DTDP-glucose goes on to form a variety of compounds in  nucleotide sugars metabolism . Many bacteria utilize dTDP-glucose to form exotic sugars that are incorporated into their  lipopolysaccharides  or into  secondary metabolites  such as  antibiotics . During the syntheses of many of these exotic sugars, dTDP-glucose undergoes a combined oxidation/reduction reaction  via the enzyme  dTDP-glucose 4,6-dehydratase , producing dTDP-4-keto-6-deoxy-glucose. [ 1 ] [ 2 ]


Thymidine monophosphate  ( TMP ), also known as  thymidylic acid  ( conjugate base   thymidylate ),  deoxythymidine monophosphate  ( dTMP ), or  deoxythymidylic acid  ( conjugate base   deoxythymidylate ), is a  nucleotide  that is used as a  monomer  in  DNA . It is an  ester  of  phosphoric acid  with the  nucleoside   thymidine . dTMP consists of a  phosphate   group , the  pentose   sugar   deoxyribose , and the  nucleobase   thymine . Unlike the other  deoxyribonucleotides , thymidine monophosphate often does not contain the "deoxy" prefix in its name; nevertheless, its symbol often includes a "d" ("dTMP"). [ 1 ]   Dorland’s Illustrated Medical Dictionary [ 2 ]  provides an explanation of the nomenclature variation at its entry for thymidine.


As a  substituent , it is called by the prefix  thymidylyl- .


This  organic chemistry  article is a  stub . You can help Wikipedia by  expanding it .


TNP-ATP  is a  fluorescent   molecule  that is able to determine whether a  protein  binds to  ATP , and the constants associated with that binding. It is primarily used in  fluorescence   spectroscopy , but is also very useful as an acceptor molecule in  FRET , and as a fluorescent  probe  in fluorescence  microscopy  and  X-ray crystallography . [ 1 ]


TNP refers to the  chemical compound  2,4,6-trinitrophenol, also known as  Picric acid . [ 2 ]  It is a primary constituent of many unexploded landmines, and is a cousin to  TNT , but less stable. [ 2 ]  It is recognized as an environmental contaminant and is  toxic  to many organisms. [ 2 ]  It is still commonly used in the manufacturing of  fireworks ,  explosives , and  rocket fuels , as well as in leather, pharmaceutical, and dye industries. [ 2 ]


ATP  is an essential mediator of life. [ 1 ]  It is used to overcome unfavorable energy barriers to initiate and fuel chemical reactions. [ 1 ]  It is also used to drive biological machinery and regulate a number of processes via protein- phosphorylation . [ 1 ]  However, the proteins that bind ATP for both regulation and  enzymatic  reactions are very diverse—many yet undiscovered—and for many proteins their relationship to ATP in terms of number of  binding sites ,  binding constants , and  dissociation constants  remain unclear. [ 1 ]


Conjugating TNP to ATP renders this nucleotide triphosphate fluorescent and colored whilst allowing it to retain its biological activity. [ 1 ]  TNP-ATP is thus a   fluorescent   analog  of ATP. [ 3 ]  This conjugation is very useful in providing information about interactions between ATP and an ATP-binding protein because TNP-ATP interacts with proteins and enzymes as a substitute for its parent nucleotide, and has a strong binding affinity for most systems that require ATP. [ 1 ]


TNP is  excited  at a  wavelength  of 408 and 470 nm, and  fluoresces  in the 530–560 nm range. [ 1 ] [ 2 ] [ 4 ] [ 5 ]  This is a very useful range of excitation because it is far from where proteins or nucleotides absorb. [ 1 ]  When TNP-ATP is in water or other aqueous solutions, this emission is very weak. [ 1 ] [ 6 ]  However, once TNP-ATP binds to a  protein , there is a dramatic increase in fluorescent intensity. [ 1 ] [ 3 ] [ 5 ] [ 6 ]  This property enables researchers to study various proteins’ binding interaction with ATP. Thus, with enhanced fluorescence, it can be seen whether a protein binds to ATP. [ 1 ]


When TNP-ATP in water is excited at 410 nm, TNP-ATP shows a single fluorescence maximum at 561 nm. [ 6 ]  This maximum shifts as the fluid's viscosity changes. For example, in  N,N-dimethylformamide , instead of having its maxima at 561 nm as in water, the maxima is instead at 533 nm. [ 6 ]


Binding to a protein will also change the wavelength of maximal emission, as well as a change in fluorescent intensity. [ 6 ]  For example, binding to the  chemotaxis  protein CheA indicates a severalfold enhancement of fluorescence intensity and a blue-shift in wavelength of the maximal emission. [ 6 ]


Using this TNP nucleotide analog has been shown in many instances to be superior to traditional  radionucleotide-labelling  based techniques. [ 1 ]  The health concerns and the cost associated with the use of radioactive  isotopes  makes TNP-ATP an attractive alternative. [ 1 ]


The first fluorescent  ribose -modified ATP is 2’,3’-O-(2,4,7-trinitrocyclohexadienylidene) adenosine 5’triphosphate (TNP-ATP), and was introduced in 1973 by Hiratsuka and Uchida. [ 1 ] [ 4 ]   TNP-ATP was originally synthesized to investigate the ATP binding site of  myosin ATPase . [ 1 ] [ 3 ]  Reports of TNP-ATP’s success in the investigation of this motor protein extended TNP-ATP’s use to other proteins and enzymes. [ 1 ]  TNP-ATP has now been used as a  spectroscopic  probe for numerous proteins suspected to have ATP interactions. [ 1 ]  These include several protein  kinases ,  ATPases ,  myosin , and other nucleotide binding proteins. [ 1 ]  Over the past twenty years, there have been hundreds of papers describing TNP-ATP’s use and applications. [ 1 ]  Many applications involving this fluorescently labeled nucleotide have helped to clarify structure-function relationships of many ATP-requiring proteins and enzymes. [ 1 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ]  There have also been a growing number of papers that display TNP-ATP use as a means of assessing the ATP-binding capacity of various mutant proteins. [ 1 ] [ 6 ]


Preparing TNP-ATP is a one-step synthesis that is relatively safe and easy. [ 1 ]  Adenosine’s ribose moiety can be trinitrophenylated by 2,4,6-trinitrobenzene-1-sulfonate ( TNBS ). [ 4 ]  The resulting compound assumes a bright orange color and has visible absorption characteristics, as is characteristic of a  Meiseinheimer spiro complex compound linking . [ 1 ] [ 4 ]


To see the exact method of preparion, please refer to T. Hiratsuka's and K. Uchida's paper  "Preparation and Properties of 2'(r 3')-O(2,4,6-trinitrophenyl) Adenosine 5'-triphosphate, an Analog of Adenosine Triphosphate,"   found in the reference section.


To revert TNP-ATP back to its constituent parts, or in other words to  hydrolyze  TNP-ATP to give equilmolar amounts of picric acid (TNP) and ATP, TNP-ATP should be treated with 1 M  HCl  at 100 degrees Celsius for 1.5 hours. [ 4 ]  This is because if TNP-ATP is acidified under mild conditions, it results in the opening of the  dioxolane  ring attached to the 2’-oxygen, leaving a 3’O-TNP derivative as the only product. [ 1 ]


TNP-ATP should be stored at −20 °C, in the dark, and used under minimal lighting conditions. [ 6 ]  When in solution, TNP-ATP has a shelf life of about 30 days.


When absorption was measured against wavelength at various pH values, the changes at wavelength 408 nm and 470 nm yielded a  sigmoidal  line with a midpoint at 5.1. [ 4 ]  This indicated that the absorbance at these two wavelengths depends upon the ionization of the  chromophoric  portion of TNP-ATP and is unaffected by ionization of ATP. [ 4 ]  Although this  ionization  constant of 5.1 is not in physiological range, it has been shown that the absorbance of TNP-ATP is sensitive enough to detect changes due to slight shifts in neutral pH. [ 4 ]   Spectroscopic  superposition indicated TNP-ATP’s  isosbestic  point to be 339 nm. [ 4 ]


At low concentrations of TNP-ATP (≤1 μM), fluorescent intensity is proportional to the concentration of TNP added. [ 6 ]  However, at concentrations exceeding 1 μM, inner filter effects cause this relationship to no longer be linear. [ 6 ]  To correct this, researchers must determine the ratio of the predicted theoretical fluorescence intensity (assuming linearity) to the observed fluorescence intensity and then apply this correction factor. [ 6 ]  However, in most cases, researchers will try to keep the concentration of TNP to lower than 1 μM. [ 1 ] [ 2 ] [ 3 ] [ 5 ] [ 6 ]


To determine binding affinities, TNP-ATP is added to a solution and then titrated with protein. [ 5 ] [ 6 ]  This produces a saturation curve from which the binding affinity can be determined. [ 5 ] [ 6 ]  The number of binding sites may also be determined through this saturation curve by looking to see if there are sudden changes in slope. [ 5 ]  One can also  titrate  a fixed amount of protein with increasing additions of TNP-ATP to obtain a saturation curve. [ 6 ]  To do so, however, may get complicated due to the inner filter effects that will need to be corrected for. [ 6 ]


To determine dissociation constants, TNP-ATP can be competed off of a protein with ATP. [ 5 ] [ 6 ]   The value of the dissociation constant K d  for a single-site binding can then be obtained by applying the  Langmuir equation  for a curve fit:


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 {\displaystyle \mathrm {RFU_{obs}} =\mathrm {RFU_{free}} +{\frac {(\mathrm {RFU_{bound}} -\mathrm {RFU_{free}} )\times \left((\mathrm {[protein]_{total}} +\mathrm {[TNP]_{total}} +\mathrm {K_{d}} )-{\sqrt {(\mathrm {[protein]_{total}} +\mathrm {[TNP]_{total}} +\mathrm {K_{d}} )^{2}-(4\times \mathrm {[protein]_{total}} \times \mathrm {[TNP]} )}}\right)}{2\mathrm {[TNP]_{total}} }}}


where RFU is relative fluorescent units, RFU obs  is the fluorescence observed, RFU free  is the fluorescence of free TNP-ATP, and RFU bound  is the fluorescence of TNP-ATP when completely bound to a protein. [ 5 ]


To measure an ATP competitor, one can add competitor to pre-incubated samples of protein:TNP-ATP. The fraction of TNP-ATP bound to the protein can be calculated via:


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 {\displaystyle \theta ={\frac {\mathrm {RFU_{obs}} -\mathrm {RFU_{free}} }{\mathrm {RFU_{max}} -\mathrm {RFU_{free}} }}}


where θ is that fraction, and RFU max  is the value of fluorescence intensity at saturation, meaning when 100% of TNP-ATP is bound. [ 5 ]


The dissociation constants for TNP and competitor can then be calculated through the equation: [ 5 ]


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 {\displaystyle \theta ={\frac {1}{2}}\mathrm {[TNP]} \times \left(\mathrm {K_{TNP}} +{\frac {\mathrm {K_{TNP}} }{\mathrm {K_{competitor}} }}\times \mathrm {[competitor]} +\mathrm {[TNP]} +\mathrm {[protein]} -{\sqrt {\left(\mathrm {K_{TNP}} +{\frac {\mathrm {K_{TNP}} }{\mathrm {K_{competitor}} }}\times \mathrm {[competitor]} +\mathrm {[TNP]} +\mathrm {[protein]} \right)^{2}-4\times \mathrm {[TNP]} \times \mathrm {[protein]} }}\right)}


For reasons not yet fully understood, TNP-ATP typically binds the ATP binding sites of proteins and enzymes anywhere from one to three times tighter than regular ATP. [ 1 ] [ 6 ]  The dissociation constants are usually around 0.3–50 μM. [ 1 ]


In addition to using TNP-ATP to determine whether a protein binds ATP, its binding affinity and dissociation constants, and number of binding sites, TNP-ATP can also be used in ligand binding studies. [ 1 ]  To do this, titrations of the protein are added to TNP-ATP. Then, ligand is added to displace the bound analog. [ 1 ]  This is measured by decreases in fluorescence. [ 1 ]  One can also do this by titrating protein with TNP-ATP in the presence and absence of varying concentrations of the ligand of interest. [ 1 ]  Using either experiment will allow the binding affinity of the ligand to protein to be measured.


TNP-ATP is also valuable fluorescence acceptor. [ 1 ] [ 2 ]  This is because, as with any good acceptor, TNP-ATP absorbs over a wide wavelength range that corresponds to the range of emission of common  FRET  donors. [ 2 ]  Thus, TNP-ATP can be used to look at the conformational changes that proteins undergo. [ 2 ]   For example, Na+/K+ ATPase, the distance between the active site and Cys457 was shown to change from 25 Angstroms to 28 Angstroms in changing from the Na+ conformation to the K+ conformation. [ 1 ]


In addition to fluorescent spectroscopy, TNP-ATP is very useful in fluorescent  microscopy . [ 1 ]  This is because it greatly increases the sensitivity of the observations when bound to proteins—the enhanced fluorescence greatly reduces the problem of background fluorescence. [ 1 ]  This is especially true under  epifluorescent  illumation (illumination and light are both on the same side of the specimen). [ 1 ]


TNP-ATP has also been used in  X-ray crystallography  because it can be used to determine binding constants of crystallized substrates. This technique also demonstrates the structure of proteins in the presence or absence of TNP-ATP, which may or may not correspond to the structure of proteins when they bind ATP. [ 1 ] [ 6 ]


Tuberculinic acid  is a noncanonical  nucleic acid  initially identified as the  poison  of  Tubercle bacillus  (= Mycobacterium tuberculosis ), the principal causative  bacterium  of  tuberculosis . Its discovery was one of the most important landmarks in understanding tuberculosis and in  molecular biology . It is regarded as the most  toxic  component of the  bacillus . It was from this compound that  DNA   methylation  was discovered as it was the first molecule found to contain  5-methylcytosine . In addition it contains  thymine ,  guanine  and  cytosine . [ 1 ]


It was first identified by a  German   chemist  W.G. Ruppel in 1898 while trying to isolate the bacterial toxin responsible for tuberculosis. From the crushed bacilli, specifically the  protein   tuberculin , he isolated two toxic substances, namely a  basic compound  which he called tuberculosamine, and a  nucleotide  he named tuberculisäure, later to be  anglicised  to tuberculinic acid. He claimed that the tuberculosamin was bound to the nucleotide. Ruppel also found that his new compound was the most toxic component of the bacillus, 2.5 to 6 times more potent than tuberculin, the  proteinaceous  toxin. [ 2 ]


In 1925,  Treat Baldwin Johnson  and Coghill were detected a minor amount of a methylated  cytosine  derivative as a product of  hydrolysis  of tuberculinic acid, from  avian  tubercle bacilli, with  sulfuric acid . [ 3 ] [ 4 ]  This report was seriously challenged because others failed to reproduce the result after a series of tests. [ 5 ]   But Johnson and Coghill were in fact proved correct. In 1948, Hotchkiss separated the nucleic acids of  DNA  from  calf   thymus  using  paper chromatography , by which he detected a unique methylated cytosine, quite distinct from cytosine and  uracil . [ 5 ] [ 6 ] [ 7 ]


Uridine diphosphate , abbreviated  UDP , is an  organic compound . It is an  ester  of  pyrophosphoric acid  with the  nucleoside   uridine . UDP consists of the  pyrophosphate   group , the  pentose   sugar   ribose , and the  nucleobase   uracil .


UDP is an important factor in  glycogenesis . Before  glucose  can be stored as  glycogen  in the  liver  and  muscles , the  enzyme   UDP-glucose pyrophosphorylase  forms a  UDP-glucose  unit by combining glucose 1-phosphate with  uridine triphosphate , cleaving a  pyrophosphate  ion in the process. Then, the enzyme  glycogen synthase  combines UDP-glucose units to form a glycogen chain. The UDP molecule is cleaved from the glucose ring during this process and can be reused by UDP-glucose pyrophosphorylase. [ 1 ] [ 2 ]


Uridine diphosphate galactose  ( UDP - galactose ) is an intermediate in the production of  polysaccharides . [ 1 ]  It is important in  nucleotide sugars metabolism , and is the substrate for the transferase  B4GALT5 .


Uridine diphosphate (UDP)-galactose is relevant in glycolysis. UDP-galactose is the activated form of Gal, a crucial monosaccharide building block for human milk oligosaccharide (HMO). [ 2 ]  The activated form of galactose (Gal) serves as a donor molecule involved in catalyzing the conversion of UDP-galactose to UDP-glucose. The conversion is a rate-limiting step essential to the pace of UDP-glucose production that determines the completion of glycosylation reactions. [ 3 ]


To further explain, UDP-galactose is derived from a galactose molecule which is an epimer of glucose, and via the  Leloir pathway , it is used be used as a precursor for the metabolism of glucose into pyruvate. [ 4 ]  When lactose is hydrolyzed, D-Galactose enters the liver via the bloodstream. There, galactokinase phosphorylates it to galactose-1-phosphate using ATP. This compound then engages in a "ping-pong" reaction with UDP-glucose, catalyzed by uridylyltransferase, yielding glucose-1-phosphate and UDP-galactose. This glucose-1-phosphate feeds into glycolysis, while UDP-galactose undergoes epimerization to regenerate UDP-glucose. [ 5 ]


Uridine diphosphate glucose  ( uracil-diphosphate glucose ,  UDP - glucose ) is a  nucleotide sugar . It is involved in  glycosyltransferase  reactions in  metabolism .


UDP-glucose is used in  nucleotide sugar metabolism  as an activated form of glucose, a substrate for enzymes called  glucosyltransferases . [ 1 ]


UDP-glucose is a precursor of  glycogen  and can be converted into  UDP-galactose  and  UDP-glucuronic acid , which can then be used as substrates by the enzymes that make  polysaccharides  containing  galactose  and  glucuronic acid .


UDP-glucose can also be used as a precursor of sucrose,  lipopolysaccharides  and  glycosphingolipids .


UDP-glucose consists of the  pyrophosphate  group,  ribose ,  glucose , and  uracil .


UDP-glucuronic acid  is a  sugar  used in the creation of  polysaccharides  and is an intermediate in the biosynthesis of  ascorbic acid  (except in  primates  and  guinea pigs ). It also participates in the  heme degradation process  of human.


It is made from  UDP-glucose  by  UDP-glucose 6-dehydrogenase  (EC 1.1.1.22) using  NAD+  as a cofactor. It is the source of the glucuronosyl group in  glucuronosyltransferase  reactions. [ 1 ] [ 2 ]


Uridine monophosphate  ( UMP ), also known as  5′-uridylic acid  ( conjugate base   uridylate ), is a  nucleotide  that is used as a  monomer  in  RNA . It is an  ester  of  phosphoric acid  with the  nucleoside   uridine . UMP consists of the  phosphate   group , the  pentose   sugar   ribose , and the  nucleobase   uracil ; hence, it is a  ribonucleotide monophosphate . As a  substituent  or  radical  its name takes the form of the prefix  uridylyl- . The deoxy form is abbreviated  dUMP . Covalent attachment of UMP (e.g., to a protein such as adenylyltransferase) is called  uridylylation  (or sometimes uridylation). [ 2 ]


Uridine monophosphate is formed from  Orotidine 5'-monophosphate  (orotidylic acid) in a decarboxylation reaction catalyzed by the  enzyme   orotidylate decarboxylase . Uncatalyzed, the decarboxylation reaction is extremely slow (estimated to occur on average one time per 78 million years). Adequately catalyzed, the reaction takes place once per second, an increase of 10 17 -fold. [ 3 ]


In humans, the orotidylate decarboxylase function is carried out by the protein  UMP synthase . [ 4 ]  Defective UMP synthase can result in  orotic aciduria , a metabolic disorder.


In a study,  gerbils  fed a combination of uridine monophosphate,  choline , and  docosahexaenoic acid  (DHA) were found to have significantly improved performance in running mazes over those not fed the supplements, implying an increase in cognitive function. [ 5 ]


In brain research studies, uridine monophosphate is used as a convenient delivery compound for  uridine . [ 6 ]   Uridine  is the active component of this compound.  Uridine is present in many foods, mainly in the form of RNA. Non-phosphorylated uridine is not bioavailable beyond first-pass metabolism, as it is almost entirely catabolised in the liver and gastrointestinal tract. [ 7 ]


Uridine-5′-triphosphate  ( UTP ) is a  pyrimidine   nucleoside triphosphate , consisting of the  organic base   uracil  linked to the 1′ carbon of the  ribose  sugar, and esterified with tri- phosphoric acid  at the 5′ position. Its main role is as substrate for the synthesis of  RNA  during  transcription . UTP is the precursor for the production of  CTP  via  CTP synthetase . [ 1 ]  UTP can be biosynthesized from UDP by Nucleoside Diphosphate Kinase after using the phosphate group from ATP. [ 2 ] [ 3 ]  UDP + ATP ⇌ UTP + ADP; [ 4 ]  both UTP and ATP are energetically equal. [ 4 ]


The homologue in DNA is  thymidine triphosphate  (TTP or dTTP). UTP also has a deoxyribose form (dUTP).


UTP also has the role of a source of energy or an activator of substrates in metabolic reactions, like that of  ATP , but more specific. When UTP activates a substrate (such as glucose-1-phosphate), UDP-glucose is formed and inorganic phosphate is released. [ 5 ]
 UDP-glucose  enters the synthesis of  glycogen . UTP is used in the metabolism of  galactose , where the activated form  UDP-galactose  is converted to UDP-glucose.  UDP-glucuronate  is used to conjugate  bilirubin  to a more water-soluble  bilirubin diglucuronide . UTP is also used to activate amino sugars like glucosamine-1-phosphate to UDP-glucosamine, and  N -acetyl-glucosamine-1-phosphate to UDP- N -acetylglucosamine. [ 6 ]


UTP also has roles in mediating responses by extracellular binding to the P2Y receptors of cells. UTP and its derivatives are still being investigated for their applications in human medicine. However, there is evidence from various model systems to suggest it has applications in pathogen defense and injury repair. In mice UTP has been found to interact with P2Y4 receptors to mediate an enhancement in antibody production. [ 7 ]  In Schwannoma cells, UTP binds to the P2YP receptors in the event of damage.  This leads to the downstream signal cascade that leads to the eventual injury repair. [ 8 ]


Vidarabine phosphate  is an  adenosine monophosphate   nucleotide  in which  ribose  is replaces by an arabinso moiety. It has antiviral and possibly antineoplastic properties. [ 1 ]


Xanthosine monophosphate  ( xanthylate ) is an intermediate in  purine metabolism . [ 1 ]  It is a  ribonucleoside monophosphate . It is formed from  IMP  via the action of  IMP dehydrogenase , and it forms  GMP  via the action of  GMP synthase . Also, XMP can be released from  XTP  by enzyme deoxyribonucleoside triphosphate pyrophosphohydrolase containing (d)XTPase activity. [ 2 ]


It is abbreviated XMP. [ 3 ]


Xanthosine 5'-triphosphate  ( XTP ) is a  nucleotide  that is not produced by - and has no known function in - living cells. Uses of XTP are, in general, limited to experimental procedures on enzymes that bind other nucleotides. Deamination of purine bases can result in accumulation of such nucleotides as  ITP ,  dITP , XTP, and  dXTP . [ 1 ]


This article about an  alkaloid  is a  stub . You can help Wikipedia by  expanding it .


Genetic engineering , also called  genetic modification  or  genetic manipulation , is the modification and  manipulation of an organism's  genes  using  technology . 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 novel  organisms .


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


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


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


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


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


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


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


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


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


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


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


Plants, animals or microorganisms that have been changed through genetic engineering are termed  genetically modified organisms  or GMOs. [ 14 ]  If genetic material from another species is added to the host, the resulting organism is called  transgenic . If genetic material from the same species or a species that can naturally breed with the host is used the resulting organism is called  cisgenic . [ 15 ]  If genetic engineering is used to remove genetic material from the target organism the resulting organism is termed a  knockout  organism. [ 16 ]  In Europe genetic modification is  synonymous  with genetic engineering while within the United States of America and Canada genetic modification can also be used to refer to more conventional breeding methods. [ 17 ] [ 18 ] [ 19 ]


Humans have altered the genomes of species for thousands of years through  selective breeding , or artificial selection [ 20 ] : 1  [ 21 ] : 1   as contrasted with  natural selection . More recently,  mutation breeding  has used exposure to chemicals or radiation to produce a high frequency of random mutations, for selective breeding purposes. Genetic engineering as the direct manipulation of DNA by humans outside breeding and mutations has only existed since the 1970s. The term "genetic engineering" was coined by the Russian-born geneticist  Nikolay Timofeev-Ressovsky  in his 1934 paper "The Experimental Production of Mutations", published in the British journal Biological Reviews. [ 22 ]   Jack Williamson  used the term in his science fiction novel Dragon's Island, published in 1951 [ 23 ]  – one year before DNA's role in  heredity  was confirmed by  Alfred Hershey  and  Martha Chase , [ 24 ]  and two years before  James Watson  and  Francis Crick  showed that the  DNA  molecule has a double-helix structure – though the general concept of direct genetic manipulation was explored in rudimentary form in  Stanley G. Weinbaum 's 1936 science fiction story  Proteus Island . [ 25 ] [ 26 ]


In 1972,  Paul Berg  created the first  recombinant DNA  molecules by combining DNA from the monkey virus  SV40  with that of the  lambda virus . [ 27 ]  In 1973  Herbert Boyer  and  Stanley Cohen  created the first  transgenic organism  by inserting  antibiotic resistance genes  into the  plasmid  of an  Escherichia coli  bacterium. [ 28 ] [ 29 ]  A year later  Rudolf Jaenisch  created a  transgenic mouse  by introducing foreign DNA into its embryo, making it the world's first  transgenic animal [ 30 ]  These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the  Asilomar Conference  in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe. [ 31 ] [ 32 ]


In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer and  Robert Swanson  and a year later the company produced a human protein ( somatostatin ) in  E. coli . Genentech announced the production of genetically engineered human  insulin  in 1978. [ 33 ]  In 1980, the  U.S. Supreme Court  in the  Diamond v. Chakrabarty  case ruled that genetically altered life could be patented. [ 34 ]  The  insulin  produced by bacteria was approved for release by the  Food and Drug Administration  (FDA) in 1982. [ 35 ]


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


The first field trials of  genetically engineered plants  occurred in France and the US in 1986, tobacco plants were engineered to be resistant to  herbicides . [ 39 ]  The People's Republic of China was the first country to commercialise transgenic plants, introducing a virus-resistant tobacco in 1992. [ 40 ]  In 1994  Calgene  attained approval to commercially release the first  genetically modified food , the  Flavr Savr , a tomato engineered to have a longer shelf life. [ 41 ]  In 1994, the European Union approved tobacco engineered to be resistant to the herbicide  bromoxynil , making it the first genetically engineered crop commercialised in Europe. [ 42 ]  In 1995,  Bt potato  was approved safe by the  Environmental Protection Agency , after having been approved by the FDA, making it the first pesticide producing crop to be approved in the US. [ 43 ]  In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the US, Brazil, Argentina, India, Canada, China, Paraguay and South Africa. [ 44 ]


In 2010, scientists at the  J. Craig Venter Institute  created the first  synthetic genome  and inserted it into an empty bacterial cell. The resulting bacterium, named  Mycoplasma laboratorium , could  replicate  and produce proteins. [ 45 ] [ 46 ]  Four years later this was taken a step further when a bacterium was developed that replicated a  plasmid  containing a unique  base pair , creating the first organism engineered to use an expanded genetic alphabet. [ 47 ] [ 48 ]  In 2012,  Jennifer Doudna  and  Emmanuelle Charpentier  collaborated to develop the  CRISPR/Cas9  system, [ 49 ] [ 50 ]  a technique which can be used to easily and specifically alter the genome of almost any organism. [ 51 ]


Creating a GMO is a multi-step process. Genetic engineers must first choose what gene they wish to insert into the organism. This is driven by what the aim is for the resultant organism and is built on earlier research.  Genetic screens  can be carried out to determine potential genes and further tests then used to identify the best candidates. The development of  microarrays ,  transcriptomics  and  genome sequencing  has made it much easier to find suitable genes. [ 52 ]  Luck also plays its part; the  Roundup Ready  gene was discovered after scientists noticed a bacterium thriving in the presence of the herbicide. [ 53 ]


The next step is to isolate the candidate gene. The  cell  containing the gene is opened and the DNA is purified. [ 54 ]  The gene is separated by using  restriction enzymes  to cut the DNA into fragments [ 55 ]  or  polymerase chain reaction  (PCR) to amplify up the gene segment. [ 56 ]  These segments can then be extracted through  gel electrophoresis . If the chosen gene or the donor organism's  genome  has been well studied it may already be accessible from a  genetic library . If the  DNA sequence  is known, but no copies of the gene are available, it can also be  artificially synthesised . [ 57 ]  Once isolated the gene is  ligated  into a  plasmid  that is then inserted into a bacterium. The plasmid is replicated when the bacteria divide, ensuring unlimited copies of the gene are available. [ 58 ]  The  RK2 plasmid  is notable for its ability to replicate in a wide variety of  single-celled organisms , which makes it suitable as a genetic engineering tool. [ 59 ]


Before the gene is inserted into the target organism it must be combined with other genetic elements. These include a  promoter  and  terminator  region, which initiate and end  transcription . A  selectable marker  gene is added, which in most cases confers  antibiotic resistance , so researchers can easily determine which cells have been successfully transformed. The gene can also be modified at this stage for better expression or effectiveness. These manipulations are carried out using  recombinant DNA  techniques, such as  restriction digests , ligations and molecular cloning. [ 60 ]


There are a number of techniques used to insert genetic material into the host genome. Some bacteria can naturally  take up foreign DNA . This ability can be induced in other bacteria via stress (e.g.  thermal  or electric shock), which increases the cell membrane's permeability to DNA; up-taken DNA can either integrate with the genome or exist as  extrachromosomal DNA . DNA is generally inserted into animal cells using  microinjection , where it can be injected through the cell's  nuclear envelope  directly into the  nucleus , or through the use of  viral vectors . [ 61 ]


Plant genomes can be engineered by physical methods or by use of  Agrobacterium  for the delivery of sequences hosted in  T-DNA binary vectors . In plants the DNA is often inserted using  Agrobacterium -mediated transformation , [ 62 ]  taking advantage of the  Agrobacterium s  T-DNA  sequence that allows natural insertion of genetic material into plant cells. [ 63 ]  Other methods include  biolistics , where particles of gold or tungsten are coated with DNA and then shot into young plant cells, [ 64 ]  and  electroporation , which involves using an electric shock to make the cell membrane permeable to plasmid DNA.


As only a single cell is transformed with genetic material, the organism must be  regenerated  from that single cell. In plants this is accomplished through the use of  tissue culture . [ 65 ] [ 66 ]  In animals it is necessary to ensure that the inserted DNA is present in the  embryonic stem cells . [ 67 ]  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. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant. [ 68 ]


Further testing using PCR,  Southern hybridization , and  DNA sequencing  is conducted to confirm that an organism contains the new gene. [ 69 ]  These tests can also confirm the chromosomal location and copy number of the inserted gene. The presence of the gene does not guarantee it will be  expressed  at appropriate levels in the target tissue so methods that look for and measure the gene products (RNA and protein) are also used. These include  northern hybridisation , quantitative  RT-PCR ,  Western blot ,  immunofluorescence ,  ELISA  and phenotypic analysis. [ 70 ]


The new genetic material can be inserted randomly within the host genome or targeted to a specific location. The technique of  gene targeting  uses  homologous recombination  to make desired changes to a specific  endogenous  gene.  This tends to occur at a relatively low frequency in plants and animals and generally requires the use of  selectable markers . The frequency of gene targeting can be greatly enhanced through  genome editing . Genome editing uses artificially engineered  nucleases  that create specific  double-stranded breaks  at desired locations in the genome, and use the cell's endogenous mechanisms to repair the induced break by the natural processes of  homologous recombination  and  nonhomologous end-joining . There are four families of engineered nucleases:  meganucleases , [ 71 ] [ 72 ]   zinc finger nucleases , [ 73 ] [ 74 ]   transcription activator-like effector nucleases  (TALENs), [ 75 ] [ 76 ]  and the Cas9-guideRNA system (adapted from  CRISPR ). [ 77 ] [ 78 ]  TALEN and CRISPR are the two most commonly used and each has its own advantages. [ 79 ]  TALENs have greater target specificity, while CRISPR is easier to design and more efficient. [ 79 ]  In addition to enhancing gene targeting, engineered nucleases can be used to introduce mutations at endogenous genes that generate a  gene knockout . [ 80 ] [ 81 ]


Genetic engineering has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and microorganisms.  Bacteria , the first organisms to be genetically modified, can have plasmid DNA inserted containing new genes that code for medicines or enzymes that process food and other  substrates . [ 82 ] [ 83 ]  Plants have been modified for insect protection,  herbicide resistance , virus resistance, enhanced nutrition, tolerance to environmental pressures and the production of  edible vaccines . [ 84 ]  Most commercialised GMOs are insect resistant or herbicide tolerant crop plants. [ 85 ]  Genetically modified animals have been used for research, model animals and the production of agricultural or pharmaceutical products. The genetically modified animals include animals with  genes knocked out ,  increased susceptibility to disease , hormones for extra growth and the ability to express proteins in their milk. [ 86 ]


Genetic engineering has many applications to medicine that include the manufacturing of drugs, creation of  model animals  that mimic human conditions and  gene therapy . One of the earliest uses of genetic engineering was to mass-produce human insulin in bacteria. [ 33 ]  This application has now been applied to human  growth hormones ,  follicle stimulating hormones  (for treating infertility),  human albumin ,  monoclonal antibodies ,  antihemophilic factors ,  vaccines  and many other drugs. [ 87 ] [ 88 ]  Mouse  hybridomas , cells fused together to create  monoclonal antibodies , have been adapted through genetic engineering to create human monoclonal antibodies. [ 89 ]   Genetically engineered viruses  are being developed that can still confer immunity, but lack the  infectious   sequences . [ 90 ]


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


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


Germline  gene therapy would result in any change being inheritable, which has raised concerns within the scientific community. [ 100 ] [ 101 ]  In 2015, CRISPR was used to edit the DNA of non-viable  human embryos , [ 102 ] [ 103 ]  leading scientists of major world academies to call for a moratorium on inheritable human genome edits. [ 104 ]  There are also concerns that the technology could be used not just for treatment, but for enhancement, modification or alteration of a human beings' appearance, adaptability, intelligence, character or behavior. [ 105 ]  The distinction between cure and enhancement can also be difficult to establish. [ 106 ]  In November 2018,  He Jiankui  announced that he had  edited the genomes  of two human embryos, to attempt to disable the  CCR5  gene, which codes for a receptor that  HIV  uses to enter cells. The work was widely condemned as unethical, dangerous, and premature. [ 107 ]  Currently, germline modification is banned in 40 countries. Scientists that do this type of research will often let embryos grow for a few days without allowing it to develop into a baby. [ 108 ]


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


Genetic engineering is an important tool for  natural scientists , with the creation of transgenic organisms one of the most important tools for analysis of gene function. [ 111 ]  Genes and other genetic information from a wide range of organisms can be inserted into bacteria for storage and modification, creating  genetically modified bacteria  in the process. Bacteria are cheap, easy to grow,  clonal , multiply quickly, relatively easy to transform and can be stored at -80 °C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research. [ 112 ]


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


Organisms can have their cells transformed with a gene coding for a useful protein, such as an enzyme, so that they will  overexpress  the desired protein. Mass quantities of the protein can then be manufactured by growing the transformed organism in  bioreactor  equipment using  industrial fermentation , and then  purifying  the protein. [ 116 ]  Some genes do not work well in bacteria, so yeast, insect cells or mammalian cells can also be used. [ 117 ]  These techniques are used to produce medicines such as  insulin ,  human growth hormone , and  vaccines , supplements such as  tryptophan , aid in the production of food ( chymosin  in cheese making) and fuels. [ 118 ]  Other applications with genetically engineered bacteria could involve making them perform tasks outside their natural cycle, such as making  biofuels , [ 119 ]  cleaning up oil spills, carbon and other toxic waste [ 120 ]  and detecting arsenic in drinking water. [ 121 ]  Certain genetically modified microbes can also be used in  biomining  and  bioremediation , due to their ability to extract heavy metals from their environment and incorporate them into compounds that are more easily recoverable. [ 122 ]


In  materials science , a genetically modified virus has been used in a research laboratory as a scaffold for assembling a more environmentally friendly  lithium-ion battery . [ 123 ] [ 124 ]  Bacteria have also been engineered to function as sensors by expressing a fluorescent protein under certain environmental conditions. [ 125 ]


One of the best-known and  controversial  applications of genetic engineering is the creation and use of  genetically modified crops  or  genetically modified livestock  to produce  genetically modified food . Crops have been developed to increase production, increase tolerance to  abiotic stresses , alter the composition of the food, or to produce novel products. [ 127 ]


The first crops to be released commercially on a large scale provided protection from insect pests or tolerance to  herbicides . Fungal and virus resistant crops have also been developed or are in development. [ 128 ] [ 129 ]  This makes the insect and weed management of crops easier and can indirectly increase crop yield. [ 130 ] [ 131 ]  GM crops that directly improve yield by accelerating growth or making the plant more hardy (by improving salt, cold or drought tolerance) are also under development. [ 132 ]  In 2016  Salmon  have been genetically modified with growth hormones to reach normal adult size much faster. [ 133 ]


GMOs have been developed that modify the quality of produce by increasing the nutritional value or providing more industrially useful qualities or quantities. [ 132 ]  The  Amflora  potato produces a more industrially useful blend of starches.  Soybeans  and  canola  have been genetically modified to produce more healthy oils. [ 134 ] [ 135 ]  The first commercialised GM food was a  tomato  that had delayed ripening, increasing its  shelf life . [ 136 ]


Plants and animals have been engineered to produce materials they do not normally make.   Pharming  uses crops and animals as bioreactors to produce vaccines, drug intermediates, or the drugs themselves; the useful product is purified from the harvest and then used in the standard pharmaceutical production process. [ 137 ]  Cows and goats have been engineered to express drugs and other proteins in their milk, and in 2009 the FDA approved a drug produced in goat milk. [ 138 ] [ 139 ]


Genetic engineering has potential applications in conservation and natural area management. Gene transfer through  viral vectors  has been proposed as a means of controlling invasive species as well as vaccinating threatened fauna from disease. [ 140 ]  Transgenic trees have been suggested as a way to confer resistance to pathogens in wild populations. [ 141 ]  With the increasing risks of  maladaptation  in organisms as a result of  climate change  and other perturbations, facilitated adaptation through gene tweaking could be one solution to reducing extinction risks. [ 142 ]  Applications of genetic engineering in conservation are thus far mostly theoretical and have yet to be put into practice.


Genetic engineering is also being used to create  microbial art . [ 143 ]  Some bacteria have been genetically engineered to create black and white photographs. [ 144 ]  Novelty items such as lavender-colored  carnations , [ 145 ]   blue roses , [ 146 ]  and  glowing fish , [ 147 ] [ 148 ]  have also been produced through genetic engineering.


The regulation of genetic engineering concerns the approaches taken by governments to assess and manage the risks associated with the development and release of GMOs. The development of a regulatory framework began in 1975, at  Asilomar , California. [ 149 ]  The  Asilomar meeting  recommended a set of voluntary guidelines regarding the use of recombinant technology. [ 31 ]  As the technology improved the US established a committee at the  Office of Science and Technology , [ 150 ]  which assigned regulatory approval of GM food to the USDA, FDA and EPA. [ 151 ]  The  Cartagena Protocol on Biosafety , an international treaty that governs the transfer, handling, and use of GMOs, [ 152 ]  was adopted on 29 January 2000. [ 153 ]  One hundred and fifty-seven countries are members of the Protocol, and many use it as a reference point for their own regulations. [ 154 ]


The legal and regulatory status of GM foods varies by country, with some nations banning or restricting them, and others permitting them with widely differing degrees of regulation. [ 155 ] [ 156 ] [ 157 ] [ 158 ]  Some countries allow the import of GM food with authorisation, but either do not allow its cultivation (Russia, Norway, Israel) or have provisions for cultivation even though no GM products are yet produced (Japan, South Korea).  Most countries that do not allow GMO cultivation do permit research. [ 159 ]  Some of the most marked differences occur between the US and Europe. The US policy focuses on the product (not the process), only looks at verifiable scientific risks and uses the concept of  substantial equivalence . [ 160 ]  The  European Union  by contrast has possibly the most stringent GMO regulations in the world. [ 161 ]  All GMOs, along with  irradiated food , are considered "new food" and subject to extensive, case-by-case, science-based food evaluation by the  European Food Safety Authority .  The criteria for authorisation fall in four broad categories: "safety", "freedom of choice", "labelling", and "traceability". [ 162 ]  The level of regulation in other countries that cultivate GMOs lie in between Europe and the United States.


One of the key issues concerning regulators is whether GM products should be labeled.  The  European Commission  says that mandatory labeling and traceability are needed to allow for informed choice, avoid potential  false advertising [ 173 ]  and facilitate the withdrawal of products if adverse effects on health or the environment are discovered. [ 174 ]  The  American Medical Association [ 175 ]  and the  American Association for the Advancement of Science [ 176 ]  say that absent scientific evidence of harm even voluntary labeling is  misleading  and will falsely alarm consumers. Labeling of GMO products in the marketplace is required in 64 countries. [ 177 ]  Labeling can be mandatory up to a threshold GM content level (which varies between countries) or voluntary. In Canada and the US labeling of GM food is voluntary, [ 178 ]  while in Europe all food (including  processed food ) or  feed  which contains greater than 0.9% of approved GMOs must be labelled. [ 161 ]


Critics have objected to the use of genetic engineering on several grounds, including ethical, ecological and economic concerns. Many of these concerns involve GM crops and whether food produced from them is safe and what impact growing them will have on the environment. These controversies have led to litigation, international trade disputes, and protests, and to restrictive regulation of commercial products in some countries. [ 179 ]


Accusations that scientists are " playing God " and other  religious issues  have been ascribed to the technology from the beginning. [ 180 ]  Other ethical issues raised include the  patenting of life , [ 181 ]  the use of  intellectual property  rights, [ 182 ]  the level of labeling on products, [ 183 ] [ 184 ]  control of the food supply [ 185 ]  and the objectivity of the regulatory process. [ 186 ]  Although doubts have been raised, [ 187 ]  economically most studies have found growing GM crops to be beneficial to farmers. [ 188 ] [ 189 ] [ 190 ]


Gene flow  between GM crops and compatible plants, along with increased use of selective  herbicides , can increase the risk of " superweeds " developing. [ 191 ]  Other environmental concerns involve potential impacts on non-target organisms, including  soil microbes , [ 192 ]  and an increase in secondary and resistant insect pests. [ 193 ] [ 194 ]  Many of the environmental impacts regarding GM crops may take many years to be understood and are also evident in conventional agriculture practices. [ 192 ] [ 195 ]  With the commercialisation of  genetically modified fish  there are concerns over what the environmental consequences will be if they escape. [ 196 ]


There are three main concerns over the safety of genetically modified food: whether they may provoke an  allergic reaction ; whether the genes could transfer from the food into human cells; and whether the genes not approved for human consumption could  outcross  to other crops. [ 197 ]  There is a  scientific consensus [ 198 ] [ 199 ] [ 200 ] [ 201 ]  that currently available food derived from GM crops poses no greater risk to human health than conventional food, [ 202 ] [ 203 ] [ 204 ] [ 205 ] [ 206 ]  but that each GM food needs to be tested on a case-by-case basis before introduction. [ 207 ] [ 208 ] [ 209 ]  Nonetheless, members of the public are less likely than scientists to perceive GM foods as safe. [ 210 ] [ 211 ] [ 212 ] [ 213 ]


Genetic engineering features in many  science fiction  stories. [ 214 ]   Frank Herbert 's novel  The White Plague  describes the deliberate use of genetic engineering to create a  pathogen  which specifically kills women. [ 214 ]  Another of Herbert's creations, the  Dune  series of novels, uses genetic engineering to create the powerful  Tleilaxu . [ 215 ]  Few films have informed audiences about genetic engineering, with the exception of the 1978  The Boys from Brazil  and the 1993  Jurassic Park , both of which make use of a lesson, a demonstration, and a clip of scientific film. [ 216 ] [ 217 ]  Genetic engineering methods are weakly represented in film; Michael Clark, writing for the  Wellcome Trust , calls the portrayal of genetic engineering and biotechnology "seriously distorted" [ 217 ]  in films such as  The 6th Day . In Clark's view, the biotechnology is typically "given fantastic but visually arresting forms" while the science is either relegated to the background or fictionalised to suit a young audience. [ 217 ]


Human genetic enhancement  or  human genetic engineering  refers to  human enhancement  by means of a  genetic modification . This could be done in order to cure diseases ( gene therapy ), prevent the possibility of getting a particular  disease [ 1 ]  (similarly to vaccines),  to improve athlete performance in sporting events ( gene doping ), or to change physical appearance, metabolism, and even improve physical capabilities and mental faculties such as memory and intelligence.
These genetic enhancements  may or may not be done in such a way that the change is heritable  (which has raised concerns within the scientific community). [ 2 ]


Genetics is the study of genes and inherited traits and while the ongoing advancements in this field have resulted in the advancement of healthcare at multiple levels, ethical considerations have become increasingly crucial especially alongside. Genetic engineering has always been a topic of moral debate among bioethicists. [ 3 ]  Even though the technological advancements in this field present exciting prospects for biomedical improvement, it also prompts the need for ethical, societal, and practical assessments to understand its impact on human biology, evolution, and the environment. [ 4 ]   Genetic testing ,  genetic engineering , and  stem cell research  are often discussed together due to the interrelated moral arguments surrounding these topics. The distinction between repairing genes and enhancing genes is a central idea in many moral debates surrounding genetic enhancement because some argue that repairing genes is morally permissible, but that genetic enhancement is not due to its potential to lead to social injustice through discriminatory eugenics initiatives. [ 5 ]


Moral questions related to  genetic testing  are often related to duty to warn family members if an inherited disorder is discovered, how physicians should navigate patient autonomy and confidentiality with regard to genetic testing, the ethics of genetic discrimination, and the moral permissibility of using genetic testing to avoid causing seriously disabled persons to exist, such as through selective abortion. [ 5 ] [ 6 ] [ 7 ]


The responsibility of public health professionals is to determine potential exposures and suggest testing for communicable diseases that require reporting. Public health professionals may encounter disclosure concerns if the extension of obligatory screening results in genetic abnormalities being classified as reportable conditions. [ 8 ]
Genetic data is personal and closely linked to a person's identity. Confidentiality concerns not only work, health care, and insurance coverage, but a family's whole genetic test results can be impacted. Affected individuals may also have their parents, children, siblings, sisters, and even extended relatives if the condition is either genetically dominant or carried by them. Moreover, a person's decisions could change their entire life depending on the outcome of a genetic test. Results of genetic testing may need to be disclosed in all facets of a person's life. [ 8 ] [ 9 ]


Non-invasive prenatal testing (NIPT) can accurately determine the sex of the fetus at an early stage of gestation, raising concerns about the potential facilitation of sex-selective termination of pregnancy (TOP) due to its ease, timing, and precision. Even though the ultrasound technology can do the same, NIPT is being explored due to its capability to accurately identify the fetus's sex at an early stage in pregnancy, with increasing precision as early as 7 weeks' gestation. This timeframe precedes the typical timing for other sex determination techniques, such as ultrasound or chorionic villus sampling (CVS). [ 10 ] [ 11 ]  The high early accuracy of NIPT reduces the uncertainty associated with other methods, such as the aforementioned, leading to more informed decisions and eliminating the risk of inaccurate results that could influence decision-making regarding sex-selective TOP. Additionally, NIPT enables sex-selective TOP in the first trimester, which is more practical, and allows pregnant women to postpone maternal-fetal bonding. These considerations may significantly facilitate the pursuit of sex-selective TOP when NIPT is utilized. Therefore, it is crucial to examine these ethical concerns within the framework of NIPT adoption. [ 12 ]


Ethical issues related to gene therapy and human genetic enhancement concern the medical risks and benefits of the therapy, the duty to use the procedures to prevent suffering, reproductive freedom in genetic choices, and the morality of practicing positive genetics, which includes attempts to improve normal functions. [ 5 ]


In every genetic based study conducted for humanity, studies must be carried out in accordance with the ethics committee approval statement, ethical, legal norms and human morality. CAR T cell therapy, which is intended to be a new treatment aims to change the genetics of T cells and transform immune system cells that do not recognize cancer into cells that recognize and fight cancer. it works with the T cell therapy method, which is arranged with palindromic repeats at certain short intervals called  CRISPR . [ 13 ]


All research involving human subjects in healthcare settings must be registered in a public database before the recruitment of the first trial. The informed consent statement should include adequate information about possible conflicts of interest, the expected benefits of the study, its potential risks, and other issues related to the discomfort it may involve. [ 14 ]


Technological advancements play an integral role in new forms of human enhancement. While phenotypic and somatic interventions for human enhancement provide noteworthy ethical and sociological dilemmas, germline heritable genetic intervention necessitates even more comprehensive deliberations at the individual and societal levels. [ 15 ]


Moral judgments are empirically based and entail evaluating prospective risk-benefit ratios particularly in the field of biomedicine. The technology of CRISPR genome editing raises ethical questions for several reasons. To be more specific, concerns exist regarding the capabilities and technological constraints of CRISPR technology. Furthermore, the long-term effects of the altered organisms and the possibility of the edited genes being passed down to succeeding generations and having unanticipated effects are two further issues to be concerned about. Decision-making on morality becomes more difficult when uncertainty from these circumstances prevents appropriate risk/benefit assessments. [ 16 ]


The potential benefits of revolutionary tools like CRISPR are endless. For example, because it can be applied directly in the embryo, CRISPR/Cas9 reduces the time required to modify target genes compared to gene targeting technologies that rely on the use of embryonic stem (ES) cells. Bioinformatics tools developed to identify the optimal sequences for designing guide RNAs and optimization of experimental conditions have provided very robust procedures that guarantee the successful introduction of the desired mutation. [ 17 ]  Major benefits are likely to develop from the use of safe and effective HGGM, making a precautionary stance against HGGM unethical. [ 18 ]


Going forward, many people support the establishment of an organization that would provide guidance on how best to control the ethical complexities mentioned above. Recently, a group of scientists founded the Association for Responsible Research and Innovation in Genome Editing (ARRIGE) to study and provide guidance on the ethical use of genome editing. [ 19 ] [ 20 ]


In addition, Jasanoff and Hurlbut have recently advocated for the establishment and international development of an interdisciplinary "global observatory for gene regulation". [ 21 ]


Researchers proposed that debates in gene editing should not be controlled by the scientific community. The network is envisioned to focus on gathering information from dispersed sources, bringing to the fore perspectives that are often overlooked, and fostering exchange across disciplinary and cultural divides. [ 22 ]


The interventions aimed at enhancing human traits from a genetic perspective are emphasized as being contingent upon the understanding of genetic engineering, and comprehending the outcomes of these interventions requires an understanding of the interactions between humans and other living beings. Therefore, the regulation of genetic engineering underscores the significance of examining the knowledge between humans and the environment. [ 15 ]


To address the ethical challenges and uncertainties arising from genetic advancements, the development of comprehensive guidelines based on universal principles has been emphasized as essential. The importance of adopting a cautious approach to safeguard fundamental values such as autonomy, global well-being, and individual dignity has been elucidated when overcoming these challenges. [ 23 ]


When contemplating genetic enhancement, genetic technologies should be approached from a broad perspective, using a definition that encompasses not only direct genetic manipulation but also indirect technologies such as biosynthetic drugs. It has been emphasized that attention should be given to expectations that can shape the marketing and availability of these technologies, anticipating the allure of new treatments. These expectations have been noted to potentially signify the encouragement of appropriate public policies and effective professional regulations. [ 24 ]


Clinical stem cell research must be conducted in accordance with ethical values. This entails a full respect for ethical principles, including the accurate assessment of the balance between risks and benefits, as well as obtaining informed and voluntary participant consent. The design of research should be strengthened, scientific and ethical reviews should be effectively coordinated, assurance should be provided that participants understand the fundamental features of the research, and full compliance with additional ethical requirements for disclosing negative findings has been addressed. [ 25 ]


Clinicians have been emphasized to understand the role of genomic medicine in accurately diagnosing patients and guiding treatment decisions. It has been highlighted that detailed clinical information and expert opinions are crucial for the accurate interpretation of genetic variants. While personalized medicine applications are exciting, it has been noted that the impact and evidence base of each intervention should be carefully evaluated. The human genome contains millions of genetic variants, so caution should be exercised and expert opinions sought when analyzing genomic results. [ 26 ]


With the discovery of various types of immune-related disorders, there is a need for diversification in prevention and treatment. Developments in the field of gene therapy are being studied to be included in the scope of this treatment, but of course more research is needed to increase the positive results and minimize the negative effects of gene therapy applications. [ 27 ]
The CRISPR/Cas9 system is also designed as a gene editing technology for the treatment of HIV-1/AIDS. CRISPR/Cas9 has been developed as the latest gene editing technique that allows the insertion, deletion and modification of DNA sequences and provides advantages in the disruption of the latent HIV-1 virus. However, the production of some vectors for HIV-1-infected cells is still limited and further studies are needed [ 28 ]
Being an HIV carrier also plays an important role in the incidence of cervical cancer. While there are many personal and biological factors that contribute to the development of cervical cancer, HIV carriage is correlated with its occurrence. However, long-term research on the effectiveness of preventive treatment is still ongoing. Early education, accessible worldwide, will play an important role in prevention. [ 29 ]
When medications and treatment methods are consistently adhered to, safe sexual practices are maintained and healthy lifestyle changes are implemented, the risk of transmission is reduced in most people living with HIV. Consistently implemented proactive prevention strategies can significantly reduce the incidence of HIV infections. Education on safe sex practices and risk-reducing changes for everyone, whether they are HIV carriers or not, is critical to preventing the disease. [ 30 ]
However, controlling the HIV epidemic and eliminating the stigma associated with the disease may not be possible only through a general AIDS awareness campaign. It is observed that HIV awareness, especially among individuals in low socio-economic regions, is considerably lower than the general population. Although there is no clear-cut solution to prevent the transmission of HIV and the spread of the disease through sexual transmission, a combination of preventive measures can help to control the spread of HIV. Increasing knowledge and awareness plays an important role in preventing the spread of HIV by contributing to the improvement of behavioral decisions with high risk perception. [ 31 ]
Genetics plays a pivotal role in disease prevention, offering insights into an individual's predisposition to certain conditions and paving the way for personalized strategies to mitigate disease risk. The burgeoning field of genetic testing and analysis has provided valuable tools for identifying genetic markers associated with various diseases, allowing for proactive measures to be taken in disease prevention  [ 32 ]  Disease prevention via genetic testing is made easier as genetic testing can unveil an individual's genetic susceptibility to certain diseases, enabling early detection and intervention which can be very crucial in disease like heritable cancers such and breast  [ 33 ] [ 34 ]  and ovarian cancer. [ 35 ] [ 36 ]  Having genetic information can inform the development of precision medicine approaches and targeted therapies for disease prevention in general. By identifying genetic factors contributing to disease susceptibility, such as specific gene mutations associated with autoimmune disorders, researchers can develop targeted therapies to modulate the immune response and prevent the onset or progression of these conditions. [ 37 ] [ 38 ] [ 39 ]


There are many types of neurodegenerative diseases. Alzheimer's disease is the one of the most common one of these diseases and it affects millions of people worldwide. The CRISPR-Cas9 techniques can be used to prevent the Alzheimer's disease. For example, it has a potential to correct the autosomal dominant mutaitons, problematic neurons, restoring the associated electrophysiological deficits and decreased the Aβ peptides. [ 40 ]  Amyotrophic Lateral Sclerosis (ALS) is another highly lethal neurodegenerative disease. And CRISPR-Cas9 technology is simple and effective for changinc specific point mutations about ALS. Also with this technology Chen and his colleagues were found some important alterations in major indicators of ALS like decreasing in RNA foci, polypeptides and haplosufficiency. [ 41 ] [ 40 ]


Some individuals experience  immunocompromise , a condition in which their immune systems are weakened and less effective in defending against various diseases, including but not limited to  influenza . This susceptibility to infections can be attributed to a range of factors, including genetic flaws and genetic diseases such as Severe Combined Immunodeficiency (SCID). Some gene therapies have already been developed or are being developed to correct these genetic flaws/diseases, hereby making these people less susceptible to catching additional diseases (i.e. influenza, ). [ 42 ]  These genetic flaws and diseases can significantly impact the body's ability to mount an effective immune response, leaving individuals vulnerable to a wide array of pathogens. However, advancements in gene therapy research and development have shown promising potential in addressing these genetic deficiencies however not without associated challenges [ 43 ] [ 44 ]


CRISPR technology is a promising tool not only for genetic disease corrections but also for the prevention of viral and bacterial infections. Utilizing CRISPR–Cas therapies, researchers have targeted viral infections like HSV-1, EBV, HIV-1, HBV, HPV, and HCV, with ongoing clinical trials for an HIV-clearing strategy named  EBT-101 . Additionally, CRISPR has demonstrated efficacy in preventing viral infections such as IAV and SARS-CoV-2 by targeting viral RNA genomes with Cas13d, and it has been used to sensitize antibiotic-resistant S. aureus to treatment through Cas9 delivered via bacteriophages. [ 45 ]


Advancements in gene editing and gene therapy hold promise for disease prevention by addressing genetic factors associated with certain conditions. Techniques like CRISPR-Cas9 offer the potential to correct genetic mutations associated with hereditary diseases, thereby preventing their manifestation in future generations and reducing disease burden. In November 2018,  Lulu and Nana  were created. [ 46 ]  By using clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9, a gene editing technique, they disabled a gene called CCR5 in the embryos, aiming to close the protein doorway that allows HIV to enter a cell and make the subjects immune to the HIV virus.


Despite existing evidence of CRISPR technology, advancements in the field persist in reducing limitations. Researchers developed a new, gentle gene editing method for embryos using nanoparticles and peptide nucleic acids. Delivering editing tools without harsh injections, the method successfully corrected genes in mice without harming development. While ethical and technical questions remain, this study paves the way for potential future use in improving livestock and research animals, and maybe even in human embryos for disease prevention or therapy. [ 47 ]


Informing prospective parents about their susceptibility to genetic diseases is crucial. Pre-implantation genetic diagnosis also holds significance for disease prevention by inheritance, as whole genome amplification and analysis help select a healthy embryo for implantation, preventing the transmission of a fatal metabolic disorder in the family. [ 48 ]


Genetic human enhancement emerges as a potential frontier in disease prevention by precisely targeting genetic predispositions to various illnesses. Through techniques like CRISPR, specific genes associated with diseases can be edited or modified, offering the prospect of reducing the hereditary risk of conditions such as cancer, cardiovascular disorders, or neurodegenerative diseases. This approach not only holds the potential to break the cycle of certain genetic disorders but also to influence the health trajectories of future generations.


Furthermore, genetic enhancement can extend its impact by focusing on fortifying the immune system and optimizing overall health parameters. By enhancing immune responses and fine-tuning genetic factors related to general well-being, the susceptibility to infectious diseases can be minimized. This proactive approach to health may contribute to a population less prone to ailments and more resilient in the face of environmental challenges.


However, the ethical dimensions of genetic manipulation cannot be overstated. Striking a delicate balance between scientific progress and ethical considerations is imperative. Robust regulatory frameworks and transparent guidelines are crucial to ensuring that genetic human enhancement is utilized responsibly, avoiding unintended consequences or potential misuse. As the field advances, the integration of ethical, legal, and social perspectives becomes paramount to harness the full potential of genetic human enhancement for disease prevention while respecting individual rights and societal values. [ 49 ]


Overall, the technology requires improvements in effectiveness, precision, and applications. Immunogenicity, off-target effects, mutations, delivery systems, and ethical issues are the main challenges that CRISPR technology faces. The safety concerns, ethical considerations, and the potential for misuse underscore the need for careful and responsible exploration of these technologies. [ 50 ]  CRISPR-Cas9 technology offers so much on disease prevention and treatment yet its future aspects, especially those that affect the next generations, should be investigated strictly.


Modification of human genes in order to treat genetic diseases is referred to as  gene therapy . Gene therapy is a medical procedure that involves inserting genetic material into a patient's cells to repair or fix a malfunctioning gene in order to treat hereditary illnesses. Between 1989 and December 2018, over 2,900  clinical trials  of gene therapies were conducted, with more than half of them in  phase I . [ 51 ]  Since that time, many gene therapy based drugs became available, such as  Zolgensma  and  Patisiran . Most of these approaches utilize  viral vectors , such as  adeno-associated viruses  (AAVs), adenoviruses (AV) and  lentiviruses  (LV), for inserting or replacing  transgenes   in vivo  or  ex vivo . [ 52 ] [ 53 ]


In 2023,  nanoparticles  that act similarly to viral vectors were created. These nanoparticles, called  bioorthgonal engineered virus-like recombinant biosomes , display strong and rapid binding capabilities to  LDL receptors  on cell surfaces, allowing them to enter cells efficiently and  deliver genes  to specific target areas, such as  tumor  and  arthritic tissues . [ 54 ]


RNA interference -based agents, such as  zilebesiran , contain  siRNA  which binds with mRNA of the target cells, modifying gene expression. [ 55 ]


Many diseases are complex and cannot be effectively treated by simple coding sequence-targeting strategies. CRISPR/Cas9 is one technology that targets double strand breaks in the human genome, modifying genes and providing a quick way to treat genetic disorders. Gene treatment employing the CRISPR/Cas genome editing method is known as CRISPR/Cas-based gene therapy. Mammalian cells can be genetically modified using the straightforward, affordable, and extremely specific CRISPR/Cas method. It can help with single-base exchanges, homology-directed repair, and non-homologous end joining. The primary application is targeted gene knockouts, involving the disruption of coding sequences to silence deleterious proteins. Since the development of the CRISPR-Cas9 gene editing method between 2010 and 2012, scientists have been able to alter genes by making specific breaks in their DNA. This technology has many uses, including  genome editing  and molecular diagnosis.


Genetic engineering has undergone a revolution because to CRISPR/Cas technology, which provides a flexible framework for building disease models in larger animals. This breakthrough has created new opportunities to evaluate possible therapeutic strategies and comprehend the genetic foundations of different diseases. But in order to fully realize the promise of CRISPR/Cas-based gene therapy, a number of obstacles must be removed. Improving CRISPR/Cas systems' editing precision and efficiency is one of the main issues. Although this technology makes precise gene editing possible, reducing off-target consequences is still a major challenge. Unintentional genetic changes resulting from off-target modifications may have unanticipated effects or difficulties. Using enhanced guide RNA designs, updated Cas proteins, and cutting-edge bioinformatics tools, researchers are actively attempting to improve the specificity and reduce off-target effects of CRISPR/Cas procedures. Moreover, the effective and specific delivery of CRISPR components to target tissues presents another obstacle. Delivery systems must be developed or optimized to ensure the CRISPR machinery reaches the intended cells or organs efficiently and safely. This includes exploring various delivery methods such as viral vectors, nanoparticles, or lipid-based carriers to transport CRISPR components accurately to the target tissues while minimizing potential toxicity or immune responses.


Despite recent progress, further research is needed to develop safe and effective CRISPR therapies. CRISPR/Cas9 technology is not actively used today, however there are ongoing clinical trials of its use in treating various disorders, including sickle cell disease, human papillomavirus (HPV)-related cervical cancer, COVID-19 respiratory infection, renal cell carcinoma, and multiple myeloma. [ 56 ]


Gene therapy  has emerged as a promising field in  medical science , aiming to address and treat various  genetic diseases  by modifying  human genes . The process involves the introduction of genetic material into a patient's cells, with the primary goal of repairing or correcting malfunctioning genes that contribute to  hereditary illnesses . This innovative medical procedure has seen significant advancements and a growing number of clinical trials since its inception.


Between 1989 and December 2018, more than 2,900 clinical trials of gene therapies were conducted, with over half of them reaching the phase I stage. Over the years, several gene therapy-based drugs have been developed and made available to the public, marking important milestones in the treatment of  genetic disorders . Examples include Zolgensma and Patisiran, which have demonstrated efficacy in addressing specific genetic conditions.


The majority of gene therapy approaches leverage viral vectors, such as adeno-associated viruses (AAVs), adenoviruses (AV), and lentiviruses (LV), to facilitate the insertion or replacement of transgenes either in vivo or ex vivo. These vectors serve as delivery vehicles for introducing the therapeutic genetic material into the patient's cells.


A notable development in 2023 was the creation of  nanoparticles  designed to function similarly to viral vectors. These bioorthogonal engineered virus-like recombinant biosomes represent a novel approach to gene delivery. They exhibit robust and rapid binding capabilities to low-density lipoprotein (LDL) receptors on cell surfaces, enhancing their efficiency in entering cells. This capability enables the targeted delivery of genes to specific areas, such as tumor and arthritic tissues. This advancement holds the potential to enhance the precision and effectiveness of gene therapy, minimizing off-target effects and improving overall therapeutic outcomes.


In addition to viral vector and nanoparticle-based approaches, RNA interference (RNAi) has emerged as another strategy in gene therapy. Agents like zilebesiran utilize small interfering RNA (siRNA) that binds with the messenger RNA ( mRNA ) of target cells, effectively modifying gene expression. This RNA interference-based approach provides a targeted and specific method for regulating gene activity, presenting further opportunities for treating  genetic disorders .


The continuous evolution of  gene therapy techniques , along with the development of innovative delivery systems and therapeutic agents, underscores the ongoing commitment of the scientific and medical communities to advance the field and provide effective treatments for a wide range of genetic diseases.
 [ 57 ]


Athletes might adopt gene therapy technologies to improve their performance. [ 58 ]   Gene doping  is not known to occur, but multiple gene therapies may have such effects. Kayser et al. argue that gene doping could  level the playing field  if all athletes receive equal access. Critics claim that any therapeutic intervention for non-therapeutic/enhancement purposes compromises the ethical foundations of medicine and sports. [ 59 ]  Therefore, this technology, which is a subfield of genetic engineering commonly referred to as gene doping in sports, has been prohibited due to its potential risks. [ 60 ]  The primary objective of gene doping is to aid individuals with medical conditions. However, athletes, cognizant of its associated health risks, resort to employing this method in pursuit of enhanced athletic performance. The prohibition of the indiscriminate use of gene doping in sports has been enforced since the year 2003, pursuant to the decision taken by the World Anti-Doping Agency (WADA). [ 61 ]  A study conducted in 2011 underscored the significance of addressing issues related to gene doping and highlighted the importance of promptly comprehending how gene doping in sports and exercise medicine could impact healthcare services by elucidating its potential to enhance athletic performance. The article elucidates, according to the World Anti-Doping Agency (WADA), how gene doping poses a threat to the fairness of sports. Additionally, the paper delves into health concerns that may arise as a consequence of the utilization of gene doping solely for the purpose of enhancing sports performance. [ 62 ]  The misuse of gene doping to enhance athletic performance constitutes an unethical practice and entails significant health risks, including but not limited to cancer, viral infections, myocardial infarction, skeletal damage, and autoimmune complications. In addition, gene doping may give rise to various health issues, such as excessive muscle development leading to conditions like hypertonic cardiomyopathy, and render bones and tendons more susceptible to injuries [ 63 ]  Several genes such as EPO, IGF1, VEGFA, GH, HIFs, PPARD, PCK1, and myostatins are prominent choices for gene doping. Particularly in gene doping, athletes employ substances such as antibodies against myostatin or myostatin blockers. These substances contribute to the augmentation of the athletes' mass, facilitation of increased muscle development, and enhancement of strength. However, the primary genes utilized for gene doping in humans may lead to complications such as excessive muscle growth, which can adversely impact the cardiovascular system and increase the likelihood of injuries. [ 64 ]  However, due to insufficient awareness of these risks, numerous athletes resort to employing gene doping for purposes divergent from its genuine intent. Within the realm of athlete health, sports ethics and the ethos of fair play, scientists have developed various technologies for the detection of gene doping. Although in its early years the technology used wasn’t reliable, more extensive research has been done for better techniques to uncover gene doping instances that have been more successful. In the beginning, scientist resorted to techniques such as PCR in its various forms. This was not successful due to the fact that such technologies rely on exon-exon junctions in the DNA. This leads to a lack of precision in its detection as results can be easily tampered using misleading primers and gene doping would go undetected. [ 65 ]  With the emerge of new technologies, more recent studies utilized Next Generation Sequencing (NGS) as a method of detection. With the help of bioinformatics, this technology surpassed previous sequencing techniques in its in-depth analysis of DNA make up. Next Generation Sequencing (NGS) focuses on using an elaborate method of analyzing sample sequence and comparing it to a pre-existing reference sequence from a gene database. This way, primer tampering is not possible as the detection is on a genomic level. Using bioinformatic visualizing tools, data can be easily read and sequences that do not align with reference sequence can be highlighted. [ 66 ] [ 67 ]  Most recently, One of the high-efficiency gene doping analysis methods conducted in the year 2023, leveraging cutting-edge technology, is HiGDA (High-efficiency Gene Doping Analysis), which employs CRISPR/deadCas9 technology. [ 68 ]


The ethical issues concerning gene doping have been present long before its discovery. Although gene doping is relatively new, the concept of genetic enhancement of any kind has always been subject to ethical concerns. Even when used in a therapeutic manner, gene therapy poses many risks due to its unpredictability among other reasons. Factors other than health issues have raised ethical questions as well. These are mostly concerned with the hereditary factor of these therapies, where gene editing in some cases can be transmitted to the next generation with higher rates of unpredictability and risks in outcomes. [ 69 ]  For this reason, non-therapeutic application of gene therapy can be seen as a riskier approach to a non-medical concern. [ 70 ]


In a study, from history to today, human beings have always been in competition. While in the past warriors competed to be stronger in wars, today there is competition to be successful in every field, and it is understood that this psychology is a phenomenon that has always existed in human history until today. It is known that although an athlete has genetic potential, he cannot become a champion if he does not comply with the necessary training and lifestyle. However, as competition increases, both more physical training and more mental performance are needed. Just as warriors in history used some herbal cures to look stronger and more aggressive, it is a fact that today, athletes resort to doping methods to increase their performance. However, this situation is against sports ethics because it does not comply with the morality and understanding of the game. [ 71 ]


One of the negative effects is the risk of cancer, and as a positive effect is taking precautions against certain pathological conditions.Altering genes could lead to unintended and unpredictable changes in the body, potentially causing unforeseen health issues. Further effects of gene doping in sports is the constant fight against drugs not approved by the World Anti doping agency and unfairness regarding athletes that take drugs and don't. The long-term health consequences of gene doping may not be fully understood, and athletes may face health problems later in life. [ 72 ]


Other hypothetical gene therapies could include changes to physical appearance, metabolism, mental faculties such as memory and intelligence, and well-being (by increasing resistance to  depression  or relieving  chronic pain , for example). [ 73 ] [ 74 ]


The exploration of challenges in understanding the effects of gene alterations on phenotypes, particularly within natural genetic diversity, is highlighted. Emphasis is placed on the potential of systems biology and advancements in  genotyping / phenotyping  technologies for studying complex traits. Despite progress, persistent difficulties in predicting the influence of gene alterations on phenotypic changes are acknowledged, emphasizing the ongoing need for research in this area. [ 75 ]


Some congenital disorders (such as  those affecting the muscoskeletal system ) may affect physical appearance, and in some cases may also cause physical discomfort. Modifying the genes causing these congenital diseases (on those diagnosed to have mutations of the gene known to cause these diseases) may prevent this.


- Phenotypic Impacts of CRISPR-Cas9 Editing in Mice Targeting the Tyr Gene:


In a comprehensive  CRISPR - Cas9  study on gene editing, the Tyr gene in mice was targeted, seeking to instigate genetic alterations. The analysis found no off-target effects across 42 subjects, observing modifications exclusively at the intended Tyr locus. Though specifics were not explicitly discussed, these alterations may potentially influence non-defined aspects, such as coat color, emphasizing the broader potential of gene editing in inducing diverse phenotype changes. [ 76 ]


Also changes in the myostatin gene [ 77 ]  may alter appearance.


Significant  quantitative genetic  discoveries were made in the 1970s and 1980s, going beyond estimating heritability. However, issues such as  The Bell Curve  resurfaced, and by the 1990s, scientists recognized the importance of  genetics  for behavioral traits such as  intelligence . The  American Psychological Association 's Centennial Conference in 1992 chose  behavioral genetics  as a theme for the past, present, and future of  psychology .  Molecular genetics  synthesized, resulting in the  DNA  revolution and behavioral  genomics , as  quantitative genetic  discoveries slowed. Individual behavioral differences can now be predicted early thanks to the  behavioral sciences'  DNA revolution. The first law of behavioral genetics was established in 1978 after a review of thirty twin studies revealed that the average heritability estimate for intelligence was 46%. [ 78 ]   Behavior  may also be modified by genetic intervention. [ 79 ]   Some people may be aggressive, selfish, and may not be able to function well in society. Mutations in GLI3 and other patterning genes have been linked to HH etiology, according to genetic research. Approximately 50%-80% of children with HH have acute wrath and violence, and the majority of patients have externalizing problems. Epilepsy may be preceded by behavioral instability and intellectual incapacity. [ 80 ]  There is currently research ongoing on genes that are or may be (in part) responsible for selfishness (e.g.  ruthlessness gene ), aggression (e.g.  warrior gene ), altruism (e.g.  OXTR ,  CD38 ,  COMT ,  DRD4 ,  DRD5 ,  IGF2 ,  GABRB2 [ 81 ] )


There has been a great anticipation of  gene editing  technology to modify genes and regulate our  biology  since the invention of recombinant DNA technology. These expectations, however, have mostly gone unmet. Evaluation of the appropriate uses of germline interventions in reproductive medicine should not be based on concerns about enhancement or eugenics, despite the fact that gene editing research has advanced significantly toward clinical application. [ 82 ]


Cystic fibrosis (CF)  is a  hereditary disease  caused by mutations in the  Cystic fibrosis transmembrane conductance regulator (CFTR)  gene. While 90% of CF patients can be treated, current treatments are not curative and do not address the entire spectrum of CFTR mutations. Therefore, a comprehensive, long-term therapy is needed to treat all  CF  patients once and for all.  CRISPR/Cas   gene editing  technologies are being developed as a viable platform for genetic treatment. [ 83 ]  However, the difficulties of delivering enough  CFTR gene  and sustaining expression in the lungs has hampered gene therapy's efficacy. Recent technical breakthroughs, including as  viral  and non-viral vector transport, alternative  nucleic acid  technologies, and new technologies like  mRNA  and  CRISPR gene editing , have taken use of our understanding of  CF  biology and airway epithelium. [ 84 ]


Human  gene transfer  has held the promise of a lasting remedy to hereditary illnesses such as  cystic fibrosis (CF)  since its conception and use. The emergence of sophisticated technologies that allow for site-specific alteration with programmable nucleases has greatly revitalized the area of  gene therapy . [ 85 ]  There is some research going on on the  hypothetical  treatment of psychiatric disorders by means of gene therapy. It is assumed that, with gene-transfer techniques, it is possible (in experimental settings using animal models) to alter CNS gene expression and thereby the intrinsic generation of molecules involved in neural plasticity and neural regeneration, and thereby modifying ultimately behaviour. [ 86 ]


In recent years, it was possible to modify ethanol intake in animal models. Specifically, this was done by targeting the expression of the aldehyde dehydrogenase gene (ALDH2), lead to a significantly altered alcohol-drinking behaviour. [ 87 ]  Reduction of p11, a serotonin receptor binding protein, in the nucleus accumbens led to depression-like behaviour in rodents, while restoration of the p11 gene expression in this anatomical area reversed this behaviour. [ 73 ]


Recently, it was also shown that the gene transfer of CBP (CREB (c-AMP response element binding protein) binding protein) improves cognitive deficits in an animal model of Alzheimer's dementia via increasing the expression of BDNF (brain-derived neurotrophic factor). [ 88 ]  The same authors were also able to show in this study that accumulation of amyloid-β (Aβ) interfered with CREB activity which is physiologically involved in memory formation.


In another study, it was shown that Aβ deposition and plaque formation can be reduced by sustained expression of the neprilysin (an endopeptidase) gene which also led to improvements on the behavioural (i.e. cognitive) level. [ 89 ]


Similarly, the intracerebral gene transfer of ECE (endothelin-converting enzyme) via a virus vector stereotactically injected in the right anterior cortex and hippocampus, has also shown to reduce Aβ deposits in a transgenic mouse model of Alzeimer's dementia. [ 90 ]