) in 2009 opened the way to the
development of a new class of efficient TAL-based gene editing tools.
TALE, proteins secreted by the Xanthomonas plant pathogen, bind with
great specificity to genes within the plant host and initiate transcription of the genes helping infection. Engineering TALE by fusing the DNA binding core to the FokI nuclease catalytic domain allowed creation of a new tool of designer nucleases, the TALE nuclease (TALEN).
They have one of the greatest specificities of all the current
engineered nucleases. Due to the presence of repeat sequences, they are
difficult to construct through standard molecular biology procedure and
rely on more complicated method of such as Golden gate cloning.
In 2011, another major breakthrough technology was developed
based on CRISPR/Cas (clustered regularly interspaced short palindromic
repeat / CRISPR associated protein) systems that function as an adaptive
immune system in bacteria and archaea.
The CRISPR/Cas system allows bacteria and archaea to fight against
invading viruses by cleaving viral DNA and inserting pieces of that DNA
into their own genome. The organism then transcribes this DNA into RNA and combines this RNA with Cas9
proteins to make double-stranded breaks in the invading viral DNA. The
RNA serves as a guide RNA to direct the Cas9 enzyme to the correct spot
in the virus DNA. By pairing Cas proteins with a designed guide RNA
CRISPR/Cas9 can be used to induce double-stranded breaks at specific
points within DNA sequences. The break gets repaired by cellular DNA
repair enzymes, creating a small insertion/deletion type mutation in
most cases. Targeted DNA repair is possible by providing a donor DNA
template that represents the desired change and that is (sometimes) used
for double-strand break repair by homologous recombination. It was
later demonstrated that CRISPR/Cas9 can edit human cells in a dish.
Although the early generation lacks the specificity of TALEN, the major
advantage of this technology is the simplicity of the design. It also
allows multiple sites to be targeted simultaneously, allowing the
editing of multiple genes at once. CRISPR/Cpf1
is a more recently discovered system that requires a different guide
RNA to create particular double-stranded breaks (leaves overhangs when
cleaving the DNA) when compared to CRISPR/Cas9.
CRISPR/Cas9 is efficient at gene disruption. The creation of
HIV-resistant babies by Chinese researcher He Jiankui is perhaps the
most famous example of gene disruption using this method.
It is far less effective at gene correction. Methods of base editing
are under development in which a “nuclease-dead” Cas 9 endonuclease or a
related enzyme is used for gene targeting while a linked deaminase
enzyme makes a targeted base change in the DNA.
The most recent refinement of CRISPR-Cas9 is called Prime Editing. This
method links a reverse transcriptase to an RNA-guided engineered
nuclease that only makes single-strand cuts but no double-strand breaks.
It replaces the portion of DNA next to the cut by the successive action
of nuclease and reverse transcriptase, introducing the desired change
from an RNA template.
A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with the most common being an organism altered in a way that "does not occur naturally by mating and/or natural recombination". A wide variety of organisms have been genetically modified (GM), including animals, plants, and microorganisms.
Genetic modification can include the introduction of new genes or enhancing, altering, or knocking outendogenous genes. In some genetic modifications, genes are transferred within the same species, across species (creating transgenic organisms), and even across kingdoms.
Creating a genetically modified organism is a multi-step process.
Genetic engineers must isolate the gene they wish to insert into the
host organism and combine it with other genetic elements, including a promoter and terminator region and often a selectable marker. A number of techniques are available for inserting the isolated gene into the host genome. Recent advancements using genome editing techniques, notably CRISPR, have made the production of GMOs much simpler. Herbert Boyer and Stanley Cohen made the first genetically modified organism in 1973, a bacterium resistant to the antibiotic kanamycin. The first genetically modified animal, a mouse, was created in 1974 by Rudolf Jaenisch, and the first plant was produced in 1983. In 1994, the Flavr Savr tomato was released, the first commercialized genetically modified food. The first genetically modified animal to be commercialized was the GloFish (2003) and the first genetically modified animal to be approved for food use was the AquAdvantage salmon in 2015.
Bacteria are the easiest organisms to engineer and have been used
for research, food production, industrial protein purification
(including drugs), agriculture, and art. There is potential to use them
for environmental purposes or as medicine. Fungi have been engineered
with much the same goals. Viruses play an important role as vectors for inserting genetic information into other organisms. This use is especially relevant to human gene therapy. There are proposals to remove the virulent
genes from viruses to create vaccines. Plants have been engineered for
scientific research, to create new colors in plants, deliver vaccines,
and to create enhanced crops. Genetically modified crops are publicly the most controversial GMOs, in spite of having the most human health and environmental benefits. Animals are generally much harder to transform and the vast majority are still at the research stage. Mammals are the best model organisms
for humans. Livestock is modified with the intention of improving
economically important traits such as growth rate, quality of meat, milk
composition, disease resistance, and survival. Genetically modified fish
are used for scientific research, as pets, and as a food source.
Genetic engineering has been proposed as a way to control mosquitos, a vector for many deadly diseases. Although human gene therapy is still relatively new, it has been used to treat genetic disorders such as severe combined immunodeficiency and Leber's congenital amaurosis.
Many objections have been raised over the development of GMOs,
particularly their commercialization. Many of these involve GM crops and
whether food produced from them is safe and what impact growing them
will have on the environment. Other concerns are the objectivity and
rigor of regulatory authorities, contamination of non-genetically
modified food, control of the food supply, patenting of life, and the use of intellectual property rights. Although there is a scientific consensus
that currently available food derived from GM crops poses no greater
risk to human health than conventional food, GM food safety is a leading
issue with critics. Gene flow,
impact on non-target organisms, and escape are the major environmental
concerns. Countries have adopted regulatory measures to deal with these
concerns. There are differences in the regulation for the release of
GMOs between countries, with some of the most marked differences
occurring between the US and Europe. Key issues concerning regulators
include whether GM food should be labeled and the status of gene-edited
organisms.
Definition
The definition of a genetically modified organism (GMO) is not clear
and varies widely between countries, international bodies, and other
communities. At its broadest, the definition of a GMO can include
anything that has had its genes altered, including by nature.
Taking a less broad view, it can encompass every organism that has had
its genes altered by humans, which would include all crops and
livestock. In 1993, the Encyclopedia Britannica defined genetic engineering as "any of a wide range of techniques ... among them artificial insemination, in vitro fertilization (e.g., 'test-tube' babies), sperm banks, cloning, and gene manipulation." The European Union (EU) included a similarly broad definition in early reviews, specifically mentioning GMOs being produced by "selective breeding and other means of artificial selection"
These definitions were promptly adjusted with a number of exceptions
added as the result of pressure from scientific and farming communities,
as well as developments in science. The EU definition later excluded
traditional breeding, in vitro fertilization, induction of polyploidy, mutation breeding, and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process.
Another approach was the definition provided by the Food and Agriculture Organization, the World Health Organization, and the European Commission, stating that the organisms must be altered in a way that does "not occur naturally by mating and/or natural recombination". Progress in science, such as the discovery of horizontal gene transfer
being a relatively common natural phenomenon, further added to the
confusion on what "occurs naturally", which led to further adjustments
and exceptions. There are examples of crops that fit this definition, but are not normally considered GMOs. For example, the grain crop triticale was fully developed in a laboratory in 1930 using various techniques to alter its genome.
Genetically engineered organism (GEO) can be considered a more
precise term compared to GMO when describing organisms' genomes that
have been directly manipulated with biotechnology. The Cartagena Protocol on Biosafety used the synonym living modified organism (LMO)
in 2000 and defined it as "any living organism that possesses a novel
combination of genetic material obtained through the use of modern
biotechnology." Modern biotechnology is further defined as "In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) and direct injection of nucleic acid into cells or organelles, or fusion of cells beyond the taxonomic family."
Originally, the term GMO was not commonly used by scientists to
describe genetically engineered organisms until after usage of GMO
became common in popular media. The United States Department of Agriculture
(USDA) considers GMOs to be plants or animals with heritable changes
introduced by genetic engineering or traditional methods, while GEO
specifically refers to organisms with genes introduced, eliminated, or
rearranged using molecular biology, particularly recombinant DNA techniques, such as transgenesis.
The definitions focus on the process more than the product, which
means there could be GMOS and non-GMOs with very similar genotypes and
phenotypes. This has led scientists to label it as a scientifically meaningless category, saying that it is impossible to group all the different types of GMOs under one common definition. It has also caused issues for organic institutions and groups looking to ban GMOs. It also poses problems as new processes are developed. The current definitions came in before genome editing became popular and there is some confusion as to whether they are GMOs. The EU has adjudged that they are changing their GMO definition to include "organisms obtained by mutagenesis",
but has excluded them from regulation based on their "long safety
record" and that they have been "conventionally been used in a number of
applications". In contrast the USDA has ruled that gene edited organisms are not considered GMOs.
Even greater inconsistency and confusion is associated with
various "Non-GMO" or "GMO-free" labeling schemes in food marketing,
where even products such as water or salt, which do not contain any
organic substances and genetic material (and thus cannot be genetically
modified by definition), are being labeled to create an impression of
being "more healthy".
A gene gun uses biolistics to insert DNA into plant tissue.
Creating a genetically modified organism (GMO) is a multi-step
process. Genetic engineers must isolate the gene they wish to insert
into the host organism. This gene can be taken from a cell or artificially synthesized. 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 a selectable marker.
As only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through tissue culture. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene.
Traditionally the new genetic material was inserted randomly within the host genome. 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.
Humans have domesticated plants and 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.
Herbert Boyer and Stanley Cohen made the first genetically modified organism in 1973. They took a gene from a bacterium that provided resistance to the antibiotic kanamycin, inserted it into a plasmid
and then induced other bacteria to incorporate the plasmid. The
bacteria that had successfully incorporated the plasmid was then able to
survive in the presence of kanamycin. Boyer and Cohen expressed other genes in bacteria. This included genes from the toad Xenopus laevis in 1974, creating the first GMO expressing a gene from an organism of a different kingdom.
In 1974, Rudolf Jaenisch created the first genetically modified animal.
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 removed (termed a knockout mouse) were created in 1989. The first transgenic livestock were produced in 1985 and the first animal to synthesize transgenic proteins in their milk were mice in 1987. The mice were engineered to produce human tissue plasminogen activator, a protein involved in breaking down blood clots.
In 1976, Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson; a year later, the company produced a human protein (somatostatin) in E. coli. Genentech announced the production of genetically engineered human insulin in 1978. The insulin produced by bacteria, branded Humulin, was approved for release by the Food and Drug Administration in 1982. In 1988, the first human antibodies were produced in plants. In 1987, a strain of Pseudomonas syringae became the first genetically modified organism to be released into the environment when a strawberry and potato field in California were sprayed with it.
The first genetically modified crop, an antibiotic-resistant tobacco plant, was produced in 1982. China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992. In 1994, Calgene attained approval to commercially release the Flavr Savr tomato, the first genetically modified food. Also in 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialized in Europe. An insect resistant Potato was approved for release in the US in 1995,
and by 1996 approval had been granted to commercially grow 8 transgenic
crops and one flower crop (carnation) in 6 countries plus the EU.
The first genetically modified animal to be commercialized 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. In 2015, AquAdvantage salmon became the first genetically modified animal to be approved for food use. Approval is for fish raised in Panama and sold in the US. 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.
Top: Bacteria transformed with pGLO under ambient light Bottom: Bacteria transformed with pGLO visualized under ultraviolet light
Bacteria were the first organisms to be genetically modified in the laboratory, due to the relative ease of modifying their chromosomes.
This ease made them important tools for the creation of other GMOs.
Genes and other genetic information from a wide range of organisms can
be added to a plasmid and inserted into bacteria for storage and modification. Bacteria are cheap, easy to grow, clonal,
multiply quickly and can be stored at −80 °C almost indefinitely. Once a
gene is isolated it can be stored inside the bacteria, providing an
unlimited supply for research. A large number of custom plasmids make manipulating DNA extracted from bacteria relatively easy.
Their ease of use has made them great tools for scientists looking to study gene function and evolution. The simplest model organisms come from bacteria, with most of our early understanding of molecular biology coming from studying Escherichia coli.
Scientists can easily manipulate and combine genes within the bacteria
to create novel or disrupted proteins and observe the effect this has on
various molecular systems. Researchers have combined the genes from
bacteria and archaea, leading to insights on how these two diverged in the past. In the field of synthetic biology, they have been used to test various synthetic approaches, from synthesizing genomes to creating novel nucleotides.
Bacteria have been used in the production of food for a long
time, and specific strains have been developed and selected for that
work on an industrial scale. They can be used to produce enzymes, amino acids, flavorings,
and other compounds used in food production. With the advent of genetic
engineering, new genetic changes can easily be introduced into these
bacteria. Most food-producing bacteria are lactic acid bacteria,
and this is where the majority of research into genetically engineering
food-producing bacteria has gone. The bacteria can be modified to
operate more efficiently, reduce toxic byproduct production, increase
output, create improved compounds, and remove unnecessary pathways. Food products from genetically modified bacteria include alpha-amylase, which converts starch to simple sugars, chymosin, which clots milk protein for cheese making, and pectinesterase, which improves fruit juice clarity.
The majority are produced in the US and even though regulations are in
place to allow production in Europe, as of 2015 no food products derived
from bacteria are currently available there.
Genetically modified bacteria are used to produce large amounts
of proteins for industrial use. The bacteria are generally grown to a
large volume before the gene encoding the protein is activated. The
bacteria are then harvested and the desired protein purified from them. The high cost of extraction and purification has meant that only high value products have been produced at an industrial scale. The majority of these products are human proteins for use in medicine.
Many of these proteins are impossible or difficult to obtain via
natural methods and they are less likely to be contaminated with
pathogens, making them safer. The first medicinal use of GM bacteria was to produce the protein insulin to treat diabetes. Other medicines produced include clotting factors to treat hemophilia, human growth hormone to treat various forms of dwarfism, interferon to treat some cancers, erythropoietin for anemic patients, and tissue plasminogen activator which dissolves blood clots. Outside of medicine they have been used to produce biofuels.
There is interest in developing an extracellular expression system
within the bacteria to reduce costs and make the production of more
products economical.
With a greater understanding of the role that the microbiome
plays in human health, there is a potential to treat diseases by
genetically altering the bacteria to, themselves, be therapeutic agents.
Ideas include altering gut bacteria so they destroy harmful bacteria,
or using bacteria to replace or increase deficient enzymes or proteins. One research focus is to modify Lactobacillus, bacteria that naturally provide some protection against HIV, with genes that will further enhance this protection. If the bacteria do not form colonies
inside the patient, the person must repeatedly ingest the modified
bacteria in order to get the required doses. Enabling the bacteria to
form a colony could provide a more long-term solution, but could also
raise safety concerns as interactions between bacteria and the human
body are less well understood than with traditional drugs. There are
concerns that horizontal gene transfer to other bacteria could have unknown effects. As of 2018 there are clinical trials underway testing the efficacy and safety of these treatments.
For over a century, bacteria have been used in agriculture. Crops have been inoculated with Rhizobia (and more recently Azospirillum) to increase their production or to allow them to be grown outside their original habitat. Application of Bacillus thuringiensis
(Bt) and other bacteria can help protect crops from insect infestation
and plant diseases. With advances in genetic engineering, these bacteria
have been manipulated for increased efficiency and expanded host range.
Markers have also been added to aid in tracing the spread of the
bacteria. The bacteria that naturally colonize certain crops have also
been modified, in some cases to express the Bt genes responsible for
pest resistance. Pseudomonas strains of bacteria cause frost damage by nucleating water into ice crystals around themselves. This led to the development of ice-minus bacteria,
which have the ice-forming genes removed. When applied to crops they
can compete with the non-modified bacteria and confer some frost
resistance.
This artwork is made with bacteria modified to express 8 different colors of fluorescent proteins.
Other uses for genetically modified bacteria include bioremediation,
where the bacteria are used to convert pollutants into a less toxic
form. Genetic engineering can increase the levels of the enzymes used to
degrade a toxin or to make the bacteria more stable under environmental
conditions. Bioart has also been created using genetically modified bacteria. In the 1980s artist Jon Davis and geneticist Dana Boyd converted the Germanic symbol for femininity (ᛉ) into binary code and then into a DNA sequence, which was then expressed in Escherichia coli. This was taken a step further in 2012, when a whole book was encoded onto DNA. Paintings have also been produced using bacteria transformed with fluorescent proteins.
Viruses are often modified so they can be used as vectors for inserting genetic information into other organisms. This process is called transduction
and if successful the recipient of the introduced DNA becomes a GMO.
Different viruses have different efficiencies and capabilities.
Researchers can use this to control for various factors; including the
target location, insert size, and duration of gene expression. Any
dangerous sequences inherent in the virus must be removed, while those
that allow the gene to be delivered effectively are retained.
While viral vectors can be used to insert DNA into almost any
organism it is especially relevant for its potential in treating human
disease. Although primarily still at trial stages, there has been some successes using gene therapy to replace defective genes. This is most evident in curing patients with severe combined immunodeficiency rising from adenosine deaminase deficiency (ADA-SCID), although the development of leukemia in some ADA-SCID patients along with the death of Jesse Gelsinger in a 1999 trial set back the development of this approach for many years. In 2009, another breakthrough was achieved when an eight-year-old boy with Leber's congenital amaurosis regained normal eyesight and in 2016 GlaxoSmithKline gained approval to commercialize a gene therapy treatment for ADA-SCID. As of 2018, there are a substantial number of clinical trials underway, including treatments for hemophilia, glioblastoma, chronic granulomatous disease, cystic fibrosis and various cancers.
The most common virus used for gene delivery comes from adenoviruses
as they can carry up to 7.5 kb of foreign DNA and infect a relatively
broad range of host cells, although they have been known to elicit
immune responses in the host and only provide short term expression.
Other common vectors are adeno-associated viruses, which have lower toxicity and longer-term expression, but can only carry about 4kb of DNA. Herpes simplex viruses
make promising vectors, having a carrying capacity of over 30kb and
providing long term expression, although they are less efficient at gene
delivery than other vectors. The best vectors for long term integration of the gene into the host genome are retroviruses, but their propensity for random integration is problematic. Lentiviruses
are a part of the same family as retroviruses with the advantage of
infecting both dividing and non-dividing cells, whereas retroviruses
only target dividing cells. Other viruses that have been used as vectors
include alphaviruses, flaviviruses, measles viruses, rhabdoviruses, Newcastle disease virus, poxviruses, and picornaviruses.
Most vaccines consist of viruses that have been attenuated, disabled, weakened or killed in some way so that their virulent
properties are no longer effective. Genetic engineering could
theoretically be used to create viruses with the virulent genes removed.
This does not affect the viruses infectivity,
invokes a natural immune response and there is no chance that they will
regain their virulence function, which can occur with some other
vaccines. As such they are generally considered safer and more efficient
than conventional vaccines, although concerns remain over non-target
infection, potential side effects and horizontal gene transfer to other viruses.
Another potential approach is to use vectors to create novel vaccines
for diseases that have no vaccines available or the vaccines that do not
work effectively, such as AIDS, malaria, and tuberculosis. The most effective vaccine against Tuberculosis, the Bacillus Calmette–Guérin (BCG) vaccine, only provides partial protection. A modified vaccine expressing a M tuberculosis antigen is able to enhance BCG protection. It has been shown to be safe to use at phase II trials, although not as effective as initially hoped. Other vector-based vaccines have already been approved and many more are being developed.
Another potential use of genetically modified viruses is to alter
them so they can directly treat diseases. This can be through
expression of protective proteins or by directly targeting infected
cells. In 2004, researchers reported that a genetically modified virus
that exploits the selfish behavior of cancer cells might offer an
alternative way of killing tumours. Since then, several researchers have developed genetically modified oncolytic viruses that show promise as treatments for various types of cancer. In 2017, researchers genetically modified a virus to express spinach defensin proteins. The virus was injected into orange trees to combat citrus greening disease that had reduced orange production by 70% since 2005.
Natural viral diseases, such as myxomatosis and rabbit hemorrhagic disease,
have been used to help control pest populations. Over time the
surviving pests become resistant, leading researchers to look at
alternative methods. Genetically modified viruses that make the target
animals infertile through immunocontraception have been created in the laboratory as well as others that target the developmental stage of the animal. There are concerns with using this approach regarding virus containment and cross species infection. Sometimes the same virus can be modified for contrasting purposes. 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.
Outside of biology scientists have used a genetically modified virus to construct a lithium-ion battery and other nanostructured materials. It is possible to engineer bacteriophages to express modified proteins on their surface and join them up in specific patterns (a technique called phage display). These structures have potential uses for energy storage and generation, biosensing and tissue regeneration with some new materials currently produced including quantum dots, liquid crystals, nanorings and nanofibres. The battery was made by engineering M13 bacteriaophages so they would coat themselves in iron phosphate and then assemble themselves along a carbon nanotube.
This created a highly conductive medium for use in a cathode, allowing
energy to be transferred quickly. They could be constructed at lower
temperatures with non-toxic chemicals, making them more environmentally
friendly.
Fungi
Fungi can be used for many of the same processes as bacteria. For
industrial applications, yeasts combine the bacterial advantages of
being a single-celled organism that is easy to manipulate and grow with
the advanced protein modifications found in eukaryotes. They can be used to produce large complex molecules for use in food, pharmaceuticals, hormones, and steroids.
Yeast is important for wine production and as of 2016 two genetically
modified yeasts involved in the fermentation of wine have been
commercialized in the United States and Canada. One has increased malolactic fermentation efficiency, while the other prevents the production of dangerous ethyl carbamate compounds during fermentation. There have also been advances in the production of biofuel from genetically modified fungi.
Fungi, being the most common pathogens of insects, make attractive biopesticides.
Unlike bacteria and viruses they have the advantage of infecting the
insects by contact alone, although they are out competed in efficiency
by chemical pesticides. Genetic engineering can improve virulence, usually by adding more virulent proteins, increasing infection rate or enhancing spore persistence. Many of the disease carrying vectors are susceptible to entomopathogenic fungi. An attractive target for biological control are mosquitos, vectors for a range of deadly diseases, including malaria, yellow fever and dengue fever. Mosquitos can evolve quickly so it becomes a balancing act of killing them before the Plasmodium they carry becomes the infectious disease, but not so fast that they become resistant to the fungi. By genetically engineering fungi like Metarhizium anisopliae and Beauveria bassiana to delay the development of mosquito infectiousness the selection pressure to evolve resistance is reduced. Another strategy is to add proteins to the fungi that block transmission of malaria or remove the Plasmodium altogether.
Agaricus bisporus the common white button mushroom, has been gene edited to resist browning, giving it a longer shelf life. The process used CRISPR to knock out a gene that encodes polyphenol oxidase.
As it didn't introduce any foreign DNA into the organism it was not
deemed to be regulated under existing GMO frameworks and as such is the
first CRISPR-edited organism to be approved for release. This has intensified debates as to whether gene-edited organisms should be considered genetically modified organisms and how they should be regulated.
Plants have been engineered for scientific research, to display new
flower colors, deliver vaccines, and to create enhanced crops. Many
plants are pluripotent,
meaning that a single cell from a mature plant can be harvested and
under the right conditions can develop into a new plant. This ability
can be taken advantage of by genetic engineers; by selecting for cells
that have been successfully transformed in an adult plant a new plant
can then be grown that contains the transgene in every cell through a
process known as tissue culture.
Much of the advances in the field of genetic engineering has come from experimentation with tobacco. Major advances in tissue culture and plant cellular mechanisms for a wide range of plants has originated from systems developed in tobacco.
It was the first plant to be altered using genetic engineering and is
considered a model organism for not only genetic engineering, but a
range of other fields. As such the transgenic tools and procedures are well established making tobacco one of the easiest plants to transform. Another major model organism relevant to genetic engineering is Arabidopsis thaliana. Its small genome and short life cycle makes it easy to manipulate and it contains many homologs to important crop species. It was the first plant sequenced, has a host of online resources available and can be transformed by simply dipping a flower in a transformed Agrobacterium solution.
In research, plants are engineered to help discover the functions
of certain genes. The simplest way to do this is to remove the gene and
see what phenotype develops compared to the wild type form. Any differences are possibly the result of the missing gene. Unlike mutagenisis, genetic engineering allows targeted removal without disrupting other genes in the organism. Some genes are only expressed in certain tissues, so reporter genes, like GUS, can be attached to the gene of interest allowing visualization of the location.
Other ways to test a gene is to alter it slightly and then return it to
the plant and see if it still has the same effect on phenotype. Other
strategies include attaching the gene to a strong promoter and see what happens when it is overexpressed, forcing a gene to be expressed in a different location or at different developmental stages.
Suntory "blue" rose
Some genetically modified plants are purely ornamental. They are modified for flower color, fragrance, flower shape and plant architecture. The first genetically modified ornamentals commercialized altered color. Carnations were released in 1997, with the most popular genetically modified organism, a blue rose (actually lavender or mauve) created in 2004. The roses are sold in Japan, the United States, and Canada.Other genetically modified ornamentals include Chrysanthemum and Petunia.
As well as increasing aesthetic value there are plans to develop
ornamentals that use less water or are resistant to the cold, which
would allow them to be grown outside their natural environments.
It has been proposed to genetically modify some plant species
threatened by extinction to be resistant to invasive plants and
diseases, such as the emerald ash borer in North American and the fungal disease, Ceratocystis platani, in European plane trees. The papaya ringspot virus devastated papaya trees in Hawaii in the twentieth century until transgenic papaya plants were given pathogen-derived resistance.
However, genetic modification for conservation in plants remains mainly
speculative. A unique concern is that a transgenic species may no
longer bear enough resemblance to the original species to truly claim
that the original species is being conserved. Instead, the transgenic
species may be genetically different enough to be considered a new
species, thus diminishing the conservation worth of genetic
modification.
Genetically modified crops are genetically modified plants that are used in agriculture.
The first crops developed were used for animal or human food and
provide resistance to certain pests, diseases, environmental conditions,
spoilage or chemical treatments (e.g. resistance to a herbicide). The second generation of crops aimed to improve the quality, often by altering the nutrient profile. Third generation genetically modified crops could be used for non-food purposes, including the production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for bioremediation.
There are three main aims to agricultural advancement; increased production, improved conditions for agricultural workers and sustainability.
GM crops contribute by improving harvests through reducing insect
pressure, increasing nutrient value and tolerating different abiotic stresses. Despite this potential, as of 2018, the commercialized crops are limited mostly to cash crops
like cotton, soybean, maize and canola and the vast majority of the
introduced traits provide either herbicide tolerance or insect
resistance. Soybeans accounted for half of all genetically modified crops planted in 2014.
Adoption by farmers has been rapid, between 1996 and 2013, the total
surface area of land cultivated with GM crops increased by a factor of
100. Geographically though the spread has been uneven, with strong growth in the Americas and parts of Asia and little in Europe and Africa. Its socioeconomic spread has been more even, with approximately 54% of worldwide GM crops grown in developing countries in 2013. Although doubts have been raised,
most studies have found growing GM crops to be beneficial to farmers
through decreased pesticide use as well as increased crop yield and farm
profit.
The majority of GM crops have been modified to be resistant to selected herbicides, usually a glyphosate or glufosinate
based one. Genetically modified crops engineered to resist herbicides
are now more available than conventionally bred resistant varieties; in the USA 93% of soybeans and most of the GM maize grown is glyphosate tolerant. Most currently available genes used to engineer insect resistance come from the Bacillus thuringiensis bacterium and code for delta endotoxins. A few use the genes that encode for vegetative insecticidal proteins. The only gene commercially used to provide insect protection that does not originate from B. thuringiensis is the Cowpeatrypsin inhibitor (CpTI). CpTI was first approved for use cotton in 1999 and is currently undergoing trials in rice.
Less than one percent of GM crops contained other traits, which include
providing virus resistance, delaying senescence and altering the plants
composition.
Golden rice is the most well known GM crop that is aimed at increasing nutrient value. It has been engineered with three genes that biosynthesisebeta-carotene, a precursor of vitamin A, in the edible parts of rice. It is intended to produce a fortified food to be grown and consumed in areas with a shortage of dietary vitamin A, a deficiency which each year is estimated to kill 670,000 children under the age of 5 and cause an additional 500,000 cases of irreversible childhood blindness. The original golden rice produced 1.6μg/g of the carotenoids, with further development increasing this 23 times. It gained its first approvals for use as food in 2018.
Plants and plant cells have been genetically engineered for production of biopharmaceuticals in bioreactors, a process known as pharming. Work has been done with duckweedLemna minor, the algaeChlamydomonas reinhardtii and the mossPhyscomitrella patens. Biopharmaceuticals produced include cytokines, hormones, antibodies, enzymes
and vaccines, most of which are accumulated in the plant seeds. Many
drugs also contain natural plant ingredients and the pathways that lead
to their production have been genetically altered or transferred to
other plant species to produce greater volume. Other options for bioreactors are biopolymers and biofuels. Unlike bacteria, plants can modify the proteins post-translationally, allowing them to make more complex molecules. They also pose less risk of being contaminated. Therapeutics have been cultured in transgenic carrot and tobacco cells, including a drug treatment for Gaucher's disease.
Vaccine production and storage has great potential in transgenic
plants. Vaccines are expensive to produce, transport, and administer, so
having a system that could produce them locally would allow greater
access to poorer and developing areas.
As well as purifying vaccines expressed in plants it is also possible
to produce edible vaccines in plants. Edible vaccines stimulate the immune system
when ingested to protect against certain diseases. Being stored in
plants reduces the long-term cost as they can be disseminated without
the need for cold storage, don't need to be purified, and have long term
stability. Also being housed within plant cells provides some
protection from the gut acids upon digestion. However the cost of
developing, regulating, and containing transgenic plants is high,
leading to most current plant-based vaccine development being applied to
veterinary medicine, where the controls are not as strict.
Genetically modified crops have been proposed as one of the ways to reduce farming-related CO2
emissions due to higher yield, reduced use of pesticides, reduced use
of tractor fuel and no tillage. According to a 2021 study, in EU alone
widespread adoption of GE crops would reduce greenhouse gas emissions by
33 million tons of CO2 equivalent or 7.5% of total farming-related emissions.
The vast majority of genetically modified animals are at the research
stage with the number close to entering the market remaining small.
As of 2018 only three genetically modified animals have been approved,
all in the USA. A goat and a chicken have been engineered to produce
medicines and a salmon has increased its own growth.
Despite the differences and difficulties in modifying them, the end
aims are much the same as for plants. GM animals 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.
Some chimeras, like the blotched mouse shown, are created through genetic modification techniques like gene targeting.
The process of genetically engineering mammals is slow, tedious, and
expensive. However, new technologies are making genetic modifications
easier and more precise. The first transgenic mammals were produced by injecting viral DNA into embryos and then implanting the embryos in females.
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 the presence of the gene in every
cell. The development of the CRISPR-Cas9 gene editing system as a cheap and fast way of directly modifying germ cells, effectively halving the amount of time needed to develop genetically modified mammals.
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) 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.
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 and is extracted from the goat's milk. Human alpha-1-antitrypsin is another protein that has been produced from goats and is used in treating humans with this deficiency. Another medicinal 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. Chimeric pigs could carry fully human organs. The first human transplant of a genetically modified pig heart occurred in 2023, and kidney in 2024.
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. A GM pig called Enviropig was created with the capability of digesting plant phosphorus more efficiently than conventional pigs. They could reduce water pollution since they excrete 30 to 70% less phosphorus in manure. Dairy cows have been genetically engineered 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. Researchers have also developed a genetically engineered cow that produces allergy-free milk.
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 visualization and localization 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 help find therapies for HIV/AIDS and other diseases as feline immunodeficiency virus is related to HIV.
There have 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 creating live
elephants and transferring all the genes and reversing years of genetic
evolution is a long way from being feasible.
It is more likely that scientists could use this technology to conserve
endangered animals by bringing back lost diversity or transferring
evolved genetic advantages from adapted organisms to those that are
struggling.
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, in an 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.
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. 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. 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 in 2003 it was
introduced for sale in the USA.
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 one-cell embryo is easy to see and microinject with transgenic DNA. Zebrafish are model organisms for developmental processes, regeneration, genetics, behavior, 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.
GM fish have been developed with promoters driving an over-production of 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, a biotechnology company, have produced a salmon (called AquAdvantage salmon) that can mature in half the time as wild salmon. It obtained regulatory approval in 2015, the first non-plant GMO food to be commercialized. As of August 2017, GMO salmon is being sold in Canada. Sales in the US started in May 2021.
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. They also have 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. The discovery of transposons, in particular the p-element, in Drosophila
provided an early method to add transgenes to their genome, although
this has been taken over by more modern gene-editing techniques.
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 approach has been taken further by using the gene drive to spread 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 a 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 each year worldwide.
The approach is similar to the sterile technique tested on mosquitoes,
where males are transformed with a gene that prevents any females born
from reaching maturity. They underwent field trials in 2017. Genetically modified moths have previously been released in field trials. In this case a strain of pink bollworm that were sterilized with radiation were genetically engineered to express a red fluorescent protein making it easier for researchers to monitor them.
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 currently developed to be 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.
Systems have been developed to create transgenic organisms in a wide
variety of other animals. 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 US regulatory approval in 2015. Genetically modified frogs, in particular Xenopus laevis and Xenopus tropicalis, are used in developmental biology research. 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.
The nematodeCaenorhabditis 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 nematodes 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 localization by attaching reporter genes. Transgenes can
also be combined with RNAi techniques to rescue phenotypes, study gene
function, image cell development in real time or control expression for
different tissues or developmental stages. Transgenic nematodes have been used to study viruses, toxicology, diseases,[297] and to detect environmental pollutants.
Transgenic Hydra expressing green fluorescent protein
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 scientists 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 synchronized 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 animals that have been genetically modified include snails, geckos, turtles, crayfish, oysters, shrimp, clams, abalone and sponges.
Genetically modified organisms are regulated by government agencies.
This applies to research as well as the release of genetically modified
organisms, including crops and food. The development of a regulatory
framework concerning genetic engineering began in 1975, at Asilomar, California. The Asilomar meeting
recommended a set of guidelines regarding the cautious use of
recombinant technology and any products resulting from that technology. The Cartagena Protocol on Biosafety was adopted on 29 January 2000 and entered into force on 11 September 2003. It is an international treaty that governs the transfer, handling, and use of genetically modified organisms.
One hundred and fifty-seven countries are members of the Protocol and
many use it as a reference point for their own regulations.
Universities and research institutes generally have a special
committee that is responsible for approving any experiments that involve
genetic engineering. Many experiments also need permission from a
national regulatory group or legislation. All staff must be trained in
the use of GMOs and all laboratories must gain approval from their
regulatory agency to work with GMOs.
The legislation covering GMOs are often derived from regulations and
guidelines in place for the non-GMO version of the organism, although
they are more severe.
There is a near-universal system for assessing the relative risks
associated with GMOs and other agents to laboratory staff and the
community. They are assigned to one of four risk categories based on
their virulence, the severity of the disease, the mode of transmission,
and the availability of preventive measures or treatments. There are
four biosafety levels
that a laboratory can fall into, ranging from level 1 (which is
suitable for working with agents not associated with disease) to level 4
(working with life-threatening agents). Different countries use
different nomenclature to describe the levels and can have different
requirements for what can be done at each level.
A label marking this peanut butter as being non-GMODetail of a French cheese box declaring "GMO-free" production (i.e., below 0.9%)
There are differences in the regulation for the release of GMOs
between countries, with some of the most marked differences occurring
between the US and Europe.
Regulation varies in a given country depending on the intended use of
the products of the genetic engineering. For example, a crop not
intended for food use is generally not reviewed by authorities
responsible for food safety.
Some nations have banned the release of GMOs or restricted their use,
and others permit them with widely differing degrees of regulation. In 2016, thirty eight countries officially ban or prohibit the
cultivation of GMOs and nine (Algeria, Bhutan, Kenya, Kyrgyzstan,
Madagascar, Peru, Russia, Venezuela and Zimbabwe) ban their importation. Most countries that do not allow GMO cultivation do permit research using GMOs. Despite regulation, illegal releases have sometimes occurred, due to weakness of enforcement.
The European Union (EU) differentiates between approval for cultivation within the EU and approval for import and processing.
While only a few GMOs have been approved for cultivation in the EU a
number of GMOs have been approved for import and processing. The cultivation of GMOs has triggered a debate about the market for GMOs in Europe. Depending on the coexistence regulations, incentives for cultivation of GM crops differ.
The US policy does not focus on the process as much as other countries,
looks at verifiable scientific risks and uses the concept of substantial equivalence.
Whether gene edited organisms should be regulated the same as
genetically modified organism is debated. USA regulations sees them as
separate and does not regulate them under the same conditions, while in
Europe a GMO is any organism created using genetic engineering
techniques.
One of the key issues concerning regulators is whether GM products should be labeled. The European Commission says that mandatory labeling and traceability are needed to allow for informed choice, avoid potential false advertising and facilitate the withdrawal of products if adverse effects on health or the environment are discovered. The American Medical Association and the American Association for the Advancement of Science say that absent scientific evidence of harm even voluntary labeling is misleading and will falsely alarm consumers. Labeling of GMO products in the marketplace is required in 64 countries.
Labeling can be mandatory up to a threshold GM content level (which
varies between countries) or voluntary. In the U.S., the National
Bioengineered Food Disclosure Standard (Mandatory Compliance Date:
January 1, 2022) requires labeling GM foods. In Canada, labeling of GM food is voluntary, while in Europe all food (including processed food) or feed which contains greater than 0.9% of approved GMOs must be labeled. In 2014, sales of products that had been labeled as non-GMO grew 30 percent to $1.1 billion.
There is controversy over GMOs, especially with regard to their
release outside laboratory environments. The dispute involves consumers,
producers, biotechnology companies, governmental regulators,
non-governmental organizations, and scientists. Many of these concerns
involve GM crops and whether food produced from them is safe and what
impact growing them will have on the environment. These controversies
have led to litigation, international trade disputes, and protests, and
to restrictive regulation of commercial products in some countries. Most concerns are around the health and environmental effects of GMOs. These include whether they may provoke an allergic reaction, whether the transgenes could transfer to human cells, and whether genes not approved for human consumption could outcross into the food supply.
A protester advocating for the labeling of GMOs
There is a scientific consensusthat currently available food derived from GM crops poses no greater risk to human health than conventional food, but that each GM food needs to be tested on a case-by-case basis before introduction. Nonetheless, members of the public are much less likely than scientists to perceive GM foods as safe.
The legal and regulatory status of GM foods varies by country, with
some nations banning or restricting them, and others permitting them
with widely differing degrees of regulation.
As late as the 1990s gene flow
into wild populations was thought to be unlikely and rare, and if it
were to occur, easily eradicated. It was thought that this would add no
additional environmental costs or risks – no effects were expected other
than those already caused by pesticide applications.
However, in the decades since, several such examples have been
observed. Gene flow between GM crops and compatible plants, along with
increased use of broad-spectrum herbicides, can increase the risk of herbicide resistant weed populations.
Debate over the extent and consequences of gene flow intensified in
2001 when a paper was published showing transgenes had been found in landrace maize in Mexico, the crop's center of diversity. Gene flow from GM crops to other organisms has been found to generally be lower than what would occur naturally.
In order to address some of these concerns some GMOs have been
developed with traits to help control their spread. To prevent the
genetically modified salmon inadvertently breeding with wild salmon, all
the fish raised for food are females, triploid, 99% are reproductively sterile, and raised in areas where escaped salmon could not survive. Bacteria have also been modified to depend on nutrients that cannot be found in nature, and genetic use restriction technology has been developed, though not yet marketed, that causes the second generation of GM plants to be sterile.
Other environmental and agronomic
concerns include a decrease in biodiversity, an increase in secondary
pests (non-targeted pests) and evolution of resistant insect pests.
In the areas of China and the US with Bt crops the overall biodiversity
of insects has increased and the impact of secondary pests has been
minimal. Resistance was found to be slow to evolve when best practice strategies were followed.
The impact of Bt crops on beneficial non-target organisms became a
public issue after a 1999 paper suggested they could be toxic to monarch butterflies. Follow up studies have since shown that the toxicity levels encountered in the field were not high enough to harm the larvae.
Accusations that scientists are "playing God" and other religious issues have been ascribed to the technology from the beginning.
With the ability to genetically engineer humans now possible there are
ethical concerns over how far this technology should go, or if it should
be used at all.
Much debate revolves around where the line between treatment and
enhancement is and whether the modifications should be inheritable. Other concerns include contamination of the non-genetically modified food supply, the rigor of the regulatory process,consolidation of control of the food supply in companies that make and sell GMOs, exaggeration of the benefits of genetic modification, or concerns over the use of herbicides with glyphosate. Other issues raised include the patenting of life and the use of intellectual property rights.
There are large differences in consumer acceptance of GMOs, with
Europeans more likely to view GM food negatively than North Americans. GMOs arrived on the scene as the public confidence in food safety, attributed to recent food scares such as Bovine spongiform encephalopathy and other scandals involving government regulation of products in Europe, was low. This along with campaigns run by various non-governmental organizations (NGO) have been very successful in blocking or limiting the use of GM crops. NGOs like the Organic Consumers Association, the Union of Concerned Scientists, Greenpeace and other groups have said that risks have not been adequately identified and managed
and that there are unanswered questions regarding the potential
long-term impact on human health from food derived from GMOs. They
propose mandatory labeling or a moratorium on such products.
