StemCell Therapy

recombinant DNA Summary

Recombinant DNA, a segment of DNA that is generated by combining genetic material from at least two different species. Such new genetic combinations are of value to science, medicine, agriculture, and industry. A fundamental goal of genetics is to isolate, characterize, and manipulate genes.

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The great gene editing debate: can it be safe and ethical?  BBC.com

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Anti-biotechnology campaigners embrace classic crops, are suspicious of hybrid varieties and claim genetic modification violates nature. Heres a primer on the differences  Genetic Literacy Project

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Will IL-11 Control Extend Human Life One Day? Early Results are Tantalizing  Securities.io

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Viewpoint: As New Zealand edges toward relaxing its ban on gene edited foods, experts weigh in  Genetic Literacy Project

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Farmers in Brazil and Argentina ramp up growing of genetically-modified drought tolerant wheat that can grow in subtropical regions  Genetic Literacy Project

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Scientist explains why we'll never have a real Jurassic Park - and people are crestfallen  indy100

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Methods used to change the DNA of organisms

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.

Early techniques randomly inserted the genes into the genome. Advances allow targeting specific locations, which reduces unintended side effects. Early techniques relied on meganucleases and zinc finger nucleases. Since 2009 more accurate and easier systems to implement have been developed. Transcription activator-like effector nucleases (TALENs) and the Cas9-guideRNA system (adapted from CRISPR) are the two most common.

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.[1]:1 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.[2]:32 Some plants were able to be propagated by vegetative cloning.[2]:31

Genetic inheritance was first discovered by Gregor Mendel in 1865, following experiments crossing peas.[3] In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which was identified as DNA in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty. Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers.

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.[4] DNA ligases, which join broken DNA together, were discovered earlier in 1967.[5] By combining the two enzymes it became possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952,[6] 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).[7] Transformation using electroporation was developed in the late 1980s, increasing the efficiency and bacterial range.[8] 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.[9] 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.[10]

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 -80C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research.[11]

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.[12]

Another option is reverse genetics. This approach involves targeting a specific gene with a mutation and then observing what phenotype develops.[12] 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.[13] 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.[12] The development of microarrays, transcriptomes and genome sequencing has made it much easier to find desirable genes.[14]

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.[15] 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 Monsanto RoundUp manufacturing facility.[16] In animals, the majority of genes used are growth hormone genes.[17]

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.[18]

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.[19]

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.[20] 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.[18]:4041 Another technique to isolate genes of known sequences involves polymerase chain reaction (PCR).[21] 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.[22] Some synthetic sequences are available commercially, forgoing many of these early steps.[23]

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.[24]

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.[25]

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.[26] 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 divalent cations (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.

In plants the DNA is often inserted using Agrobacterium-mediated recombination,[27] taking advantage of the Agrobacteriums T-DNA sequence that allows natural insertion of genetic material into plant cells.[28] 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.[29]

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.[30][31] 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.[32][33]

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.[34] 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.[citation needed]

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.[35] 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.[36] These synthetic vectors have the ability to bind DNA and accommodate large genetic transfers.[37] 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.[38] 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.[36] They form lipoplexes and polyplexes respectively, which are then up-taken by the cells. Other techniques include using electroporation and biolistics.[39] In some cases, transfected cells may stably integrate external DNA into their own genome, this process is known as stable transfection.[40]

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.[26] 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.[41] 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.[42]

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector.[43] Genetically modified viruses can be used as viral vectors to transfer target genes to another organism in gene therapy.[44] 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.

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.[47] In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells.[27] 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.[citation needed] 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.[48]

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.[49] 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.[50] 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).[51] 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.[52]

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.[52]

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.[53] There are four families of engineered nucleases: meganucleases,[54][55] ZFNs,[56][57] transcription activator-like effector nucleases (TALEN),[58][59] the CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPRassociated protein (e.g. CRISPR/Cas9).[60][61] Among the four types, TALEN and CRISPR/Cas are the two most commonly used.[62] 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).[63] Recent research has also focused on developing strategies to create gene knock-out or corrections without creating double stranded breaks (base editors).[62]

Meganucleases were first used in 1988 in mammalian cells.[64] 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.[65][53]

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.[53] 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.[65][66][53] 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.[65]

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).[67] 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.[62]

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.[62]

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.[68] 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.[69] 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.[70]

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Genetic engineering techniques - Wikipedia

Genetic engineering is the alteration of an organisms genotype using recombinant DNA technology to modify an organisms DNA to achieve desirable traits. The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering. The organism that receives the recombinant DNA is called a genetically modified organism (GMO). If the foreign DNA that is introduced comes from a different species, the host organism is called transgenic. Bacteria, plants, and animals have been genetically modified since the early 1970s for academic, medical, agricultural, and industrial purposes. In the US, GMOs such as Roundup-ready soybeans and borer-resistant corn are part of many common processed foods.

Although classical methods of studying the function of genes began with a given phenotype and determined the genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask: What does this gene or DNA element do? This technique, called reverse genetics, has resulted in reversing the classic genetic methodology. This method would be similar to damaging a body part to determine its function. An insect that loses a wing cannot fly, which means that the function of the wing is flight. The classical genetic method would compare insects that cannot fly with insects that can fly, and observe that the non-flying insects have lost wings. Similarly, mutating or deleting genes provides researchers with clues about gene function. The methods used to disable gene function are collectively called gene targeting. Gene targeting is the use of recombinant DNA vectors to alter the expression of a particular gene, either by introducing mutations in a gene, or by eliminating the expression of a certain gene by deleting a part or all of the gene sequence from the genome of an organism.

The process of testing for suspected genetic defects before administering treatment is called genetic diagnosis by genetic testing. Depending on the inheritance patterns of a disease-causing gene, family members are advised to undergo genetic testing. For example, women diagnosed with breast cancer are usually advised to have a biopsy so that the medical team can determine the genetic basis of cancer development. Treatment plans are based on the findings of genetic tests that determine the type of cancer. If the cancer is caused by inherited gene mutations, other female relatives are also advised to undergo genetic testing and periodic screening for breast cancer. Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.

Gene therapy is a genetic engineering technique used to cure disease. In its simplest form, it involves the introduction of a good gene at a random location in the genome to aid the cure of a disease that is caused by a mutated gene. The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA (Figure (PageIndex{1})). More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID).

Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response. Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen. The antigen is then introduced into the body to stimulate the primary immune response and trigger immune memory. Genes cloned from the influenza virus have been used to combat the constantly changing strains of this virus.

Antibiotics are a biotechnological product. They are naturally produced by microorganisms, such as fungi, to attain an advantage over bacterial populations. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells.

Recombinant DNA technology was used to produce large-scale quantities of human insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product. Currently, the vast majority of diabetes sufferers who inject insulin do so with insulin produced by bacteria.

Human growth hormone (HGH) is used to treat growth disorders in children. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector. Bacterial HGH can be used in humans to reduce symptoms of various growth disorders.

Although several recombinant proteins used in medicine are successfully produced in bacteria, some proteins require a eukaryotic animal host for proper processing. For this reason, the desired genes are cloned and expressed in animals, such as sheep, goats, chickens, and mice. Animals that have been modified to express recombinant DNA are called transgenic animals. Several human proteins are expressed in the milk of transgenic sheep and goats, and some are expressed in the eggs of chickens. Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.

Manipulating the DNA of plants (i.e., creating GMOs) has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life (Figure (PageIndex{3})). Plants are the most important source of food for the human population. Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established.

Plants that have received recombinant DNA from other species are called transgenic plants. Because they are not natural, transgenic plants and other GMOs are closely monitored by government agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life. Because foreign genes can spread to other species in the environment, extensive testing is required to ensure ecological stability. Staples like corn, potatoes, and tomatoes were the first crop plants to be genetically engineered.

Gene transfer occurs naturally between species in microbial populations. Many viruses that cause human diseases, such as cancer, act by incorporating their DNA into the human genome. In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by transfer of DNA from the bacterium to the plant. Although the tumors do not kill the plants, they make the plants stunted and more susceptible to harsh environmental conditions. Many plants, such as walnuts, grapes, nut trees, and beets, are affected by A. tumefaciens. The artificial introduction of DNA into plant cells is more challenging than in animal cells because of the thick plant cell wall.

Researchers used the natural transfer of DNA from Agrobacterium to a plant host to introduce DNA fragments of their choice into plant hosts. In nature, the disease-causing A. tumefaciens have a set of plasmids, called the Ti plasmids (tumor-inducing plasmids), that contain genes for the production of tumors in plants. DNA from the Ti plasmid integrates into the infected plant cells genome. Researchers manipulate the Ti plasmids to remove the tumor-causing genes and insert the desired DNA fragment for transfer into the plant genome. The Ti plasmids carry antibiotic resistance genes to aid selection and can be propagated in E. coli cells as well.

Bacillus thuringiensis (Bt) is a bacterium that produces protein crystals during sporulation that are toxic to many insect species that affect plants. Bt toxin has to be ingested by insects for the toxin to be activated. Insects that have eaten Bt toxin stop feeding on the plants within a few hours. After the toxin is activated in the intestines of the insects, death occurs within a couple of days. Modern biotechnology has allowed plants to encode their own crystal Bt toxin that acts against insects. The crystal toxin genes have been cloned from Bt and introduced into plants. Bt toxin has been found to be safe for the environment, non-toxic to humans and other mammals, and is approved for use by organic farmers as a natural insecticide.

The first GM crop to be introduced into the market was the Flavr Savr Tomato produced in 1994. Antisense RNA technology was used to slow down the process of softening and rotting caused by fungal infections, which led to increased shelf life of the GM tomatoes. Additional genetic modification improved the flavor of this tomato. The Flavr Savr tomato did not successfully stay in the market because of problems maintaining and shipping the crop.

Unless otherwise noted, images on this page are licensed under CC-BY 4.0 by OpenStax.

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Moen I, Jevne C, Kalland K-H, Chekenya M, Akslen LA, Sleire L, Enger P, Reed RK, Oyan AM, Stuhr LEB. 2012.Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation.BMC Cancer. 12:21. doi:10.1186/1471-2407-12-21 PDF

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20.3: Genetic Engineering - Biology LibreTexts

Most recombinant DNA technology involves the insertion of foreign genes into the plasmids of common laboratory strains of bacteria. Plasmids are small rings of DNA; they are not part of the bacteriums chromosome (the main repository of the organisms genetic information). Nonetheless, they are capable of directing protein synthesis, and, like chromosomal DNA, they are reproduced and passed on to the bacteriums progeny. Thus, by incorporating foreign DNA (for example, a mammalian gene) into a bacterium, researchers can obtain an almost limitless number of copies of the inserted gene. Furthermore, if the inserted gene is operative (i.e., if it directs protein synthesis), the modified bacterium will produce the protein specified by the foreign DNA.

A subsequent generation of genetic engineering techniques that emerged in the early 21st century centred on gene editing. Gene editing, based on a technology known as CRISPR-Cas9, allows researchers to customize a living organisms genetic sequence by making very specific changes to its DNA. Gene editing has a wide array of applications, being used for the genetic modification of crop plants and livestock and of laboratory model organisms (e.g., mice).

The correction of genetic errors associated with disease in animals suggests that gene editing has potential applications in gene therapy for humans. Gene therapy is the introduction of a normal gene into an individuals genome in order to repair a mutation that causes a genetic disease. When a normal gene is inserted into a mutant nucleus, it most likely will integrate into a chromosomal site different from the defective allele; although this may repair the mutation, a new mutation may result if the normal gene integrates into another functional gene. If the normal gene replaces the mutant allele, there is a chance that the transformed cells will proliferate and produce enough normal gene product for the entire body to be restored to the undiseased phenotype.

Genetic engineering has advanced the understanding of many theoretical and practical aspects of gene function and organization. Through recombinant DNA techniques, bacteria have been created that are capable of synthesizing human insulin, human growth hormone, alpha interferon, a hepatitis B vaccine, and other medically useful substances. Plants may be genetically adjusted to enable them to fix nitrogen, and genetic diseases can possibly be corrected by replacing dysfunctional genes with normally functioning genes.

Genes for toxins that kill insects have been introduced in several species of plants, including corn and cotton. Bacterial genes that confer resistance to herbicides also have been introduced into crop plants. Other attempts at the genetic engineering of plants have aimed at improving the nutritional value of the plant.

In 1980 the new microorganisms created by recombinant DNA research were deemed patentable, and in 1986 the U.S. Department of Agriculture approved the sale of the first living genetically altered organisma virus, used as a pseudorabies vaccine, from which a single gene had been cut. Since then several hundred patents have been awarded for genetically altered bacteria and plants. Patents on genetically engineered and genetically modified organisms, particularly crops and other foods, however, were a contentious issue, and they remained so into the first part of the 21st century.

Grains of golden rice, a genetically modified rice (Oryza sativa) that contains beta-carotene.(more)

Special concern has been focused on genetic engineering for fear that it might result in the introduction of unfavourable and possibly dangerous traits into microorganisms that were previously free of theme.g., resistance to antibiotics, production of toxins, or a tendency to cause disease. Indeed, possibilities for misuse of genetic engineering were vast. In particular, there was significant concern about genetically modified organisms, especially modified crops, and their impacts on human and environmental health. For example, genetic manipulation may potentially alter the allergenic properties of crops. In addition, whether some genetically modified crops, such as golden rice, deliver on the promise of improved health benefits was also unclear. The release of genetically modified mosquitoes and other modified organisms into the environment also raised concerns.

In the 21st century, significant progress in the development of gene-editing tools brought new urgency to long-standing discussions about the ethical and social implications surrounding the genetic engineering of humans. The application of gene editing in humans raised significant ethical concerns, particularly regarding its potential use to alter traits such as intelligence and beauty. More practically, some researchers attempted to use gene editing to alter genes in human sperm, which would enable the edited genes to be passed on to subsequent generations, while others sought to alter genes that increase the risk of certain types of cancer, with the aim of reducing cancer risk in offspring. The impacts of gene editing on human genetics, however, were unknown, and regulations to guide its use were largely lacking.

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Genetic engineering - DNA Modification, Cloning, Gene Splicing

Global Gene Editing Market Poised for Significant Growth, Projected to Reach $14.28 Billion by 2027  EIN News

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Global Gene Editing Market Poised for Significant Growth, Projected to Reach $14.28 Billion by 2027 - EIN News

Abstract

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

Keywords: CRISPR/Cas9, embryonic stem (ES) cells, genetic engineering, gene targeting, homologous recombination, microinjection, retroviruses, transgenic mice, transgenic rats, transposons, vectors

Since the identification of DNA as the unit of heredity and the basis for the central dogma of molecular biology [1] that DNA makes RNA and RNA makes proteins, scientists have pursued experiments and methods to understand how DNA controls heredity. With the discovery of molecular biology tools such as restriction enzymes, DNA sequencing, and DNA cloning, scientists quickly turned to experiments to change chromosomal DNA in cells and animals. In that regard, initial experiments that involved the co-incubation of viral DNA with cultured cell lines progressed to the use of selectable markers in plasmids. Delivery methods for random DNA integration have progressed from transfection by physical co-incubation of DNA with cultured cells, to electroporation and microinjection of cultured cells [2,3,4]. Moreover, the use of viruses to deliver DNA to cultured cells has progressed in tandem with physical methods of supplying DNA to cells [5,6,7]. Homologous recombination in animal cells [8] was rapidly exploited by the mouse genetics research community for the production of gene-modified mouse ES cells, and thus gene-modified whole animals [9,10].

This impetus to understand gene function in intact animals was ultimately manifested in the international knockout mouse project, the purpose of which was to knock out every gene in the mouse genome, such that researchers could choose to make knockout mouse models from a library of gene-targeted knockout ES cells [11,12,13]. Thousands of mouse models have resulted from that effort and have been used to better understand gene function and the bases of human genetic diseases [14]. This project required high-throughput pipelines for the construction of vectors, including bacterial artificial chromosome (BAC) recombineering technology [13,15,16,17]. BACs contain long segments of cloned genomic DNA. For example, the C57BL/6J mouse BAC library, RPCI-23, has an average insert size of 197 kb of genomic DNA per clone [18]. Because of their size, BACs often carry all of the genetic regulatory elements to faithfully recapitulate the expression of genes contained in them, and thus can be used to generate BAC transgenic mice [19,20]. Recombineering can be used to insert reporters in BACs that are then used to generate transgenic mice to accurately label cells and tissues according to the genes in the BACs [21,22,23,24,25,26]. A panoply of approaches to genetic engineering are available for researchers to manipulate the genome. ES cell and BAC transgene engineering have given way to directly editing genes in zygotes, consequently avoiding the need for ES cell or BAC intermediates on the way to an animal model.

Prior to the adaptation of Streptococcus pyogenes Cas9 protein to cause chromosome breaks, three other endonuclease systems were used: (1) rare-cutting meganucleases, (2) zinc finger nucleases (ZFNs), and (3) transcription activator-like effector (TALE) nucleases (TALENs) [27]. The I-CreI meganuclease recognizes a 22 bp DNA sequence [28,29]. Proof-of-concept experiments demonstrated that the engineered homing endonuclease I-CreI can be used to generate transgenic mice and transgenic rats [30]. I-CreI specificity can be adjusted to target specific sequences in DNA by protein engineering methodology, although this limits its widespread application to genetic engineering [31]. Subsequently, ZFN technology was developed to cause chromosome breaks [32]. A single zinc finger is made up of 30 amino acids that bind three base pairs. Thus, three zinc fingers can be combined to specifically recognize nine base pairs on one DNA strand and a triplet of zinc fingers is made to bind nine base pairs on the opposite strand. Each zinc finger is fused to the DNA-cutting domain of the FokI restriction endonuclease. Because FokI domains only cut DNA when they are present as dimers, a ZFN monomer binding to a chromosome cannot induce a DNA break [32], instead requiring ZFN heterodimers for sequence-specific chromosome breaks. It is estimated that 1 in every 500 genomic base pairs can be cleaved by ZNFs [33]. Compared with meganucleases, ZFNs are easier to construct because of publicly available resources [34]. Additionally, the value of ZFNs in mouse and rat genome engineering was demonstrated in several studies that produced knockout, knockin, and floxed (described below) animal models [35,36,37]. The development of transcription activator-like effector nucleases (TALENs) followed after ZFN technology [38]. TALENs are made up of tandem repeats of 34 amino acids. The central amino acids at positions 12 and 13, named repeat variable di-residues (NVDs), determine the base to which the repeat will bind [38]. To achieve a specific chromosomal break, 15 TALE repeats assembled and fused to the FokI endonuclease domain (TALEN monomer) are required. Thus, one TALEN monomer binds to 15 base pairs on one DNA strand, and a second TALEN monomer binds to bases on the opposite strand [38]. When the FokI endonuclease domains are brought together, a double-stranded DNA break occurs. In this way, a TALEN heterodimer can be used to cause a sequence-specific chromosome break. It has been estimated that, within the entire genome, TALENs have potential target cleavage sites every 35 bp [39]. Compared with ZFNs, TALENs are easier to construct with publicly available resources [40,41], and TALENs have been adopted for use in mouse and rat genome engineering in several laboratories that have produced knockout and knockin animal models [42,43,44,45,46].

The efficiencies of producing specific double-strand chromosome breaks, using prior technologies such as meganucleases, ZFNs, and TALENs [28,32,38], were surpassed when CRISPR/Cas9 technology was shown to be effective in mammalian cells [47,48,49]. The essential feature that all of these technologies have in common is the production of a chromosome break at a specific location to facilitate genetic modifications [50]. In particular, the discovery of bacterial CRISPR-mediated adaptive immunity, and its application to genetic modification of human and mouse cells in 2013 [47,48,49], was a watershed event to modern science. Moreover, the introduction of CRISPR/Cas9 methodology has revolutionized transgenic mouse generation. This paradigm shift can be seen by changes in demand for nucleic acid microinjections into zygotes, and ES cell microinjections into blastocysts at the University of Michigan Transgenic Core (). While previously established principles of genetic engineering using mouse ES cell technology [51,52,53] remain applicable, CRISPR/Cas9 methodologies have made it much easier to produce genetically engineered model organisms in mice, rats, and other species [54,55]. Herein, we discuss principles in genetic engineering for the design and characterization of targeted alleles in mouse and rat zygotes, or in cultured cell lines, for the production of animal and cell culture models for biomedical research.

Recent trends in nucleic acid microinjection in zygotes, and embryonic stem (ES) cell microinjections into blastocysts, for the production of genetically engineered mice at the University of Michigan Transgenic Core. As shown, prior to the introduction of CRISPR/Cas9, the majority of injections were of ES cells, to produce gene-targeted mice, and DNA transgenes, to produce transgenic mice. After CRISPR/Cas9 became available, adoption was slow until 2014, when it was enthusiastically embraced, and the new technology corresponded to a reduced demand for ES cell and DNA microinjections.

There are many types of genetic modifications that can be made to the genome. The ability to specifically target locations in the genome has expanded our ability to make changes that include knockouts (DNA sequence deletions), knockins (DNA sequence insertions), and replacements (replacement of DNA sequences with exogenous sequences). Deletions in the genome can be used to knockout gene expression [56,57]. Short deletions in the genome can be used to remove regulatory elements that knockout gene expression [58], activate gene expression [59], or change protein structure/function by changing coding sequences [60].

Insertion of new genomic information can be used to knock in a variety of genetic elements. Knockins are also powerful approaches for modifying genes. Just as genomic deletions can be used to change gene function, knockins can be used to block gene function by inserting fluorescent reporter genes such as eGFP or mCherry, in such a way as to knock out the gene at the insertion point [61,62]. It is also possible to knock in fluorescent protein reporter genes, without knocking out the targeted gene [63,64]. Just as fluorescent proteins can be used to label proteins and cells, short knockins of epitope tags in proteins can be used to label proteins for detection with antibodies [64,65].

Replacement of DNA sequences in the genome can be used to achieve two purposes at the same time, such as blocking gene function, while activating the function of a new gene such as the lacZ reporter [66]. Large-scale sequence replacements are possible with mouse ES cell technology, such as the replacement of the mouse immunoglobulin locus with the human immunoglobulin locus to produce a humanized mouse [67]. Furthermore, very small replacements of single nucleotides can be used to model point mutations that are suspected of causing human disease [68,69,70].

A special type of DNA sequence replacement is the conditional allele. Conditional alleles permit normal gene expression until the site-specific Cre recombinase removes a loxP-flanked critical exon to produce a floxed (flanked by loxP) exon. Cre recombinase recognizes 34 bp loxP (locus of recombination) elements, and catalyzes recombination between the two loxP sites [71,72]. Therefore, deletion of the critical exon causes a premature termination codon to occur in the mRNA transcript, triggering its nonsense-mediated decay and failure to make a protein [13,73]. Engineering conditional alleles was the approach used by the international knockout mouse project [13]. Mice with cell- and tissue-specific Cre recombinase expression are an important resource for the research community [74].

Other site-specific recombinases, such as FLP, Dre, and Vika, that work on the same principle have also been applied to mouse models [75,76,77,78,79,80]. Recombinase knockins can be designed to knock out the endogenous gene or preserve its function [81,82]. A variation in the conditional allele is the inducible allele, which is silent until its expression is activated by Cre recombinase [79]. For example, reporter models can activate the expression of a fluorescent protein [83], change fluorescent reporter protein colors from red to green [84], or use a combinatorial approach to produce up to 90 fluorescent colors [85]. Another type of inducible allele is the FLEX allele. FLEX genes are Cre-dependent gene switches based on the use of heterotypic loxP sites [86]. In one application that combined Cre and FLP recombinases, it was demonstrated that a gene inactivated in ES cells by a gene trap could be switched back on and then switched off again [87]. In another application of heterotypic loxP sites in mouse ES cells, it was demonstrated that genes could be made conditional by inversion (COIN) [88]. This application has been used to produce mice with conditional genes for point mutations [89] and has been applied to produce conditional single exon genes that lack critical exons by definition [90].

The central principle of gene targeting with CRISPR/Cas9, or other directed DNA endonucleases, is that a double-strand DNA break is generated in the cell of interest. Following a chromosomal break, the principal outcomes of interest are nonhomologous end joining (NHEJ) repair [91] or homology-directed repair (HDR) [92]. When the break is directed to a coding exon in a gene, the outcome of NHEJ is usually a small insertion or deletion of DNA sequence at the break (indel), causing frame shifts in mRNA transcripts that lead to premature termination codons, causing nonsense-mediated mRNA decay and loss of protein expression [73]. The HDR pathway copies a template during DNA repair, and thus the insertion of modified genetic sequences in the form of a DNA donor. This DNA donor can introduce new information into the genome flanked by homology arms on either side of the chromosome break. Typical applications of HDR include the use of genetic engineering to abrogate gene expression (gene knockouts), to modify amino acid codons (i.e.; point mutations), to replace genes with new genes (e.g.; knockins of fluorescent reporters, Cre recombinase, cDNA coding sequences), to produce conditional genes (floxed genes that are normally expressed until they are inactivated by Cre recombinase), to produce Cre-inducible genes (genes that are only expressed after Cre recombinase activates them), and to delete DNA from chromosomes (e.g.; delete regulatory elements that control gene expression, delete entire genes, or delete up to a megabase of chromosome segments). The simplest of these modifications is abrogation of gene expression. Multifunctional alleles, such as FLEX alleles, require the cloning or synthesis of multi-element plasmid DNA donors for HDR.

The processes of CRISPR/Cas9-mediated modifications of genes (gene editing) to produce a new cell line or animal model have in common a series of steps to achieve the final product. First, a gene of interest is identified and the final desired allele is specified. The next step is to identify single guide RNA(s) (gRNAs) that will be used to target a chromosomal break in one or more places. There are numerous online websites that can be used for this purpose [93]. One of the most up-to-date and versatile sites is CRISPOR (http://crispor.tefor.net) [94]. Interestingly, the authors provide evidence that the predictive powers of algorithms vary depending on whether they were based on the analysis of gRNAs delivered as RNA molecules, versus gRNAs delivered as U6-transcribed DNA molecules [94]. In any event, the selection of a gRNA target (20 nucleotides), adjacent to a protospacer-adjacent motif (PAM; NGG motif), should not be done without the aid of a computer algorithm that minimizes the possibility of off-target hits. After a gRNA target is identified, a decision is made to obtain gRNAs. While it is possible to produce in vitro-transcribed gRNAs, this may be inadvisable in so much as in vitro-transcribed RNAs can trigger innate immune responses and cause cytotoxicity in cells [95]. Chemically synthesized gRNAs using phosphorothioate modifications that improve gRNA stability may be preferable alternatives to in vitro-transcribed molecules [96,97]. With a gRNA in hand, a Cas9 protein is then selected. There are numerous forms of Cas9 that can be used for different purposes [98]. For practical purposes, we limit our discussion to Cas9 varieties that are on the market. A number of commercial entities sell wild-type Cas9 protein. When wild type Cas9 is used to target the genome with nonspecific guides, the frequency of off-target genomic hits, besides the desired Cas9 target, is very likely to increase [94,99]. Alternatives to the wild-type protein include enhanced specificity Cas9 from Sigma-Aldrich [100], and high-fidelity Cas9 from Integrated DNA Technologies [101]. In addition, there are other versions such as HF1 Cas9 [102], hyperaccurate Cas9 [103], and evolved Cas9 [104], all available in plasmid format from Addgene.org. As may be inferred from the names of these engineered Cas9 versions, they are designed to be more specific than wild type Cas9. Once the gRNAs and Cas9 protein are on hand, then it is a simple matter to combine them and deliver them to the target cell to produce a chromosome break and achieve a gene knockout by introducing premature termination codons or DNA sequence deletion of regulatory regions or entire genes.

The most challenging type of genetic engineering is the insertion (i.e.; knockin) of a long coding sequence to express a fluorescent reporter protein, Cre recombinase, or conditional allele (floxed gene). In addition to these genetic modifications, numerous other types of specialized reporters can be introduced, each designed to achieve a different purpose. There is great interest in achieving rapid and efficient gene insertions of reporters in animal models with CRISPR/Cas9 technology. It is generally recognized that, the longer the insertion, the less efficient it is to produce a knockin animal. Additional challenges are allele-specific differences that affect efficiency. For example, it is fairly efficient to produce knockins into the genomic ROSA26 locus in mice, while other loci are targeted less efficiently, and thus refractory to knockins. This accessibility to CRISPR/Cas9 complexes mirrors observations in mouse ES cell gene targeting technology, in which it was reported that some genes are not as efficiently targeted as others [105].

When the purpose of the experiment is to specifically modify the DNA sequence by changing amino acid codons, or introducing new genetic information, then a DNA donor must be delivered to the cells with Cas9 reagents. After the selected gRNAs and Cas9 proteins are demonstrated to produce the desired chromosome break, the DNA donor is designed and procured. The donor should be designed to insert into the genome such that it will not be cleaved by Cas9, usually by mutating the PAM site. The DNA donor may take the form of short oligonucleotides (<200 nt) [106,107], long single-stranded DNA molecules (>200 nt) [108], or double-stranded linear or circular DNA molecules of varying lengths [109,110].

DNA donor design principles should include the following: (1) nucleotide changes that prevent CRISPR/Cas9 cleavage of the chromosome, after introduction of the DNA donor; (2) insertion of restriction enzyme sites unique to the donor, to simplify downstream genotyping; (3) insertions of reporters or coding sequences, at least 1.5 kb in length, that can be introduced as long single-stranded DNA templates with short 100 base pair arms of homology [111], or as circular double-stranded DNA plasmids with longer (1.5 or 2 kb) arms of homology [63,110]; and (4) insertions of longer coding sequences, such as Cas9, that use circular double-stranded DNA donors with longer arms of homology [63,112]. It is also possible to use linear DNA fragments as donors [63,110,113], although random integration of linear DNA molecules is much higher than those of circular donors, thus requiring careful quality control.

The establishment of genetically modified mouse and rat models can be divided into three phases, after potential founder animals are born from CRISPR/Cas9-treated zygotes. In the first phase, animals with genetic modifications are identified. The first phase requires a sensitive and specific genotyping assay to identify cells or animals harboring the desired knockin. Genotyping potential founder mice for knockins typically begins with a PCR assay using a primer that recognizes the exogenous DNA sequence and a primer in genomic DNA outside of the homology arm in the targeting vector. Accordingly, PCR assays are designed to specifically detect the upstream and downstream junctions of the inserted DNA in genomic DNA. Subsequent assays may be used to confirm that the entire exogenous sequence is intact. Conditional genes represent a special case of insertion, as PCR assays designed to detect correct insertion of loxP-flanked exons will also detect genomic DNA [108]. In the second phase, founders are mated and G1 pups are identified that inherited the desired mutation [114]. In the third phase, it is essential to sequence additional genomic regions upstream and downstream of the inserted targeting vector DNA, because Cas9 is very efficient at inducing chromosomal breaks, but has no repair function. Thus, it is not unusual to identify deletions/insertions that flank the immediate vicinity of the Cas9 cut site or inserted targeting vector DNA sequences [115,116]. If such deletions affect nearby exons, gene expression can be disrupted, and confounding phenotypes may arise.

For gene knockouts, PCR amplicons from primers that span the chromosome break site are analyzed by DNA sequencing. Any animals that are wild-type at the allele are not further characterized or used, so as to prevent any off-target hits from entering the animal colony or confounding phenotypes. Animals that show disrupted DNA sequences at the Cas9 cut site are mated with wild-type animals for the transmission of mutant alleles that produce premature termination codons, for gene knockout models [57,73]. As founders from Cas9-treated zygotes are genetic mosaics [55,115], it is essential to mate them to wild-type breeding partners, such that obligate heterozygotes are produced. In the heterozygotes, the wild-type sequence and the mutant sequence can be precisely identified by techniques such as TOPO TA cloning (Invitrogen, CA, USA) or next-generation sequencing (NGS) methods [117,118,119,120]. Animals carrying a defined indel, with the desired properties, are then used to establish lines for phenotyping. The identical approach is used when short DNA sequences are deleted by two guide RNAs [58]. Intercrossing mosaic founders will produce offspring carrying two different mutations with different effects on gene expression. These animals are not suitable for line establishment.

CRISPR/Cas9 gene editing in immortalized cell lines presents a set of challenges unique from those used in the generation of transgenic animals. Cell lines encompass a wide range of characteristics, resulting in each line being handled differently. Some of these characteristics include phenotype heterogeneity, aberrant chromosome ploidy, varying growth rates, DNA damage response efficiency, transfection efficiency, and clonability. While the principles of CRISPR/Cas9 experimental design, as stated above, remain the same, three major considerations must be taken into account when using cell lines: (1) copy number variation, or the number of alleles of the gene of interest; (2) transfection efficiency of the cell line; and (3) clonal isolation of the modified cell line. In cell lines, all alleles need to be modified in the generation of a null phenotype, or in the creation of a homozygous genotype. Unlike transgenic animals, where single allele gene edits can be bred to homozygosity, CRISPR/Cas9-edited cells must be screened for homozygous gene edits. Copy number variations within the cell line can decrease the efficiency and add labor and time (i.e.; editing 3 or 4 copies versus editing 1 or 2). Furthermore, an aberrant number of chromosomes, deletions, duplications, pseudogenes, and repetitive regions complicate genetic backgrounds for PCR analysis of the CRISPR edits. To help with some of these issues, one common approach is to use NGS on all the clonal isolates for a complete understanding of copy number variations for each clonal cell line generated, and the exact sequence for each allele.

As all cell types are not the same, different CRISPR/Cas9 delivery techniques may need to be tested to identify which method works best. One approach is to use viruses or transposons to deliver CRISPR/Cas9 reagents (detailed below). However, the viruses and transposons themselves will integrate into the genome, as well as allowing long-term expression of CRISPR/Cas9 in the cell. This prolonged expression of gRNAs and Cas9 protein may lead to off-target effects. Moreover, transfection and electroporation can have varying efficiencies, depending on the cell lines and the form of CRISPR/Cas9 reagents (e.g.; DNA plasmids or ribonucleoprotein particles (RNPs)).

Following delivery, clonal isolation is required to identify the edited cell line, and at times, can result in the isolation of a cell phenotype different than that expected, arising from events apart from the desired gene edit. While flow cytometry can aid in isolating individual cells, specific flow conditions, such as pressure, may require adjustment to ensure cell viability. Furthermore, one clonal isolate from a cell line may possess a different number of alleles for the targeted gene than another clonal isolate. Additionally, not all cell lines will grow from a single cell, thus complicating isolation. Growth conditions and cell viability can also change when isolating single cells.

Despite these challenges, new advances in CRISPR technology can likely alleviate some of these difficulties when editing cell lines. For example, fluorescently tagged Cas9 and RNAs help to isolate only transfected cells, which helps to eliminate time wasted on screening untransfected cells. Cas9-variants that harbor mutations that only create single-strand nicks (Cas9-nickases) complexed with two different, but proximal gRNAs can increase HDR-mediated knockin [48,121]. Similarly, fusing Cas9 with base-editing enzymes can also increase the efficiency of editing, without causing double-strand breaks [121].

Viral and transposon vectors have been engineered to be safe, efficient delivery systems of exogenous genetic material into cells. The natural lifecycle of some viruses and transposons includes the stable integration into the host genome. In the field of genome engineering, these vectors can be used to modify the genome in a non-directed fashion, by inserting cassettes expressing any cDNA, shRNA, miRNA, or any non-coding RNA. The most widely used vectors capable of integrating ectopic genetic material into cells are retroviruses, lentiviruses, and adeno-associated virus (AAV). These viruses are flanked by terminal repeats that mark the boundaries of the integration. In engineering these viruses into recombinant vector systems, all the viral genes are removed from the flanking terminal repeats and supplied in trans for the recombinant virus to be packaged. These gutted, nonreplicable viral vectors allow for the packaging, delivery, integration, and expression of cDNAs of interest, shRNAs, and CRISPR/Cas9, without viral replication in various biological targets.

Similar to recombinant viruses, transposon vectors are also gutted, separating the transposase from the terminal repeat-flanked genetic material to be inserted into the genome. DNA transposons are mobile elements (jumping genes) that integrate into the host genome through a cut-and-paste mechanism [122]. Transposons, much like viral vectors, are flanked by repeats that mark the region to be transposed [123]. The enzyme transposase binds the flanking DNA repeats and mediates the excision and integration into the genome. Unlike viral vectors, transposons are not packaged into viral particles, but form a DNA-protein complex that stays in the host cell. Thus, the transgene to be integrated can be much larger than the packaging limits of some viruses.

Two transposons, Sleeping Beauty (SB) and piggybac (PB), have been engineered and optimized for high activity for generating transgenic mammalian cell lines [124,125,126]. Sleeping Beauty is a transposable element resurrected from fish genomes. The SB system has been used to generate transgenic HeLa cell lines, T-cells expressing chimeric antigen receptors that recognize tumor-specific antigens, and transgenic primary human stem cells [127,128,129]. The insect-derived PB system also has been used to generate transgenic cell lines [126,130,131]. The PB system was used to generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts, by linking four or five cDNAs of the reprogramming (Yamanaka) factors [132] with intervening peptide self-cleavage (P2A) sites, thus delivering all of the factors in one vector [130]. Furthermore, once reprogrammed, the transgene may be removed by another round of PB transposase activity, leaving no genetic trace of integration or excision (i.e.; transgene-free iPSCs). Following PB transposase activity, epigenetic differences remaining at the endogenous promoters of the reprogramming factor genes result in sustained expression and pluripotency, despite transgene removal.

Aside from transgene insertion, Sleeping Beauty (SB) and piggyback (PB) have both been engineered to deliver CRISPR/Cas9 reagents into cells [133,134,135]. Similar to lentivirus, the stable integration of CRISPR/Cas9 by transposons could increase the efficacy of targeting and modifying multiple alleles. SB and PB have been used to deliver multiple gRNAs to target multiple genes (instead of just one), aiding in high-throughput screening. Furthermore, owing to the nature of PB excision stated above, the integrated CRISPR/Cas9 can be removed once a clonal cell line is established, to limit off-target effects. However, engineered transposons must be transfected into cells. As stated above, efficiencies vary between different cell lines and transfection methods. One potential solution to overcome this challenge is to merge technologies. For example, instead of transfecting cells with a plasmid harboring a gRNA flanked by SB terminal repeats (SB-CRISPR), the SB-CRISPR may be flanked by recombinant AAV (rAAV) terminal repeats (AAV-SB-CRISPR), allowing for packaging into rAAV. To that end, rAAV-SB-CRISPR has been used to infect primary murine T-cells, and deliver the SB-CRISPR construct [136].

Retroviruses are RNA viruses that replicate through a DNA intermediate [137]. They belong to a large family of viruses including both onco-retroviruses, such as the Moloney murine leukemia virus (MMLV) (simply referred to as retrovirus), and lentiviruses, including human immunodeficiency virus (HIV). In all retroviruses, the RNA genome is flanked on both sides by long terminal repeats (LTRs); packaged with viral reverse transcriptase, integrase, and protease, surrounded by a protein capsid; and then enveloped into a lipid-based particle [138]. Envelope proteins interact with specific host cell surface receptors to mediate entry into host cells through membrane fusion. Then, the RNA genome is reverse-transcribed by the associated viral reverse transcriptase. The proviral DNA is then transported into the nucleus, along with viral integrase, resulting in integration into the host cell genome [139]. By contrast, the retroviral MMLV pre-integration complex is incapable of crossing the nuclear membrane, thus requiring the cell to undergo mitosis to gain access to chromatin [139], while lentiviral pre-integration complexes can cross nuclear membrane pores, allowing genome integration in both dividing and non-dividing cells.

Large-scale assessments of genomic material composition have uncovered features associated with retroviral insertion into mammalian genomes [140]. Although determination of integration target sites remains ill-defined, it does depend on both cellular and viral factors. For retroviruses such as MMLV, integration is preferentially targeted to promoter and regulatory regions [140,141,142]. Such preferences can be genotoxic owing to insertional activation of proto-oncogenes in patients undergoing gene therapy treatments for X-linked severe combined immunodeficiency [143,144], WiskottAldrich syndrome [143], and chronic granulomatous disease [145]. Likewise, retroviral integration can generate chimeric and read-through transcripts driven by strong retroviral LTR promoters, post-transcriptional deregulation of endogenous gene expression by introducing retroviral splice sites (leading to aberrant splicing), and retroviral polyadenylation signals that lead to premature termination of endogenous transcripts [142,146,147].

Unlike retroviruses, lentiviruses prefer to integrate into transcribed portions of expressed genes in gene-rich regions, distanced from promoters and regulatory elements [140,142,148]. The cellular protein LEDGF/p75 aids in the target site selection by binding directly to both the active gene and the viral integrase within the HIV pre-integration complex [149]. Although the propensity of lentivirus to integrate into the body of expressed genes should increase the incidence of post-transcriptional deregulation, deletion of promoter elements from the lentiviral LTR (self-inactivating (SIN) vectors) has been reported to decrease transcriptional termination, but increase the generation of chimeric transcripts [149]. Overall, it appears that lentiviral SIN vectors are less likely to cause tumors than retroviral vectors with an active LTR promoter [148,150,151,152].

The 7.510 kb packaging limit of lentiviruses can accommodate the packaging, delivery, and stable integration of Cas9 cDNA, gRNAs, or Cas9 and gRNAs (all-in-one) to cells [153,154]. Often, a selectable marker, such as drug resistance, can also be included to isolate transduced cells. The high transduction efficiency of lentivirus can result in an abundance of CRISPR/Cas9-expressing cells to screen, compared with more traditional transfection methods. Stable and prolonged expression of CRISPR/Cas9 can facilitate targeting of multiple alleles of the gene of interest, resulting in more cells harboring homozygous gene modifications. Conversely, stable integration of CRISPR/Cas9 increases potential off-target effects. Moreover, lentiviral integration itself is a factor that may confound cellular phenotypes and should be considered when characterizing CRISPR-edited cell lines.

Adeno-associated virus (AAV) is a human parvovirus with a single-stranded DNA genome of 4.7 kb, which was originally identified as a contaminant of adenoviral preparations [155]. The genome is flanked on both sides by inverted terminal repeats (ITR) and contains two genes, rep and cap [156,157]. Different capsid proteins confer serotype and tissue-specific targeting of distinct AAVs, in vivo. AAV cannot replicate on its own, and requires a helper virus, such as adenovirus or herpes simplex virus (HSV), to provide essential proteins in trans. AAV is the only known virus to integrate into the human genome in a site-specific manner at the AAVS1 site on chromosome 19q13.3-qter [158,159,160]. Although the precise mechanism is not well understood, the Rep protein functions to tether the virus to the host genome through direct binding of the AAV ITR and the AAVS1 site [158,160,161]. In the recombinant AAV (rAAV) vector system, the rep and cap genes are removed from the packaged virus, resulting in the loss of site-specific integration into the AAVS1 site. Despite removal of Rep, it has been shown that rAAV can still integrate, albeit randomly, into the host genome, via nonhomologous recombination, at low frequencies [162,163,164]. Furthermore, numerous clinical trials, to date, have shown that rAAV integration is safe and has no genotoxicity [165,166,167]. However, this safety is controversial, owing to preclinical studies suggesting genotoxicity in mouse models [168,169,170,171]. More studies are needed to understand the cellular impact of rAAV integration.

rAAVs have been used to deliver one or two CRISPR guide RNAs (gRNAs), in cells and model animals, by taking advantage of different rAAV serotypes to target specific cells or tissue types. Owing to the packaging capacity of rAAV, SpCas9 must be delivered as a separate virus, unlike lentivirus, which can be delivered as an all-in-one CRISPR/Cas9 vector. However, alternate, smaller Cas9s can be packaged into rAAVs [172]. Furthermore, rAAVs can be used to deliver repair templates or single-stranded donor oligonucleotides (ssODNs) for homology-directed repair (HDR), relying on the single-stranded nature of the AAV genome [173,174]. It has also been observed that rAAVs can integrate into the genome at CRISPR/Cas9-induced breaks in various cultured mouse tissue types, including neurons and muscle [175]. This observation goes against the notion of rAAVs integrating only at the AAVS1 locus, and should be considered when analyzing and characterizing rAAV-mediated CRISPR-edited cells.

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Principles of Genetic Engineering - PMC - National Center for ...

Quitting: A Life Strategy: The Myth of Perseveranceand How the New Science of Giving Up Can Set You Free  Next Big Idea Club Magazine

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Quitting: A Life Strategy: The Myth of Perseveranceand How the New Science of Giving Up Can Set You Free - Next Big Idea Club Magazine

Melissa Nolan

By the end of this chapter, students should be able to:

Those beautiful blue eyes you inherited from your mother are actually a result of a complex science known as Genetics. The scientific field of genetics studies genes in our DNA. Genes are units of heredity transferred from a parent to offspring and determine some characteristic of offspring. Your genes are responsible for coding all of your traits- including hair color, eye color, and so on. In recent years, scientists began exploring the concept of gene editing, which is the deliberate manipulation of genetic material to achieve desired results. Gene editing can potentially alter any given trait in an organism- from height to hair texture to susceptibility for certain diseases.

Gene editing applied to humans is referred to as Human Genetic Engineering, or HGE. There is extensive debate in and out of the scientific community regarding the ethics of HGE. Much of this debate stems from how this technology will affect society, and vice versa. Individuals may harbor concerns about the rise of designer babies or scientists playing God by determining the traits of an individual. On the contrary, HGE presents potential cures to diseases caused by genetic mutations. Human Genetic Engineering (HGE) is a novel technology which presents various ethical concerns and potential consequences. HGE should be approached cautiously and with extensive governmental regulation given its history, its current state, and the potential it has to change the world in the future.

Genetic Encoding of Proteins by MIT OpenCourseWare is licensed under CC BY-NC-SA 2.0

HGE utilizes CRISPR/Cas9 gene editing tools to cut out specific genes and replace them with a newly designed gene.

HGE encompasses a variety of methods which all work to produce a deliberate change in the human genome. The most common and prevalent way to edit the human genome is via CRISPR/Cas9. CRISPR stands for clustered regularly interspaced short palindromic repeats, and Cas9 is a protein that functions as scissors to cut DNA/genes. The CRISPR/Cas9 system originally developed as a part of a bacterias immune system, which can recognize repeats in DNA of invading viruses, then cut them out. Since then, scientists have harnessed the CRISPR/Cas9 system to cut DNA sequences of their choice and then insert new DNA sequences in their place.

The CRISPR/Cas9 system allows for designer genomes, and rapid engineering of any cells programming. With the use of CRISPR/Cas9, scientists can cut out certain traits from an individuals cells and insert new traits into those same cells.

CRISPR Cas9 System by Marius Walter is licensed under CC-BY-SA-4.0

Gene therapy is a recently-developed technology which can be applied to both somatic and germline genome editing.

Gene therapy concepts were initially introduced in the 1960s, utilizing outdated methods, such as recombinant DNA technology and viral vectors, to edit microorganisms genomes. Recombinant DNA consists of genetic material from multiple sources. The first experiments involved transferring a genome from one bacteria to another via a viral vector. Soon after was the first successful transformation of human cells with foreign DNA. The success of the experiment prompted public concern over the ethics of gene therapy, and led to political regulation. In the gene therapy report of the Presidents Commission in the United States, germline genome editing was deemed problematic over somatic genome editing. Also, non-medical genome editing was deemed problematic over medical genome editing. Germline genome editing occurs when scientists alter the genome of an embryo, so that the entire organism has altered genes and the traits can be passed to offspring. Somatic genome editing involves editing only a few cells in the entire organism so that traits can not be passed down to offspring. In response to the report, the rDNA Advisory Committee of the National Institutes of Health was formed and proposed the first guidelines for the gene therapy clinical trials. This is an example of technological determinism, in which technology determines the development of its social structure and cultural values or regulations.

In the past few decades, gene editing has advanced exponentially, introducing state-of-the-art technologies such as the CRISPR/Cas9 system, which was developed to induce gene modifications at very specific target sites. Thus, gene editing became a major focus for medical research (Tamura, 2020). Gene editing has led to the potential for development of treatment strategies for a variety of diseases and cancers. So far, somatic genome editing has shown promise in treating leukemia, melanoma, and a variety of other diseases. In this way, HGE may be demonstrative of cultural determinism, in which the culture we are raised presents certain issues which necessitate the development of a specific technology.

DNA CRISPR Scissors by Max Pixel is licensed under CC0 1.0

CRISPR/Cas9 is the primary technology proposed for use in HGE. HGE presents a variety of pros and cons to society.

Somatic genome editing in HGE via the CRISPR/Cas9 system has proven to be effective at editing specific genome sites. Since 2015, genome editing technologies have been used in over 30 human clinical trials and have shown positive patient outcomes. The treatment of disease may be a positive benefit of HGE, but there are also various potential risks. Various forms of deliberative democracies formed in recent years to address scientific and ethical concerns in HGE. Deliberative democracies afrm the need to justify technological decisions made by citizens and their representatives with experts in the field via deliberation. Overall, the consensus remains that the pros and cons of HGE are not equivalent enough to justify widespread use of the technology.

Current human clinical trials show successful transformation of human immune cells to HIV-resistant cells. This implies that HGE may be the cure for HIV(Hu, 2019). Other successful somatic genome editing trials treated myeloma, leukemia, sickle cell disease, various forms of epithelial cancers, and hemophilia. Thus, gene editing has provided novel treatment options for congenital diseases and cancers (Tamaura, 2020). Congenital diseases are those present from birth, and typically have a genetic cause. For these reasons, scientific summits concluded HGE is ethical for research regarding somatic genome editing in congenital diseases and cancers.

There are many safety concerns regarding CRISPR applications, mainly in germline genome editing. As a result of technological determinism, a leading group of CRISPR/Cas9 scientists and ethicists met for the international Summit on Human Gene Editing. The summit determined that heritable genome research trials may be permitted only following extensive research on risks and benefits of HGE. However, the summit concluded that federal funding cannot be used to support research involving human embryos with germline editing techniques. These decisions were made to avoid potential risks such as the following.

The major concerns regarding germline genome editing in HGE include: serious injury or disability, a blurry line between therapeutic applications of HGE and medical applications, misapplications, potential for eugenics ( the study of how to arrange reproduction within a human population to increase the occurrence of heritable characteristics regarded as desirable), and inequitable access to the technology.

HGE is a complex technology which presents a variety of risk factors for the coming decades. Deliberative democracy is necessary to keep this technology in check, ethically.

The future of HGE is uncertain and requires immense forethought. The American Society of Human Genetics workgroup developed a position statement on human germline engineering. The statement argues that it is inappropriate to perform germline gene editing that culminates in human pregnancy; and that in vitro(outside of an organism) germline editing should be permitted with appropriate oversight. It also states future clinical human germline editing requires ethical justification, compelling medical rationale, and evidence that supports its clinical usage. Many of these decisions were made based on the potential concerts over the future possibilities of the technology.

At the societal level, there may be concerns related to eugenics, social justice, and accessibility to technology. Eugenics could potentially reinforce prejudice and enforce exclusivity in certain physical traits. Traits can be preselected for, thus labeling some as good and others as unfavorable. This may perpetuate existing racist ideals, for example.

Moreover, germline genome editing may also increase the amount of inequality in a society. Human germline editing is likely to be very expensive and access may be limited to certain geographic regions, health systems, or socioeconomic statuses. Even if human genetic engineering is only used for medical purposes, genetic disease could become an artifact of class, location, or ethnic group. Therefore, preclinical trials are necessary to establish validity, safety, and efficacy before any wide scale studies are initiated.

Others argue that HGE may lessen genetic diversity in a human population, creating a biological monoculture that could lead to disease susceptibility and eventual extinction. Analyses have predicted that there will be negligible effect on diversity and will more likely ensure the health and longevity of humans (Russel, 2010). Legacy thinking may be responsible for the hesitations towards continuing forward with HGE, as there are also many potential pros for genetic engineering. Legacy thinking is using outdated thinking strategies and actions which may not be useful anymore.

In an alternative modernity, we can imagine HGE as an end-all for most congenital diseases and cancers. Moreover, it may be used in germline gene editing to prevent certain birth defects or heritable diseases. So, although HGE has a variety of potential risk factors, there is also great promise for novel medical therapies in the coming decades. The continued use of this technology should be approached cautiously and with extensive governmental regulation, allowing for research regarding its medical applications only.

In 2016, germline gene editing was proven feasible and effective in chickens by leading researchers in genetic engineering, Dimitrov and colleagues. In this study, scientists used CRISPR/Cas9 to target the gene for an antibody/ immunoglobulin commonly produced in chickens. Antibodies are proteins produced in immune response. In the resulting population, the chickens grew normally and healthily with modified antibodies which conferred drug resistance. This study was the first to prove that germline editing is both feasible and effective.

HGE is a rapidly expanding field of research which presents novel possibilities for the coming decades. HGE utilizes CRISPR/Cas9 gene editing tools to cut out specific genes and replace them with a newly designed gene. As important as this technology is, it is also important to recognize how new it is. Gene therapy research began in the 1960s, with somatic cell editing only commencing in the past two decades. This has presented many advantages for the potential treatment of congenital diseases, but also presents various risks. Those risks stem from germline gene editing and include eugenics and inequitable access to the technology creating large socio economic divides. In the future, more regulation should be placed on the advancement of HGE research before larger-scale studies take place.

1. What is the primary technology proposed for use in HGE?

A. Recombinant DNA technology

B. CRISPR/Cas9

C. Bacterial Transformation

D. Immunoglobulin

2. When was gene therapy concepts first introduced?

A. 1920s

B. 1940s

C. 1960s

D. 1980s

3. What is a major ethical concern regarding HGE addressed in this chapter?

A. Potential for ageism

B. Gene editing is only 50% effective

C. HGE can only be used in Caucasians

D. Potential for eugenics

Answers:

Baltimore, D. et. al.(2015). A prudent path forward for genomic engineering and germline gene modification. Science. https://doi.org/10.1126/science.aab1028

Brokowski, C., & Adli, M. (2019). CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool. Journal of Molecular Biology. https://doi.org/10.1016/j.jmb.2018.05.044

Cong, L., Ran, F., & Zhang, F. (2013). Multiplex Genome Engineering Using CRISPR/Cas9 Systems. Science. https://doi.org/10.1126/science.1231143

Dimitrov, L., et. al. (2016). Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells. Plos One. https://doi.org/10.1371/journal.pone.0154303

Hu, C. (2019). Safety of Transplantation of CRISPR CCR5 Modified CD34+ Cells in HIV-Infected Subjects with Hematological Malignancies. U.S National Library of Medicine. https://clinicaltrials.gov/ct2/show/NCT03164135

Ormond, K., et. al.(2017). Human Germline Genome Editing. AJHG. https://doi.org/10.1016/j.ajhg.2017.06.012

Russell P.(2010) The Evolutionary Biological Implications of Human Genetic Engineering, The Journal of Medicine and Philosophy: A Forum for Bioethics and Philosophy of Medicine. https://doi.org/10.1093/jmp/jhq004

Tamura, R., & Toda, M. (2020). Historic Overview of Genetic Engineering Technologies for Human Gene Therapy. Neurologia medico-chirurgica. https://doi.org/10.2176/nmc.ra.2020-0049

Thomas, C. (2020). CRISPR-Edited Allogeneic Anti-CD19 CAR-T Cell Therapy for Relapsed/Refractory B Cell Non-Hodgkin Lymphoma. ClinicalTrials. https://clinicaltrials.gov/show/NCT04637763

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18 Human Genetic Engineering - Clemson University

Genetic engineering is the process of altering the genetic composition of plants, animals, and humans. The most practical application of genetic engineering is to create a more sustainable food system for the people of Earth, but there are other ways we can use it to our advantage as well.

Unfortunately, there are both pros and cons of genetic engineering. For every benefit, there is a list of concerns and potential problems we need to consider. There is a substantive argument on both sides of genetic engineering, and well explore both ahead.

Most people tend to focus on the negatives of genetic engineering, but there are some substantial positive we need to consider as well. Genetic engineering is a debate, and there are some good points on each side. You have to look at both the pros and cons of genetic engineering if you want to make an informed decision on the matter.

Evolution takes thousands of years to adapt to our surroundings, but genetic engineering offers a quicker path forward. With the assistance of genetic engineering, we could force our bodies to adapt to the changing climate of our planet.

Additionally, we could tack-on some extra years to our lives by altering our cells, so our bodies dont deteriorate as quickly as they currently do. The fountain of youth might be within our reach, and many look forward to advancements in the area of genetic engineering.

If we choose to go down this path, well feel better as we age and be able to outlast some of the diseases that currently take us down. We still wont be able to live forever, but genetic engineering shows promise in extending the prime of our lives.

Food shortage is a massive problem in the world, especially with the growing population. Were destroying natural habitats to make way for farmland, and overgrazing is causing current pastures to become dry and uninhabited.

The answer to this problem could come in the form of genetic engineering. If we can alter the composition of vegetables and animals, we can create new foods that might have more nutritional value than nature creates on its own.

We might even be able to advance to a point where foods give us medicines we need to combat widespread viruses and illnesses. Food is one of the most promising spaces when considering the prospect of genetic engineering.

A lot of diseases depend on genetic predisposition. Some people are more likely to get cancer, Alzheimers and other diseases than their neighbor. With genetic engineering, we can get rid of these genetic predispositions once and for all.

There will likely still be some environmental concerns that will cause diseases, but if we start altering the genes of humans, we may become resistant to genetic abnormalities. Family history wont mean anything when it comes to things like cancer, and we can start eliminating diseases that are completely based on genetics.

There are already a handful of diseases and illnesses we can detect while a baby is still in the womb. We even can genetically engineer some diseases and illnesses out of a babys system before theyre born.

Finding out your baby has a disease can be devastating, and some parents make the difficult choice to spare their child possible pain. If you know that your baby might suffer and die a few months after theyre born, you have to decide whether or not you want to roll the dice.

In the future, we might be able to eliminate the chances of unhealthy babies. Diseases like Huntingtons offer a substantial chance that the carrier will pass it onto their child. If the child isnt positive for the disease, theyll still be a carrier and have to deal with the same dilemma when it comes time to have kids of their own.

Genetic engineering has the potential to stop these threats in their tracks. Parents wont have to worry about birthing a healthy son or daughter. Science will guarantee that every baby is happy and healthy when they come into this world.

Of course, genetic engineering isnt entirely positive. There is an upside to the ability to genetically alter humans and animals, but only in ideal situations.

Our world isnt perfect, and scientists make mistakes all the time. We cant assume that genetic engineering will be available to the entirety of the human population, which is a flaw in itself.

The negatives of genetic engineering seem to outweigh the positives, especially since there is so much room for error. We dont know what were tampering with, which opens the door to a host of potential problems.

There are a couple of ethical problems with genetic engineering that we need to consider as a society. Those who subscribe to religion will see genetic engineering as blasphemy, for instance. Wed be playing God, in a sense. Anyone who believes in creation will be expressly against genetic engineering especially in human children.

Those who are on the opposite side of the spectrum from religious people probably wont love genetic engineering either. Genetically engineered food might work, but changing the genes of people will add to the overpopulation problem were currently experiencing.

Diseases are one of the most effective forms of population control. We dont have the heart to eliminate other humans in the name of population control, so disease does it for us. If we eliminate diseases, humans will have virtually no threat left on this planet.

Living longer lives might be ideal, but it isnt practical. If we extend the prime of our lives, were opening the door to having more children. Since all children would be in perfect health, well see a population increase that could have devastating consequences.

If genetic engineering becomes a reality, it will likely only be available to the richest members of society. Theyll be able to extend their lives, limit diseases, and make sure their children are always healthy when theyre born

When this happens, natural selection is completely obsolete. Instead, the wealthiest in society will thrive while the poor will die-out. Eventually, genetic diversity will completely disappear as genetically engineered children all express the most desirable characteristics

This problem also arises in nature if we decide to engineer plants and animals genetically. These organisms might start as food, but could introduce themselves to the wild and take over. Theyll decimate natural species, and eventually be the only thing left.

One of the biggest hurdles in genetic engineering is the possibility of errors or genetic defects, especially in humans. Scientists have a general understanding of what creates a functioning human, but they dont yet have all the pieces to the puzzle.

When it comes down to changing humans at a cellular level, scientists dont yet have the understanding of how small changes can affect the development of a growing baby. Changing genes could result in more damaging birth defects or even miscarriages.

Furthermore, tampering with diseases could end up creating a super-disease that is even harder to combat. There are too many variables in the human body for genetic engineering to work to the fullest potential. Even if it could, people will probably be too nervous to trust scientists tampering with the cells of their future children.

Science still isnt at a point where they can alter the genes of humans to prevent all diseases in unborn children, but it might be there soon. When that time comes, some might take genetic engineering to its logical extreme.

Our priority will be to create healthy children. Once we perfect this process, though, where to, we go? The next logical step is the ability to pick certain traits that our children will have. We might be able to select whether we have a boy or girl. Then, we can decide what eye color and hair color they have.

Pretty soon, were selecting every trait that our child has before they leave the womb. Nature will be virtually out of the question at this point, and people with enough money will design their babies from scratch.

Since the pros and cons of genetic engineering are compelling, its worth it to explore the possibility further. We still havent reached a place where scientists fully understand the opportunities genetic engineering presents, so they still have years of research on their hands.

In the end, though, no system of genetically altering humans, animals, or plants will be perfect. There is a massive potential for errors, and we likely wont have equal opportunities if and when scientists ever crack the case.

Although the positives of genetic engineering are convincing, the negatives can be terrifying. If we ever get to the point where we can genetically alter humans, we need to consider the moral, ethical, and practical application of technology before going any further.

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Pros and Cons of Genetic Engineering - Benefits and Risks

How artificial skin is made and its uses, from treating burns to skin cancer  South China Morning Post

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How artificial skin is made and its uses, from treating burns to skin cancer - South China Morning Post

Genetic engineering, also calledgenetic modification, is the direct manipulation of an organismsgenomeusing biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novelorganisms. Read important facts about Genetic Engineering in this article for the IAS Exam.

NewDNAmay be inserted in the host genome by first isolating and copying the genetic material of interest usingmolecular cloningmethods to generate a DNA sequence, or by synthesizing the DNA and then inserting this construct into the host organism.Genesmay be removed, or knocked out, using anuclease.Gene targetingis a different technique that useshomologous recombinationto change an endogenous gene and can be used to delete a gene, removeexons, add a gene, or introducepoint mutations.

Aspirants reading, GEAC can also refer to topics lined below:

Medicine, research, industry and agriculture are a few sectors where genetic engineering applies. It can be used on various plants, animals and microorganisms. The first microorganism to be genetically modified is bacteria.

Genetic Engineering Appraisal Committee (GEAC) is the biotech regulator in India. It is created under the Ministry of Environment and Forests. Read more about GEAC in the linked article.

There are five bodies that are authorized to handle rules noted underEnvironment Protection Act 1986 Rules for Manufacture, Use, Import, Export and Storage of Hazardous Microorganisms/Genetically Engineered Organisms or Cells 1989. These are:

Soybean-Herbicide tolerance,Canola-Altered fatty acid composition,Plum-Virus resistance,Corn-Insect resistance

Pros:Tackling and Defeating Diseases,Getting Rid of All Illnesses in Young and Unborn Children,Potential to Live Longer,Produce New Foods,Faster Growth in Animals and Plants,Pest and Disease Resistance.Cons:May Lead to Genetic Defects,Limits Genetic Diversity,Reduced Nutritional Value,Risky Pathogens,Negative Side Effects

UPSC Preparation:

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Genetic Engineering - Meaning, Applications, Advantages and Challenges ...

Revolutionary Specialty Enzymes Transform Industries, Projected to Reach $2.2 Billion by 2031 - Billion-Dollar  EIN News

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Revolutionary Specialty Enzymes Transform Industries, Projected to Reach $2.2 Billion by 2031 - Billion-Dollar - EIN News

On Wednesday, the central government paved the way for easy introduction of genome edited crops. The government has clearly distinguished such crops from genetically modified crops and has prescribed relatively easier norms for their introduction. The Indian Express explains what genome editing is and how it is different from genetically modified crops.

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A decade ago, scientists in Germany and the US discovered a technique which allowed them to cut DNA strands and edit genes. For agriculture scientists this process allowed them to bring about desired changes in the genome by using site directed nuclease (SDN) or sequence specific nuclease (SSN). Nuclease is an enzyme which cleaves through nucleic acid the building block of genetic material.

Advanced research has allowed scientists to develop the highly effective clustered regularly interspaced palindromic repeat (CRISPR) -associated proteins based systems. This system allows for targeted intervention at the genome sequence. This tool has opened up various possibilities in plant breeding. Using this tool, agricultural scientists can now edit genome to insert specific traits in the gene sequence. Depending on the nature of the edit that is carried out, the process is divided into three categories SDN 1, SDN 2 and SDN 3.

SDN1 introduces changes in the host genomes DNA through small insertions/deletions without introduction of foreign genetic material. In the case of SDN 2, the edit involves using a small DNA template to generate specific changes. Both these processes do not involve alien genetic material and the end result is indistinguishable from conventionally bred crop varieties. On the other hand, SDN3 process involves larger DNA elements or full length genes of foreign origin which makes it similar to Genetically modified organisms (GMO) development.

Genetically modified organisms (GMO) involves modification of the genetic material of the host by introduction of a foreign genetic material. In the case of agriculture, soil bacteria is the best mining source for such genes which are then inserted into the host genome using genetic engineering. For example, in case of cotton, introduction of genes cry1Ac and cry2Ab mined from the soil bacterium Bacillus Thuringiensis (BT) allow the native cotton plant to generate endotoxins to fight pink bollworm naturally. BT Cotton uses this advantage to help farmers naturally fight pink bollworm which is the most common pest for cotton farmers.

The basic difference between genome editing and genetic engineering is that while the former does not involve the introduction of foreign genetic material, the latter does. In the case of agriculture, both the techniques aim to generate variants which are better yielding and more resistant to biotic and abiotic stress. Before the advent of genetic engineering, such variety improvement was done through selective breeding which involved carefully crossing plants with specific traits to produce the desired trait in the offspring. Genetic engineering has not only made this work more accurate but has also allowed scientists to have greater control on trait development.

Across the world, GM crop has been a topic of debate, with many environmentalists opposing it on the grounds of bio safety and incomplete data. In India, the introduction of GM crops is a laborious process which involves multiple levels of checks. The Genetic Engineering Appraisal Committee (GEAC), a high power committee under the Ministry of Environment, Forest and Climate Change, is the regulator for introduction of any GM material and in case of agriculture multiple field trials, data about biosafety and other information is necessary for getting the nod before commercial release of any GM crop. Till date the only crop which has crossed the regulatory red tape is Bt cotton.

Scientists both in India and across the world have been quick to draw the line between GM crops and genome edited crops. The latter, they have pointed out, has no foreign genetic material in them which makes them indistinguishable from traditional hybrids. Globally, European Union countries have bracketed genome edited crops with GM crops. Countries like Argentina, Israel, US, Canada, etc have liberal regulations for genome edited crops.

Last year, a group of eminent agricultural scientists had written to Prime Minister Narendra Modi voicing their concern about what they said was a move to put the issue of genome edited crops to the back burner. Back then, the central government had invited suggestions and objections from states and Union Territories about the issue and put on hold field trials of such crops. The signatories, many of whom were Padma awardees, had categorically said that the variants developed through SDN1 and SDN2 techniques do not have any alien DNA and as such can be treated as other hybrids.

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On Wednesday, the Environment Ministry put a lid to the topic by issuing fresh guidelines. The Wednesdays notification has exempted SDN 1 and SDN 2 genmoe from the same and instead it would rely on reports of Institutional Biosafety Committee to exclude exogenous genetic material.

The institutional biosafety committees are expert committees constituted under the Act to deal with research and release of GM material. Such committees would now be entrusted to certify that the genome edited crop is devoid of any foreign DNA This would be a less cumbersome and time consuming process for commercial release of genome edited crops.

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Explained: What is genome editing technology and how is it different from GM technology? - The Indian Express

San Antonio Zoo is in discussions with Colossal Laboratories & Biosciences on several projects, one of which is the return of the Woolly Mammoth through DNA de-extinction technology. The ultimate goal is for Woolly Mammoths to be re-introduced to the wild to decelerate the melting of arctic permafrost, prevent greenhouse emissions trapped within the permafrost layer, revert now-overshrubbed forest into natural arctic grasslands, and more. San Antonio Zoo could be the first location of this genetically engineered elephant-mammoth hybrid.

One of Colossals main goals is the de-extinction of species. According to leading scientists, on average, 30,000 species are being driven to extinction. That is 6 per hour, 150 per day, and up to 55,000 per year. The United Nations has declared that 1 million plants and animal species are threatened with extinction.

CRISPR, the most advanced, applicable technique in genetic engineering, is an engineered cellular technology used for recognizing and cutting a specific code of DNA inside the nucleus. In mammalian cells, such as an elephant or a Woolly Mammoth, CRISPR works with an enzyme called Cas9 to modify genes. A CRISPR-Cas9 complex will use a single guide RNA from CRISPR to guide and recognize a specific sequence of DNA, where the Cas9 molecule will cleave those strands that are complementary to the CRISPR sequence. This allows for the reinsertion of the laboratory-engineered DNA, giving the ability to insert cold-resistant characteristics into elephant DNA. Ultimately, leading to the de-extinction of the Woolly Mammoth.

Our goal is to have our first calves in the next four to six years, said Austinite tech entrepreneur Ben Lamm, Cofounder of Colossal. This is going to change everything.

There are times when technology and nature collide - however, through DNA work and the de-extinction science behind Colossal, this is a time when nature and humankind will benefit, said Tim Morrow, President & CEO of San Antonio Zoo. Through this science, we hope to find a vaccine for EEHV, a virus that strikes elephants, and restore a balance to nature by saving and de-extincting species on the brink.

Prior to reintroduction into the wild, San Antonio Zoo is positioning itself to utilize zoo grounds on the west side of Highway 281 with a landbridge, similar to the recently opened Hardberger Park Land Bridge, to allow the Woolly Mammoths to have access to large acreage as well as viewability for guests in the current elephant habitat.

According to Colossal, genetic engineering is used to help humanity advance treatments for genetic disorders, gene therapies, DNA fingerprinting, vaccines, and pharmaceutical products. Additional applications include sustainable plant and animal food production, diagnosing diseases and conditions, medical treatment improvement, and producing vaccines and other useful drugs. Genetic engineering applications for animals include advancing human health, enhancing food production, reducing environmental impact, optimizing animal health and welfare, and producing cutting-edge industrial applications.

Other world-bettering uses include eradicating malaria, organ donorship, and of course, the slowing of and reversal of the extinction of a species. To learn more about Association of Zoos & Aquariums program Saving Animals From Extinction (SAFE) visit: http://www.aza.org/safe-species

To learn more about International Elephant Foundation programs visit: https://elephantconservation.org/

Colossal Background presented by CNN: https://www.cnn.com/2021/09/13/world/woolly-mammoth-resurrect-deextinction-scn/index.html

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San Antonio Zoo In Discussions on Woolly Mammoth Project - iHeart