Genetic engineering

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Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's

organisms
.

New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus.

As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome.[1]

An organism that is generated through genetic engineering is considered to be genetically modified (GM) and the resulting entity is a

bacterium generated by Herbert Boyer and Stanley Cohen in 1973. Rudolf Jaenisch created the first GM animal when he inserted foreign DNA into a mouse in 1974. The first company to focus on genetic engineering, Genentech, was founded in 1976 and started the production of human proteins. Genetically engineered human insulin was produced in 1978 and insulin-producing bacteria were commercialised in 1982. Genetically modified food has been sold since 1994, with the release of the Flavr Savr tomato. The Flavr Savr was engineered to have a longer shelf life, but most current GM crops are modified to increase resistance to insects and herbicides. GloFish, the first GMO designed as a pet, was sold in the United States in December 2003. In 2016 salmon
modified with a growth hormone were sold.

Genetic engineering has been applied in numerous fields including research, medicine, industrial biotechnology and agriculture. In research, GMOs are used to study gene function and expression through loss of function, gain of function, tracking and expression experiments. By knocking out genes responsible for certain conditions it is possible to create animal model organisms of human diseases. As well as producing hormones, vaccines and other drugs, genetic engineering has the potential to cure genetic diseases through gene therapy. Chinese hamster ovary (CHO) cells are used in industrial genetic engineering. Additionally mRNA vaccines are made through genetic engineering to treat viruses such as COVID-19. The same techniques that are used to produce drugs can also have industrial applications such as producing enzymes for laundry detergent, cheeses and other products.

The rise of commercialised genetically modified crops has provided economic benefit to farmers in many different countries, but has also been the source of most of the controversy surrounding the technology. This has been present since its early use; the first field trials were destroyed by anti-GM activists. Although there is a scientific consensus that currently available food derived from GM crops poses no greater risk to human health than conventional food, critics consider GM food safety a leading concern. Gene flow, impact on non-target organisms, control of the food supply and intellectual property rights have also been raised as potential issues. These concerns have led to the development of a regulatory framework, which started in 1975. It has led to an international treaty, the Cartagena Protocol on Biosafety, that was adopted in 2000. Individual countries have developed their own regulatory systems regarding GMOs, with the most marked differences occurring between the US and Europe.

IUPAC definition

Genetic engineering: Process of inserting new genetic information into existing cells in order to modify a specific organism for the purpose of changing its characteristics.

Note: Adapted from ref.[2][3]

Overview

cisgenic
genetic modification

Genetic engineering is a process that alters the genetic structure of an organism by either removing or introducing DNA, or modifying existing genetic material in situ. Unlike traditional animal and plant breeding, which involves doing multiple crosses and then selecting for the organism with the desired phenotype, genetic engineering takes the gene directly from one organism and delivers it to the other. This is much faster, can be used to insert any genes from any organism (even ones from different domains) and prevents other undesirable genes from also being added.[4]

Genetic engineering could potentially fix severe genetic disorders in humans by replacing the defective gene with a functioning one.[5] It is an important tool in research that allows the function of specific genes to be studied.[6] Drugs, vaccines and other products have been harvested from organisms engineered to produce them.[7] Crops have been developed that aid food security by increasing yield, nutritional value and tolerance to environmental stresses.[8]

The DNA can be introduced directly into the

host organism or into a cell that is then fused or hybridised with the host.[9] This relies on recombinant nucleic acid techniques to form new combinations of heritable genetic material followed by the incorporation of that material either indirectly through a vector system or directly through micro-injection, macro-injection or micro-encapsulation
.

Genetic engineering does not normally include traditional breeding, in vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process.[9] However, some broad definitions of genetic engineering include selective breeding.[10] Cloning and stem cell research, although not considered genetic engineering,[11] are closely related and genetic engineering can be used within them.[12] Synthetic biology is an emerging discipline that takes genetic engineering a step further by introducing artificially synthesised material into an organism.[13]

Plants, animals or microorganisms that have been changed through genetic engineering are termed

synonymous with genetic engineering while within the United States of America and Canada genetic modification can also be used to refer to more conventional breeding methods.[17][18][19]

History

Humans have altered the genomes of species for thousands of years through

James Watson and Francis Crick showed that the DNA molecule has a double-helix structure – though the general concept of direct genetic manipulation was explored in rudimentary form in Stanley G. Weinbaum's 1936 science fiction story Proteus Island.[25][26]

In 1974 Rudolf Jaenisch created a genetically modified mouse, the first GM animal.

In 1972,

transgenic animal[30] These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe.[31][32]

In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer and

U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.[34] The insulin produced by bacteria was approved for release by the Food and Drug Administration (FDA) in 1982.[35]

In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S. government authorisation to perform field tests with the ice-minus strain of Pseudomonas syringae to protect crops from frost, but environmental groups and protestors delayed the field tests for four years with legal challenges.[36] In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment[37] when a strawberry field and a potato field in California were sprayed with it.[38] Both test fields were attacked by activist groups the night before the tests occurred: "The world's first trial site attracted the world's first field trasher".[37]

The first field trials of

Environmental Protection Agency, after having been approved by the FDA, making it the first pesticide producing crop to be approved in the US.[43] In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the US, Brazil, Argentina, India, Canada, China, Paraguay and South Africa.[44]

In 2010, scientists at the

synthetic genome and inserted it into an empty bacterial cell. The resulting bacterium, named Mycoplasma laboratorium, could replicate and produce proteins.[45][46] Four years later this was taken a step further when a bacterium was developed that replicated a plasmid containing a unique base pair, creating the first organism engineered to use an expanded genetic alphabet.[47][48] In 2012, Jennifer Doudna and Emmanuelle Charpentier collaborated to develop the CRISPR/Cas9 system,[49][50] a technique which can be used to easily and specifically alter the genome of almost any organism.[51]

Process

Polymerase chain reaction is a powerful tool used in molecular cloning.

Creating a GMO is a multi-step process. Genetic engineers must first choose what gene they wish to insert into the organism. This is driven by what the aim is for the resultant organism and is built on earlier research.

transcriptomics and genome sequencing has made it much easier to find suitable genes.[52] Luck also plays its part; the Roundup Ready gene was discovered after scientists noticed a bacterium thriving in the presence of the herbicide.[53]

Gene isolation and cloning

The next step is to isolate the candidate gene. The

single-celled organisms, which makes it suitable as a genetic engineering tool.[59]

Before the gene is inserted into the target organism it must be combined with other genetic elements. These include a

antibiotic resistance, so researchers can easily determine which cells have been successfully transformed. The gene can also be modified at this stage for better expression or effectiveness. These manipulations are carried out using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning.[60]

Inserting DNA into the host genome

biolistics
to insert DNA into plant tissue.

There are a number of techniques used to insert genetic material into the host genome. Some bacteria can naturally

viral vectors.[61]

Plant genomes can be engineered by physical methods or by use of

biolistics, where particles of gold or tungsten are coated with DNA and then shot into young plant cells,[64] and electroporation
, which involves using an electric shock to make the cell membrane permeable to plasmid DNA.

As only a single cell is transformed with genetic material, the organism must be

Selectable markers are used to easily differentiate transformed from untransformed cells. 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.[68]

A. tumefaciens
attaching itself to a carrot cell

Further testing using PCR,

RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis.[70]

The new genetic material can be inserted randomly within the host genome or targeted to a specific location. The technique of

zinc finger nucleases,[73][74] transcription activator-like effector nucleases (TALENs),[75][76] and the Cas9-guideRNA system (adapted from CRISPR).[77][78] TALEN and CRISPR are the two most commonly used and each has its own advantages.[79] TALENs have greater target specificity, while CRISPR is easier to design and more efficient.[79] In addition to enhancing gene targeting, engineered nucleases can be used to introduce mutations at endogenous genes that generate a gene knockout.[80][81]

Applications

Genetic engineering has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and microorganisms.

genes knocked out, increased susceptibility to disease, hormones for extra growth and the ability to express proteins in their milk.[86]

Medicine

Genetic engineering has many applications to medicine that include the manufacturing of drugs, creation of

Genetic engineering is also used to create animal models of human diseases. Genetically modified mice are the most common genetically engineered animal model.[91] They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease.[92] Potential cures can be tested against these mouse models.

Gene therapy is the

genetic engineering of humans, generally by replacing defective genes with effective ones. Clinical research using somatic gene therapy has been conducted with several diseases, including X-linked SCID,[93] chronic lymphocytic leukemia (CLL),[94][95] and Parkinson's disease.[96] In 2012, Alipogene tiparvovec became the first gene therapy treatment to be approved for clinical use.[97][98] In 2015 a virus was used to insert a healthy gene into the skin cells of a boy suffering from a rare skin disease, epidermolysis bullosa, in order to grow, and then graft healthy skin onto 80 percent of the boy's body which was affected by the illness.[99]

human embryos,[102][103] leading scientists of major world academies to call for a moratorium on inheritable human genome edits.[104] There are also concerns that the technology could be used not just for treatment, but for enhancement, modification or alteration of a human beings' appearance, adaptability, intelligence, character or behavior.[105] The distinction between cure and enhancement can also be difficult to establish.[106] 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. The work was widely condemned as unethical, dangerous, and premature.[107] Currently, germline modification is banned in 40 countries. Scientists that do this type of research will often let embryos grow for a few days without allowing it to develop into a baby.[108]

Researchers are altering the genome of pigs to induce the growth of human organs, with the aim of increasing the success of pig to human organ transplantation.[109] Scientists are creating "gene drives", changing the genomes of mosquitoes to make them immune to malaria, and then looking to spread the genetically altered mosquitoes throughout the mosquito population in the hopes of eliminating the disease.[110]

Research

Knockout mice
Human cells in which some proteins are fused with green fluorescent protein to allow them to be visualised

Genetic engineering is an important tool for

natural scientists, with the creation of transgenic organisms one of the most important tools for analysis of gene function.[111] 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.[112]

Organisms are genetically engineered to discover the functions of certain genes. This could be the effect on the phenotype of the organism, where the gene is expressed or what other genes it interacts with. These experiments generally involve loss of function, gain of function, tracking and expression.

Industrial