The  hepatitis B virus  (HBV) genome encodes its own  DNA polymerase  for replication. Biomics Biotechnologies has evaluated around 5000 siRNA sequences of this gene for effective knockdown; five sequences were chosen for further investigation and were shown to have silencing activity when converted into shRNA expression cassettes. A multi-cassette construct, Hepbarna, is under preclinical development for delivery by an  adeno-associated virus  8 (AAV-8) liver-targeting vector. [ citation needed ]


Classified as an  orphan disease , there is currently no therapy for  oculopharyngeal muscular dystrophy  (OPMD), as it is caused by a mutation in the  poly(A) binding protein nuclear 1  (PABPN1) gene. Silencing the mutant gene using ddRNAi offers a potential therapeutic approach. [ 11 ]


Besides the ex vivo approach discussed above, the Center for Infection and Immunity Amsterdam (CINIMA) of the  University of Amsterdam , Netherlands, is extensively researching the therapeutic potential of multi-cassette DNA constructs for HIV. [ 12 ]


As with all  gene therapies , a number of safety and toxicity issues must be evaluated during the development of ddRNAi therapeutic techniques.


Some gene therapy vectors integrate into the host genome, thereby acting as insertional  mutagens . This was a particular issue with early  retroviral vectors , where insertions adjacent to  oncogenes  resulted in the development of  lymphoid  tumors. [ 13 ]


Adeno-associated virus (AAV) vectors are considered low-risk for host-genome integration, as AAV infection has not been associated with the induction of cancers in humans despite widespread prevalence across the general population. Moreover, extensive clinical use of AAV vectors has provided no evidence of  carcinogenicity . While lentiviral vectors do integrate into the genome, they do not appear to show a propensity to activate oncogene expression. [ 14 ]


An  immunological response  to an adenoviral vector resulted in the death of a patient in an early human trial. Careful monitoring of potential toxicities in preclinical testing and analysis of any pre-existing antibodies to gene therapy vectors in patients minimizes such risks. [ citation needed ]


siRNAs have been shown to activate immune responses through interaction with  toll-like receptors  (TLRs), leading to  interferon  responses. These TLRs reside on the cell's outer surface, so ddRNAi constructs, which are delivered directly into the intracellular space, are not expected to induce such a response. [ 15 ]


High-level expression of shRNAs has been shown to be toxic. Strategies to minimize levels of shRNA expression [ 4 ]  or promote precise processing of shRNAs [ 16 ]  can mitigate this issue.


The unintended silencing of genes that share  sequence homology  with expressed shRNAs could theoretically occur. [ 17 ]  Careful selection of shRNA sequences and thorough preclinical testing of constructs can mitigate this risk.


DTA GVeiw  is a  program  in which  genomes  of several  organisms  can be pulled up on the program to immediately show a list of known genes and where they are located in the genome. It has been referred to as a " google maps " of genomes. [ 1 ] [ 2 ]


The purpose of DTA GVeiw is to collect  data  of organisms, in the hope of one day engineering  microbial organisms  to help make mars a suitable place for life. [ 1 ]


DARPA , the creator of DTA GVeiw, Hopes one day they will be enabled to "pick and choose" desired  genes  to create new organisms capable of living on a foreign planet. [ 1 ]


The  Enlist Weed Control System  is an  agricultural  system that includes seeds for  genetically modified crops  that are resistant to Enlist (a broadleaf  herbicide  with two active agents,   2,4-Dichlorophenoxyacetic acid  (2,4-D) and  glyphosate ) and the Enlist herbicide; spraying the herbicide will kill weeds but not the resulting crop. [ 1 ] [ 2 ]  The system was developed by  Dow AgroSciences , part of   Dow Chemical Company . [ 1 ]  In October 2014 the system was registered for restricted use in  Illinois, Indiana, Iowa, Ohio, South Dakota and Wisconsin by the  US Environmental Protection Agency . [ 3 ]  In 2013, the system was approved by Canada for the same uses. [ 4 ]


The Enlist approach was developed to replace the "Roundup-Ready" system that was introduced in 1996 by  Monsanto  and which has become less useful with the rise of glyphosate-resistant weeds. [ 1 ]


Enlist Duo  is an  herbicide  that contains the choline form of  2,4-Dichlorophenoxyacetic acid  (2,4-D) and  glyphosate  plus an unknown number of unlisted ingredients.  Dow added chemicals to the mixture in what it termed  "Colex-D technology". [ 5 ]


2,4-D is one of the most widely used herbicides in the world. [ 6 ] [ 7 ]  2,4-D is volatile and by EPA assessment is a hazardous air pollutant that is difficult to contain. [ citation needed ]  According to Dow, the Colex-D formulation reduces drift and damage from evaporation. [ 8 ]  As of 2013 glyphosate was the world's largest-selling herbicide, with sales driven by glyphosate-resistant  genetically modified crops . [ 9 ]


Other countries assessing the system include Brazil, Argentina and various food importing countries. [ 10 ]


As of April 2014 maize and soybeans resistant to 2,4-D and glyphosate had been approved in Canada, [ 10 ]  and in September 2014 the  USDA  approved the same two crops. [ 11 ] [ 12 ]


2,4-D was one of the main ingredients of  Agent Orange , a defoliant used during the  Vietnam War  that was blamed for many health problems. [ 13 ]  According to a Reuters article the main health problems arose from  TCDD  contamination created in the synthesis of the other Agent Orange component,  2,4,5-T [ 14 ]


The  U.S. Environmental Protection Agency  has moved to rescind its approval due to conflicting claims from the manufacturer about synergistic effects from mixing the two herbicides. Dow had told the EPA that the combination of the two herbicides didn't enhance their toxicity to plants, but an earlier  patent application  from Dow claimed that it did. [ 15 ]


Epigenome editing  or  epigenome engineering  is a type of genetic engineering in which the  epigenome  is modified at specific sites using engineered molecules targeted to those sites (as opposed to whole-genome modifications). Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.


The engineered proteins used for epigenome editing are composed of a DNA binding domain that target specific sequences and an effector domain that modifies epigenomic features. Currently, three major groups of DNA binding proteins have been predominantly used for epigenome editing:  Zinc finger  proteins,  Transcription Activator-Like Effectors (TALEs)  and nuclease deficient Cas9 fusions ( CRISPR ).


Comparing genome-wide  epigenetic  maps with  gene expression  has allowed researchers to assign either activating or repressing roles to specific modifications. The importance of DNA sequence in regulating the epigenome has been demonstrated by using DNA motifs to predict epigenomic modification. [ 1 ]  Further insights into mechanisms behind  epigenetics  have come from  in vitro  biochemical and structural analyses. Using  model organisms , researchers have been able to describe the role of many  chromatin  factors through  knockout  studies. However knocking out an entire chromatin modifier has massive effects on the entire genome, which may not be an accurate representation of its function in a specific context. As one example of this,  DNA methylation  occurs at  repeat regions ,  promoters ,  enhancers , and  gene  bodies. Although  DNA methylation  at gene promoters typically correlates with gene repression, methylation at gene bodies is correlated with gene activation, and DNA methylation may also play a role in gene splicing. [ 2 ]    The ability to directly target and edit individual methylation sites is critical to determining the exact function of DNA methylation at a specific site. Epigenome editing is a powerful tool that allows this type of analysis. For site-specific DNA methylation editing as well as for histone editing, genome editing systems have been adapted into epigene editing systems. In short, genome homing proteins with engineered or naturally occurring nuclease functions for gene editing, can be mutated and adapted into purely delivery systems. An epigenetic modifying enzyme or domain can be fused to the homing protein and local epigenetic modifications can be altered upon protein recruitment. Exceptionally for  DNA methylation , the homing domain itself can be enough to interfere with normal epigenetic processes to lead to targeted epigenetic editing. [ 3 ]


The  Transcription Activator-Like Effector (TALE)  protein recognizes specific DNA sequences based on the composition of its  DNA binding domain . [ 4 ]   This allows the researcher to construct different TALE proteins to recognize a target DNA sequence by editing the TALE's primary protein structure. The binding specificity of this protein is then typically confirmed using  Chromatin Immunoprecipitation  (ChIP) and  Sanger sequencing  of the resulting DNA fragment. [ 5 ] [ 6 ] [ 7 ]  This confirmation is still required on all TALE sequence recognition research. [ 8 ]
When used for epigenome editing, these DNA binding proteins are attached to an effector protein. Effector proteins that have been used for this purpose include  Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) , [ 6 ]   Lysine (K)-specific demethylase 1A (LSD1) [ 7 ]  and  Calcium and integrin binding protein 1 (CIB1) . [ 5 ]


The use of  zinc finger -fusion proteins to recognize sites for epigenome editing has been explored as well. Maeder et al. has constructed a ZF-TET1 protein for use in DNA demethylation. [ 6 ]  These zinc finger proteins work similarly to  TALE proteins  in that they are able to bind to sequence specific sites in on the DNA based on their protein structure which can be modified. Chen et al. have successfully used a zinc finger DNA binding domain coupled with the  TET1  protein to induce demethylation of several previously silenced genes. [ 9 ]  Kungulovski and Jeltsch successfully used ZFP-guided deposition of DNA methylation gene to cause gene silencing but the DNA methylation and silencing were lost when the trigger signal stopped. The authors suggest for stable epigenetic changes, there must be either multiple depositions of DNA methylation of related epigenetic marks, or long-lasting trigger stimuli. [ 10 ]  ZFP epigenetic editing has shown potential to treat various neurodegenerative diseases. [ 11 ]


The  Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) -Cas system functions as a DNA site-specific nuclease. [ 12 ]  In the well-studied type II CRISPR system, the Cas9 nuclease associates with a chimera composed of tracrRNA and crRNA. This chimera is frequently referred to as a guide RNA (gRNA). When the Cas9 protein associates with a DNA region-specific gRNA, the Cas9 cleaves DNA at targeted DNA loci. However, when the D10A and H840A point mutations are introduced, a catalytically-dead Cas9 (dCas9) is generated that can bind DNA but will not cleave. [ 13 ]  The dCas9 system has been utilized for targeted epigenetic reprogramming in order to introduce site-specific DNA methylation. By fusing the  DNMT3a  catalytic domain with the dCas9 protein, dCas9-DNMT3a is capable of achieving targeted DNA methylation of a targeted region as specified by the present guide RNA. [ 14 ]  Similarly, dCas9 has been fused with the catalytic core of the human acetyltransferase  p300 . dCas9-p300 successfully catalyzes targeted acetylation of histone H3 lysine 27. [ 15 ] [ 16 ]  Alternatively, the dCas9 protein alone is sufficient to physically interfere with normal processes which maintain  DNA methylation  at the site to which it is targeted in dividing cells; this results in targeted DNA demethylation. The primary benefit of this approach is that it is free of epigenetic-modifying enzymes, which may affect epigenetic marks over large distances and act independently throughout the genome despite being tethered to a targeted dCas9 protein, often leading to widespread off-target effects. [ 3 ]


A variant in CRISPR epigenome editing (called FIRE-Cas9) allows to reverse the changes made, in case something went wrong. [ 17 ] [ 18 ]


CRISPRoff  is a dead Cas9 fusion protein that can be used to heritably silence the gene expression of "most genes" and allows for reversible modifications. [ 19 ] [ 20 ]


TET1  induces demethylation of cytosine at  CpG sites . This protein has been used to activate genes that are repressed by CpG methylation and to determine the role of individual CpG methylation sites. [ 6 ]  It is widely believed that targeted demethylation is typically better achieved by dCas9 alone (by targeted interference with normal DNA methylation machinery) as introduction of dCas9-TET into cells leads to widespread off-target activity of the over-expressed TET enzyme.  [ 3 ]   LSD1  induces the demethylation of  H3K4me1 /2, which also causes an indirect effect of deacetylation on H3K27. This effector can be used on  histones  in enhancer regions, which can changes the expression of neighboring genes. [ 7 ]   CIB1  is a light sensitive  cryptochrome , this cryptochrome is fused to the TALE protein. A second protein contains an interaction partner ( CRY2 ) fused with a  chromatin /DNA modifier (ex.  SID4X ).  CRY2  is able to interact with  CIB1  when the cryptochrome has been activated by illumination with blue light. [ 21 ]  The interaction allows the chromatin modifier to act on the desired location. This means that the modification can be performed in an inducible and reversible manner, which reduces long-term secondary effects that would be caused by constitutive epigenetic modification. [ 5 ]


Editing of gene enhancer regions in the genome through targeted epigenetic modification has been demonstrated by Mendenhall et al. (2013). [ 7 ]  This study utilized a TALE-LSD1 effector fusion protein in order to target enhancers of genes, to induce enhancer silencing in order to deduce enhancer activity and gene control. Targeting specific enhancers followed by locus specific  RT-qPCR  allows for the genes affected by the silenced enhancer to be determined. Alternatively, inducing enhancer silencing in regions upstream of genes allows for gene expression to be altered.  RT-qPCR  can then be utilized to study effects of this on gene expression. This allows for enhancer function and activity to be studied in detail. [ 7 ]


It is important to understand the role specific methylation sites play regulating in gene expression. To study this, one research group used a TALE-TET1 fusion protein to demethylate a single CpG methylation site. [ 6 ]  Although this approach requires many controls to ensure specific binding to target loci, a properly performed study using this approach can determine the biological function of a specific CpG methylation site. [ 6 ]


Epigenetic editing using an inducible mechanism offers a wide array of potential use to study epigenetic effects in various states. One research group employed an  optogenetic  two-hybrid system which integrated the sequence specific TALE DNA-binding domain with a light-sensitive cryptochrome 2 protein ( CIB1 ). [ 5 ]  Once expressed in the cells, the system was able to inducibly edit histone modifications and determine their function in a specific context. [ 5 ]


Targeted regulation of disease-related genes may enable novel therapies for many diseases, especially in cases where adequate gene therapies are not yet developed or are inappropriate. [ 22 ]  While transgenerational and population level consequences are not fully understood, it may become a major tool for applied functional genomics and  personalized medicine . [ 23 ]  As with  RNA editing , it does not involve genetic changes and their accompanying risks. [ 22 ]  One example of a potential functional use of epigenome editing was described in 2021: repressing  Na v 1.7   gene expression  via  CRISPR-dCas9  which showed therapeutic potential in three mouse models of chronic pain. [ 24 ] [ 25 ]


In 2022, research assessed its usefulness in reducing  tau protein  levels, regulating a protein involved in  Huntington's disease , targeting an inherited form of obesity, and  Dravet syndrome . [ 26 ]


Sequence specificity is critically important in epigenome editing and must be carefully verified (this can be done using  chromatin immunoprecipitation  followed by  Sanger sequencing  to verify the targeted sequence). [ 8 ]  It is unknown if the TALE fusion may cause effects on the catalytic activity of the epigenome modifier. This could be especially important in effector proteins that require multiple subunits and complexes such as the  Polycomb  repressive complex. [ 8 ]  Proteins used for epigenome editing may also obstruct ligands and substrates at the target site. [ 8 ]  The TALE protein itself may even compete with  transcription factors  if they are targeted to the same sequence. [ 8 ]  In addition, DNA repair systems could reverse the alterations on the chromatin and prevent the desired changes from being made. [ 8 ]  Finally, enzymes fused to dCas9 typically are able to act independently of the dCas9 protein that they are fused to. When these fusions are over-expressed in cells, these enzymes tend to modify large spans of the genome in what constitutes dramatic off-target activity. [ 3 ] [ 27 ]  It is therefore necessary for fusion constructs and targeting mechanisms to be optimized for reliable and repeatable epigenome editing.


The  eradication  or  abolition of suffering  is the concept of using  biotechnology  to create a permanent absence of involuntary  pain  and  suffering  in all  sentient beings .


The  discovery of modern anesthesia  in the 19th century was an early breakthrough in the elimination of pain during surgery, but acceptance was not universal. Some medical practitioners at the time believed that  anesthesia  was an artificial and harmful intervention in the body's natural response to injury. [ 1 ]  Opposition to anesthesia has since dissipated; however, the prospect of eradicating pain raises similar concerns about interfering with life's natural functions. [ 2 ]


People who are naturally incapable of feeling pain or unpleasant sensations due to rare conditions like  pain asymbolia  or  congenital insensitivity to pain  have been studied to discover the biological and genetic reasons for their pain-free lives. [ 3 ]  A Scottish woman with a previously unreported genetic mutation in a  FAAH   pseudogene  (dubbed FAAH-OUT) with resultant elevated  anandamide  levels was reported in 2019 to be immune to anxiety, unable to experience fear, and insensitive to pain. The frequent burns and cuts she had due to her full  hypoalgesia  healed quicker than average. [ 4 ] [ 5 ] [ 6 ]


In 1990,  Medical Hypotheses  published an article by L. S. Mancini on the "genetic engineering of a world without pain": [ 7 ]


A hypothesis is presented to the effect that everything adaptive which is achievable with a mind capable of experiencing varying degrees of both pleasure and pain (the  human condition  as we know it) could be achieved with a mind capable of experiencing only varying degrees of pleasure.


The development of  gene editing  techniques like  CRISPR  has raised the prospect that "scientists can identify the causes of certain unusual people's physical  superpowers  and use gene editing to grant them to others." [ 8 ]  Geneticist  George Church  has commented on the potential future of replacing pain with a painless sensory system: [ 9 ]


I imagine what this would be like on another planet and in the future, and... given that imagined future, whether we would be willing to  come back to where we are now . Rather than saying whether we're willing to go forward... ask whether you're willing to come back.


Ethicists and philosophers in the schools of  hedonism  and  utilitarianism , especially  negative utilitarianism , have debated the merits of eradicating suffering. [ 10 ]   Transhumanist  philosopher  David Pearce , in  The Hedonistic Imperative  (1995), argues that the abolition of suffering is both technically feasible and an issue of moral urgency, [ 11 ]  stating that: "It is predicted that the world's last unpleasant experience will be a precisely dateable event." [ 12 ]


The philosopher  Nick Bostrom , director of the  Future of Humanity Institute , advises a more cautious approach due to pain's function in protecting individuals from harm. However, Bostrom supports the core idea of using biotechnology to get rid of "a huge amount of unnecessary and undeserved suffering." [ 10 ]  It has also been argued that the eradication of suffering through biotechnology may bring about unwanted consequences, and arguments have been made that transhumanism is not the only philosophy worthy of consideration regarding the question of suffering — many people view suffering as one aspect in a   dualist  understanding of psychological and physical functioning, without which  pleasure  could not exist. [ 13 ]


In 2009, Adam Shriver suggested replacing animals in  factory farming  with genetically engineered animals with a reduced or absent capacity to suffer and feel pain. [ 14 ]  Shriver and McConnachie argued that people who wish to improve  animal welfare  should support gene editing in addition to  plant-based diets  and  cultured meat . [ 15 ]


Katrien Devolder and Matthias Eggel proposed gene editing  research animals  to remove pain and suffering. This would be an intermediate step towards eventually stopping all experimentation on animals and adopting  alternatives . [ 16 ]


Concerning  wild-animal suffering , CRISPR-based  gene drives  have been suggested as a cost-effective way of spreading benign  alleles  in  sexually reproducing  species. [ 17 ] [ 18 ] [ 19 ]  To limit gene drives spreading indefinitely (for test programmes, for example), the Sculpting Evolution group at the  MIT Media Lab  developed a self-exhausting form of CRISPR-based gene drive called a "daisy-chain drive." [ 20 ] [ 21 ]  For potential adverse effects of a gene drive, "[s]everal genetic mechanisms for limiting or eliminating gene drives have been proposed and/or developed, including synthetic resistance, reversal drives, and immunizing reversal drives." [ 22 ]


EUginius  is an Internet-based database application for  Genetically modified organisms  (GMOs). The name EUginius is an acronym and stands for  EU ropean  G MO  Ini tiative for a  U nified Database  S ystem.


The EUginius database was created on the initiative of the German  Federal Office of Consumer Protection and Food Safety  (BVL) and the  Dutch research Institute Wageningen Food Safety Research  (WFSR, formerly RIKILT). Building on parallel preparatory work by both cooperation partners, EUginius has been jointly developed and maintained since 2010, and has been online since October 2014. The information is provided in English.


EUginius aims to assist competent authorities as well as interested private users in finding accurate information on the presence, detection and identification of GMOs. Data on the GMOs’ molecular characterisation and traits, detection methods, reference materials, and authorisation status (currently limited to the EU) are provided. EUginius is tax-financed and therefore offers its information on the GMOs freely accessible. Information on releases carried out and their geographical location are not provided in EUginius.


Most of the GMOs present in EUginius are used for genetically modified food and feed, the majority plants (e.g. pest resistant  Bt maize ,  Golden rice  with enhanced synthesis of ß-carotene), and thus come from the field of green biotechnology. There is also information on genetically modified animals. EUginius provides for example information on a fast-growing genetically modified salmon ( AquAdvantage ) as well as information on genetically modified insects that have been developed to combat vectors of pathogens (e.g. Aedes aegypti OX5034, used to reduce the yellow fever mosquito population). In addition, in some cases, information is provided for the detection of genetically modified microbial production strains of food or feed additives (white biotechnology).


Since the European Union classifies organisms developed using  New Breeding Techniques  (NBTs) as GMOs, EUginius provides information about commercialised NBT-organisms, including genome edited organisms such as the high-oleic soybean, the larger growing pufferfish or the heat-tolerant cattle. Furthermore, EUginius lists, to some extent, published NGT organisms which present market-relevant traits.


As of August 2022, EUginius contains


The database and web servers are located in Germany and are mirrored on servers in the Netherlands. Further development and troubleshooting are decided jointly by the cooperation partners.


Since 2018, there have also been partnerships with the Institute of Plant Breeding and Acclimatization (Instytut Hodowli i Aklimatyzacji Roślin – IHAR, based in Blonie, Poland), the Austrian Agency for Health and Food Safety (AGES, based in Vienna, Austria) and the Experimental ZooProphylactic Institute of Lazio and Tuscany (Istituto Zooprofilattico Sperimentale – IZS, based in Rome, Italy).


Beyond that, EUginius uses the element thesaurus GMO-GET1 [ 1 ]   (GMO Genetic Element Thesaurus) developed in collaboration with the Biosafety Clearing House (BCH, Montreal, Canada).


EUginius is continuously maintained and further developed. In this way, the work of the control laboratories using EUginius is supported in a timely manner. The adaptation of the contents and their provision (e.g. information on organisms developed by NBTs and sequencing information) is carried out on an ongoing basis. The renewal of the module for accessing GMO authorisation applications is under development. Finally, an optimisation of the design to improve usability and make navigation more intuitive is planned (2023 – 2024).


The  Fanzor  (Fz) protein is an  eukaryotic ,  RNA -guided  DNA   endonuclease , which means it is a type of DNA cutting enzyme that uses RNA to target genes of interest. It has been recently [ when? ]  discovered and explored in a number of studies. [ 1 ] [ 2 ] [ 3 ]  In  bacteria ,  RNA -guided  DNA   endonuclease  systems, such as the  CRISPR/Cas system , serve as an  immune system  to prevent infection by cutting viral genetic material. [ 4 ]  Currently,  CRISPR / Cas9 -mediated's DNA cleavage has extensive application in  biological research , and wide-reaching medical potential in  human gene editing . [ 4 ]


Fanzor belongs to the OMEGA system. [ 1 ] [ 2 ] [ 4 ]  Evolutionarily, it shares a  common ancestor , OMEGA TnpB, with the  CRISPR / Cas12  system. [ 1 ] [ 5 ]  Due to the shared ancestry between the OMEGA system and the CRISPR system, the  protein structure  and  DNA cleavage function of Fanzor and  Cas12  remain largely conserved. [ 1 ] [ 6 ]  Combined with the widespread presence of Fanzor across the diverse  genomes  of different  eukaryotic  species, [ 6 ]  this raises the possibility of OMEGA Fanzor being an alternative to  CRISPR/Cas system  with better efficiency and compatibility in other complex  eukaryotic  organisms, such as  mammals .


Due to its  eukaryotic  origin, the OMEGA Fanzor system may have some advantages over the better studied  CRISPR/Cas gene editor  in terms of human genome editing applications. [ 1 ]  In a CRISPR/Cas9 system,  Cas9  proteins are guided by the  guide RNA (gRNA)  and  protospacer adjacent motif (PAM)  for DNA cleavage. Interestingly, Fanzor genes in the soil fungus  S. punctatus   [ 1 ] [ 5 ]  also contain non-coding sequences called  ωRNA . Similar to CRISPR/Cas9, Fanzor protein is shown to cleave DNA in test tubes under the guidance of  ωRNA  and Target-adjacent motif (TAM). [ 1 ]


In human cells, the Fanzor protein of  Spizellomyces punctatus  was successfully tested and shown to cleave DNA effectively. [ 1 ]  However, its efficiency is lower compared to the closely related CRISPR/Cas12a system. [ 1 ]  By modifying and tweaking the  ωRNA  and the amino acid sequence, a second version of the  S. punctatus  Fanzor protein with improved cleavage efficiency - comparable to that of the CRISPR/Cas12a system -  was engineered. [ 1 ]  This shows that, with better modifications and more research, OMEGA Fanzor has the potential to match the CRISPR system in human genome editing in the future.


Studies conclude that Fanzor has great potential for efficient human genome editing [ 1 ] [ 6 ]  with a higher chance of not getting attacked by the immune system. [ 6 ]  For example, Fanzor could be used in personalized  cancer treatments  where the patient's own  T-cells  - important cells of the immune system that recognize and fight foreign  pathogens   - are edited in order to recognize and destroy cancer cells. [ 2 ] [ 8 ]  In the field of  regenerative medicine , it offers hope for an application in  stem cell therapy  to treat many disease of genetic origin like  type 1 diabetes  or  neurodegenerative diseases . [ 2 ]


Furthermore, Fanzor could potentially be used for genome editing in  eggs  and  sperm [ 2 ]  for  disease prevention  and  infertility treatment . However, the intervention in such cells' DNA comes with risks and requires strict  ethical guidelines . [ 9 ]


One major advantage of Fanzor in comparison to the CRISPR/Cas9 system is its small size. Therefore, it can be delivered with  viral vectors , which are modified dead bodies of viruses engineered to safely deliver genetic material, such as  adenoviruses . [ 4 ]  Adenoviruses are commonly used in medical applications like gene deliveries or  vaccines [ 10 ]  that do not elicit  immune responses  within the human body. [ 4 ]


However, researchers caution that further research is necessary to improve the editing efficiency [ 1 ] [ 6 ]  and precision. [ 1 ]


Next to the application in human cells, Fanzor is a prospective tool for specific genome editing in plants, because of the aforementioned advantages of the  protein  being a small size. [ 2 ]  Thereby, the  nutrient content , the resistance to diseases and the affordability of  crops  could be improved. [ 11 ]  Moreover, in regard to the current and arising challenges caused by  climate change , crops could be adjusted to better endure stress factors such as  drought ,  salinity  and increasing temperatures. [ 12 ]


Four Core Genotypes  ( FCG ) mice are laboratory mice produced by  genetic engineering  that allow biomedical researchers to determine if a sex difference in phenotype is caused by effects of  gonadal   hormones  or sex chromosome genes. [ 1 ] [ 2 ]  The four genotypes include XX and XY mice with  ovaries , and XX and XY mice with  testes . The comparison of XX and XY mice with the same type of gonad reveals sex differences in phenotypes that are caused by sex chromosome genes. The comparison of mice with different gonads but the same sex chromosomes reveals sex differences in phenotypes that are caused by gonadal hormones.


The FCG model was created by  Paul Burgoyne [ 3 ]  and  Robin Lovell-Badge  at the National Institute for Medical Research, London (now  Francis Crick Institute ). The model involves deleting the testis-determining gene  Sry  from the Y chromosome, and inserting Sry onto  chromosome  3. Therefore the sex chromosomes no longer determine the type of gonad, so that XX and XY mice can have the same type of gonad and gonadal hormones.


The FCG model has been used to discover that the XX and XY animals respond differently in models of human  physiology  and  disease , including  autoimmunity ,  metabolism ,  cardiovascular disease ,  cancer ,  Alzheimer’s disease , and neural and behavioral processes. [ 2 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ]  These findings imply that some sex chromosome genes may protect from disease, rationalizing the search for therapies that enhance such protective factors. [ 8 ]


Gene therapy for color blindness  is an experimental  gene therapy of the human retina  aiming to grant typical  trichromatic  color vision to individuals with congenital  color blindness  by introducing typical  alleles  for  opsin  genes. Animal testing for  gene therapy  began in 2007 with a 2009 breakthrough in  squirrel monkeys  suggesting an imminent gene therapy in humans. While progress in gene therapy for red-green color blindness has slowed since then, successful human trials are currently underway for achromatopsia, a different form of color vision deficiency. Congenital color vision deficiency affects over 200 million people worldwide, highlighting the significant demand for effective gene therapies targeting this condition.


The  retina  of the  human eye  contains photoreceptive cells called  cones  that allow color vision.  A normal trichromat possesses three different types of cones to distinguish different colors within the  visible spectrum .  The three types of cones are designated L, M, and S cones, each containing an opsin sensitive to a different portion of the visible spectrum.  More specifically, the L cone absorbs around 560 nm, the M cone absorbs near 530 nm, and the S cone absorbs near 420 nm. [ 1 ]   These cones  transduce  the absorbed light into electrical information to be relayed through other cells along the  phototransduction pathway , before reaching the  visual cortex  in the  brain . [ 1 ]


The signals from the 3 cones are compared to each other to generate 3  opponent process  channels. The channels are perceived as balances between red-green, blue-yellow and black-white. [ 1 ]


Color vision deficiency (CVD) is the deviation of an individual's color vision from typical human trichromatic vision. Relevant to gene therapy, CVD can be classified in 2 groups.


Dichromats have partial color vision. The most common form of dichromacy is red-green colorblindness. Dichromacy usually arises when one of the three opsin genes is deleted or otherwise fully nonfunctional. The effects and diagnosis depend on the missing opsin.  Protanopes  (very common) have no L-opsin,  Deuteranopes  (very common) have no M-opsin, and  Tritanopes  (rare) have no S-opsin. Accordingly, a missing cone means one of the opponent channels is inactive: red-green for protanopes/deuteranopes and blue-yellow for tritanopes. They therefore perceive a much reduced color space. Although dichromacy poses few critical problems in daily life, a lack of access to many occupations (where color vision may be safety-critical) is a large disadvantage.


Anomalous Trichromats  are not missing an opsin gene, but rather have a mutated (or  chimeric ) gene. They have trichromatic vision, but with a smaller color gamut than typical color vision. Regarding gene therapy, they are equivalent to dichromats.


Blue Cone Monochromats  are missing both the L- and M-opsin and therefore have no color vision. They are treated as a subset of dichromacy since a combination of gene therapies for protanopia and deuteranopia would be used.


Individuals with congenital achromatopsia tend to have typical opsin genes, but have a mutation in another gene downstream in the phototransduction pathway (e.g.  GNAT2  protein) that prevents their cones (and therefore  photopic vision ) from functioning. Achromats rely solely on their  scotopic vision . The severity of achromatopsia is much higher than dichromacy, not only in the lack of color vision, but also in co-occurring symptoms  photophobia ,  nystagmus  and poor  visual acuity .


Gene therapies aim to inject functional copies of missing or mutated genes into affected individuals by the use of viral vectors.  Using a replication-defective recombinant  adeno-associated virus  (rAAV) as a vector, the  cDNA  of the affected gene can be delivered to the cones at the back of the retina typically via subretinal injection.  Intravitreal injections  are much less invasive, but not yet as effective as subretinal injections. Upon gaining the gene, the cone begins to express the new photopigment. The effect is ideally permanent.


The first retinal gene therapy to be approved by the FDA was  Voretigene neparvovec  in 2017, which treats  Leber's congenital amaurosis , a genetic disorder that can lead to blindness.  These treatments also use subretinal injections of AAV vector and are therefore foundational to research in gene therapy for color blindness. [ 2 ] [ 3 ]


Human L-cone photopigment have been introduced into  mice .  Since the mice possess only S cones and M cones, they are dichromats. [ 4 ]   M-opsin was replaced with a cDNA of L-opsin in the X chromosome of some mice.  By breeding these "knock-in"  transgenic  mice, they generated heterozygous females with both an M cone and an L cone.  These mice had improved range of color vision and have gained trichromacy, as tested by  electroretinogram  and behavioral tests.  However, this is more difficult to apply in the form of gene therapy.


Recombinant AAV vector was to introduce the  green fluorescent protein  (GFP) gene in the cones of  gerbils . [ 5 ]   The genetic insert was designed to only be expressed in S or M cones, and the expression of GFP  in vivo  was observed over time.  Gene expression could stabilize if a sufficiently high dose of the viral vector is given.


In 2009, adult dichromatic  squirrel monkeys  were converted into trichromats using gene therapy. [ 6 ]   New world monkeys  are polymorphic in their M-opsin, such that females can be trichromatic, but all males are dichromatic. [ 6 ]   Recombinant AAV vector was used to deliver a human L-opsin gene subretinally. A subset of the monkey's M-cones gained the L-opsin genes and began co-expressing the new and old photopigments. [ 6 ]   Electroretinograms  demonstrated that the cones were expressing the new opsin and after 20 weeks a  pseudoisochromatic color vision test  demonstrated that the treated monkeys had indeed developed functional trichromatic vision. [ 6 ]


Gene therapy was to restore some of the sight of mice with  achromatopsia . The results were positive for 80% of the mice treated. [ 7 ]


In 2010, gene therapy for a form of  achromatopsia  was performed in dogs. Cone function and day vision have been restored for at least 33 months in two young dogs with achromatopsia. However, this therapy was less efficient for older dogs. [ 8 ]


In 2022, 4 young human ACHM2 and ACHM3 achromats were shown to have neurological responses (as measured with  fMRI ) to  photopic vision  that matched patterns generated by their  scotopic vision  after gene therapy. This inferred a photopic cone-driven system that was at least marginally functional. The methodology did not investigate novel color vision, though one respondent claimed to more easily interpret traffic lights. [ 9 ]  This may be considered the first case of a cure for colorblindness in humans.


In July 2023, a study found positive but limited improvements on congenital CNGA3 achromatopsia. [ 10 ] [ 11 ]


While the benefits of gene therapy to achromats typically outweigh the current risks, there are several challenges before large acceptance of gene therapy in dichromats can occur.


The procedure – namely the subretinal injection – is quite invasive, requiring several incisions and punctures in the eyeball. This poses a significant risk of infection and other complications. Subretinal injections methods promise to become less invasive with their application in other retinal gene therapies. They could also be replaced by intravitreal injections, which are significantly less invasive and can in theory be performed by a family doctor, but are less effective. [ 12 ]


The permanence of these therapies is also in question. Mancuso et al. reported that the treated squirrel monkeys maintained 2 years of color vision after the treatment. [ 6 ]  However, if repeat injections are needed, there is also the concern of the body developing an immune reaction to the virus.  If a body develops sensitivity to the viral vector, the success of the therapy could be jeopardized and/or the body may respond unfavorably. An editorial by J. Bennett points to Mancuso et al.'s use of an "unspecified postinjection corticosteroid therapy". [ 13 ]   Bennett suggests that the monkeys may have experienced inflammation due to the injection. [ 13 ]   However, the AAV virus that is commonly used for this study is non-pathogenic, and the body is less likely to develop an immune reaction. [ 14 ]


According to research by  David H. Hubel  and  Torsten Wiesel , suturing shut one eye of monkeys at an early age resulted in an irreversible  loss of vision  in that eye, even after the suture was removed. [ 1 ] [ 15 ]   The study concluded that the  neural circuitry  for vision is wired during a "critical period" in childhood, after which the visual circuitry can no longer be rewired to process new sensory input.  Contrary to this finding, Mancuso et al.’s success in conferring trichromacy to adult squirrel monkeys suggests that it is possible to adapt the preexisting circuit to allow greater acuity in color vision.  The researchers concluded that integrating the stimulus from the new photopigment as an adult was not analogous to vision loss following visual deprivation. [ 6 ]


It is yet unknown how the animals that gain a new photopigment are perceiving the new color.  While the article by Mancuso et al. states that the monkey has indeed gained trichromacy and gained the ability to discriminate between red and green, they claim no knowledge of how the animal internally perceives the sensation. [ 6 ]


As a way to introduce new genetic information to change a person's phenotype, a gene therapy for color blindness is open to the same  ethical questions  and criticisms as gene therapy in general.  These include issues around the governance of the therapy, whether treatment should be available only to those who can afford it, and whether the availability of treatment creates a stigma for those with color blindness. Given the large number of people with color blindness, there is also the question of whether color blindness is a disorder. [ 16 ]   Furthermore, even if gene therapy succeeds in converting incomplete colorblind individuals to trichromats, the degree of satisfaction among the subjects is unknown. It is uncertain how the quality of life will improve (or worsen) after the therapy.


The gene therapy for converting dichromats to trichromats can also be used hypothetically to "upgrade" typical trichromats to  tetrachromats  by introducing a new opsin genes. This begs the ethics of  designer babies  that contain genes not available naturally in the human gene pool. In 2022, the lab of  Jay Neitz  engineered a novel opsin sensitive to wavelengths between the typical human S- (420 nm) and M- (530 nm) opsins, i.e. the novel opsin at 493 nm. This allowed the opsin to be clearly visible in  ERGs , but could be used to create tetrachromacy. [ 12 ]


Cost and accessibility : The high costs of gene therapy development and treatment could limit access to those who need it, especially in low-resource settings.


Technical complexity : Precisely delivering functional opsin genes to the retina without causing immune reactions or unintended side effects remains a significant hurdle.


Long-term efficacy : Ensuring the lasting effectiveness of gene therapy, as retinal cells have limited regeneration potential, is a key concern.


Genetic engineering techniques  allow the modification of  animal  and  plant genomes . Techniques have been devised to insert, delete, and modify  DNA  at multiple levels, ranging from a specific  base pair  in a specific  gene  to entire genes. There are a number of steps that are followed before a  genetically modified organism  (GMO) is created. Genetic engineers must first choose what  gene  they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable  vector . This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.


The ability to genetically engineer organisms is built on years of research and discovery on gene function and manipulation. Important advances included the discovery of  restriction enzymes ,  DNA ligases , and the development of  polymerase chain reaction  and  sequencing .


Added genes are often accompanied by  promoter  and  terminator  regions as well as a  selectable marker  gene. The added gene may itself be modified to make it  express  more efficiently. This vector is then inserted into the host organism's genome. For animals, the gene is typically inserted into  embryonic stem cells , while in plants it can be inserted into any tissue that can be  cultured  into a fully developed plant.


Tests are carried out on the modified organism to ensure stable integration,  inheritance  and expression. First generation offspring are  heterozygous , requiring them to be inbred to create the homozygous pattern necessary for stable inheritance. Homozygosity must be confirmed in second generation specimens.


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


Many different discoveries and advancements led to the development of  genetic engineering . Human-directed genetic manipulation began with the  domestication  of plants and animals through  artificial selection  in about 12,000 BC. [ 1 ] : 1   Various techniques were developed to aid in breeding and selection.  Hybridization  was one way rapid changes in an organism's genetic makeup could be introduced. Crop hybridization most likely first occurred when humans began growing genetically distinct individuals of related species in close proximity. [ 2 ] : 32   Some plants were able to be propagated by  vegetative cloning . [ 2 ] : 31


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


After discovering the existence and properties of  DNA , tools had to be developed that allowed it to be manipulated. In 1970  Hamilton Smiths  lab discovered  restriction enzymes , enabling scientists to isolate genes from an organism's genome. [ 4 ]   DNA ligases , which join broken DNA together, were discovered earlier in 1967. [ 5 ]  By combining the two enzymes it became possible to "cut and paste" DNA sequences to create  recombinant DNA .  Plasmids , discovered in 1952, [ 6 ]  became important  tools  for transferring information between cells and  replicating  DNA sequences.   Polymerase chain reaction  (PCR), developed by  Kary Mullis  in 1983, allowed small sections of DNA to be amplified (replicated) and aided identification and isolation of genetic material.


As well as manipulating DNA, techniques had to be developed for its insertion into an organism's genome. Griffith's experiment had already shown that some bacteria had  the ability to naturally uptake and express foreign DNA . Artificial competence was induced in  Escherichia coli  in 1970 by treating them with  calcium chloride  solution (CaCl 2 ). [ 7 ]  Transformation using  electroporation  was developed in the late 1980s, increasing the efficiency and bacterial range. [ 8 ]  In 1907 a bacterium that caused plant tumors,  Agrobacterium tumefaciens , had been discovered. In the early 1970s it was found that this bacteria inserted its DNA into plants using a  Ti plasmid . [ 9 ]  By removing the genes in the plasmid that caused the tumor and adding in novel genes, researchers were able to infect plants with  A. tumefaciens  and let the bacteria insert their chosen DNA into the genomes of the plants. [ 10 ]


The first step is to identify the target gene or genes to insert into the host organism. This is driven by the goal for the resultant organism. In some cases only one or two genes are affected. For more complex objectives entire  biosynthetic pathways  involving multiple genes may be involved. Once found genes and other genetic information from a wide range of organisms can be inserted into bacteria for storage and modification, creating  genetically modified bacteria  in the process. Bacteria are cheap, easy to grow,  clonal , multiply quickly, relatively easy to transform and can be stored at -80 °C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research. [ 11 ]


Genetic screens  can be carried out to determine potential genes followed by other tests that identify the best candidates. A simple screen involves randomly  mutating  DNA with chemicals or radiation and then selecting those that display the desired trait. For organisms where mutation is not practical, scientists instead look for individuals among the population who present the characteristic through naturally-occurring mutations. Processes that look at a  phenotype  and then try and identify the gene responsible are called  forward genetics . The gene then needs to be  mapped  by comparing the inheritance of the phenotype with known  genetic markers . Genes that are close together are likely to be inherited together. [ 12 ]


Another option is  reverse genetics . This approach involves targeting a specific gene with a mutation and then observing what phenotype develops. [ 12 ]  The mutation can be designed to  inactivate the gene  or only allow it to become active under certain conditions.  Conditional mutations  are useful for identifying genes that are normally lethal if non-functional. [ 13 ]  As genes with similar functions share similar sequences ( homologous ) it is possible to predict the likely function of a gene by comparing its sequence to that of well-studied genes from  model organisms . [ 12 ]  The development of  microarrays ,  transcriptomes  and  genome sequencing  has made it much easier to find desirable genes. [ 14 ]


The bacteria  Bacillus thuringiensis  was first discovered in 1901 as the causative agent in the death of  silkworms . Due to these insecticidal properties, the bacteria was used as a  biological insecticide , developed commercially in 1938. The  cry proteins  were discovered to provide the insecticidal activity in 1956, and by the 1980s, scientists had successfully cloned the gene that encodes this protein and expressed it in plants. [ 15 ]   The gene that provides resistance to the herbicide  glyphosate  was found after seven years of searching in bacteria living in the outflow pipe of a  Monsanto   RoundUp  manufacturing facility. [ 16 ]  In animals, the majority of genes used are  growth hormone  genes. [ 17 ]


All genetic engineering processes involve the modification of DNA. Traditionally DNA was isolated from the cells of organisms. Later, genes came to be  cloned  from a DNA segment after the creation of a  DNA library  or  artificially synthesised . Once isolated, additional genetic elements are added to the gene to allow it to be expressed in the host organism and to aid selection.


First the cell must be gently  opened , exposing the DNA without causing too much damage to it. The methods used vary depending on the type of cell. Once it is open, the DNA must be separated from the other cellular components. A ruptured cell contains proteins and other cell debris. By mixing with  phenol and/or chloroform , followed by  centrifuging , the nucleic acids can be separated from this debris into an upper  aqueous phase . This aqueous phase can be removed and further purified if necessary by repeating the phenol-chloroform steps. The nucleic acids can then be  precipitated  from the aqueous solution using  ethanol  or  isopropanol . Any  RNA  can be removed by adding a  ribonuclease  that will degrade it. Many companies now sell kits that simplify the process. [ 18 ]


The gene researchers are looking to modify (known as the gene of interest) must be separated from the extracted DNA. If the sequence is not known then a common method is to break the DNA up with a random digestion method. This is usually accomplished using  restriction enzymes  (enzymes that cut DNA). A partial  restriction digest  cuts only some of the restriction sites, resulting in overlapping DNA fragment segments. The DNA fragments are put into individual  plasmid vectors  and grown inside bacteria. Once in the bacteria the plasmid is copied as the  bacteria divides . To determine if a useful gene is present in a particular fragment, the DNA library is screened for the desired  phenotype . If the phenotype is detected then it is possible that the bacteria contains the target gene.


If the gene does not have a detectable phenotype or a DNA library does not contain the correct gene, other methods must be used to isolate it. If the position of the gene can be determined using  molecular markers  then  chromosome walking  is one way to isolate the correct DNA fragment. If the gene expresses close  homology  to a known gene in another species, then it could be isolated by searching for genes in the library that closely match the known gene. [ 19 ]


For known DNA sequences, restriction enzymes that cut the DNA on either side of the gene can be used.  Gel electrophoresis  then sorts the fragments according to length. [ 20 ]  Some gels can separate sequences that differ by a single  base-pair . The DNA can be visualised by staining it with  ethidium bromide  and photographing under  UV light . A  marker  with fragments of known lengths can be laid alongside the DNA to estimate the size of each band. The DNA band at the correct size should contain the gene, where it can be excised from the gel. [ 18 ] : 40–41   Another technique to isolate genes of known sequences involves  polymerase chain reaction  (PCR). [ 21 ]  PCR is a powerful tool that can amplify a given sequence, which can then be isolated through gel electrophoresis. Its effectiveness drops with larger genes and it has the potential to introduce errors into the sequence.


It is possible to artificially  synthesise genes . [ 22 ]  Some synthetic sequences are available commercially, forgoing many of these early steps. [ 23 ]


The gene to be inserted must be combined with other genetic elements in order for it to work properly. The gene can be modified at this stage for better expression or effectiveness. As well as the gene to be inserted most  constructs  contain a  promoter  and  terminator  region as well as a  selectable marker  gene. The promoter region initiates  transcription  of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. A selectable marker, which in most cases confers  antibiotic resistance  to the organism it is expressed in, is used to determine which cells are transformed with the new gene. The constructs are made using  recombinant DNA  techniques, such as  restriction digests ,  ligations  and  molecular cloning . [ 24 ]


Once the gene is constructed it must be stably integrated into the genome of the target organism or exist as  extrachromosomal DNA . There are a number of techniques available for inserting the gene into the host genome and they vary depending on the type of organism targeted. In multicellular  eukaryotes , if the  transgene  is incorporated into the host's  germline  cells, the resulting host cell can pass the transgene to its  progeny . If the transgene is incorporated into  somatic  cells, the transgene can not be inherited. [ 25 ]


Transformation is the direct alteration of a cell's genetic components by passing the genetic material through the  cell membrane . About 1% of bacteria are naturally able to  take up foreign DNA , but this ability can be induced in other bacteria. [ 26 ]  Stressing the bacteria with a  heat shock  or  electroporation  can make the  cell membrane  permeable to DNA that may then be incorporated into the genome or exist as extrachromosomal DNA. Typically the cells are incubated in a solution containing  divalent   cations  (often  calcium chloride ) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. It is suggested that exposing the cells to divalent cations in cold condition may change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.  Electroporation  is another method of promoting competence.  In this method the cells are briefly shocked with an  electric field  of 10-20  kV /cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter.  After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms. Up-taken DNA can either integrate with the bacterials genome or, more commonly, exist as  extrachromosomal DNA .


In plants the DNA is often inserted using  Agrobacterium -mediated recombination , [ 27 ]  taking advantage of the  Agrobacterium s  T-DNA  sequence that allows natural insertion of genetic material into plant cells. [ 28 ]  Plant tissue are cut into small pieces and soaked in a fluid containing suspended  Agrobacterium . The bacteria will attach to many of the plant cells exposed by the cuts. The bacteria uses  conjugation  to transfer a DNA segment called  T-DNA  from its plasmid into the plant. The transferred DNA is piloted to the plant cell nucleus and integrated into the host plants genomic DNA.The plasmid T-DNA is integrated semi-randomly into the  genome  of the host cell. [ 29 ]


By modifying the plasmid to express the gene of interest, researchers can insert their chosen gene stably into the plants genome. The only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. [ 30 ] [ 31 ]  The genes to be introduced into the plant are cloned into a  plant transformation vector  that contains the T-DNA region of the  plasmid . An alternative method is  agroinfiltration . [ 32 ] [ 33 ]


Another method used to transform plant cells is  biolistics , where particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. [ 34 ]  Some genetic material enters the cells and transforms them. This method can be used on plants that are not susceptible to  Agrobacterium  infection and also allows transformation of plant  plastids . Plants cells can also be transformed using electroporation, which uses an electric shock to make the cell membrane permeable to plasmid DNA. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial transformation. [ citation needed ]


Transformation has a  different meaning  in relation to animals, indicating progression to a cancerous state, so the process used to insert foreign DNA into animal cells is usually called transfection. [ 35 ]  There are many ways to directly introduce DNA into animal cells  in vitro . Often these cells are  stem cells  that are used for  gene therapy . Chemical based methods uses natural or synthetic compounds to form particles that facilitate the transfer of genes into cells. [ 36 ]  These synthetic vectors have the ability to bind DNA and accommodate large genetic transfers. [ 37 ]  One of the simplest methods involves using  calcium phosphate   to bind the DNA and then exposing it to cultured cells. The solution, along with the DNA, is  encapsulated  by the cells. [ 38 ]   Liposomes  and  polymers  can be used as vectors to deliver DNA into cultured animal cells. Positively charged liposomes bind with DNA, while polymers can designed that interact with DNA. [ 36 ]  They form lipoplexes and polyplexes respectively, which are then up-taken by the cells. Other techniques include using electroporation and biolistics. [ 39 ]  In some cases, transfected cells may stably integrate external DNA into their own genome, this process is known as  stable transfection . [ 40 ]


To create  transgenic animals  the DNA must be inserted into viable embryos or eggs. This is usually accomplished using  microinjection , where DNA is injected through the cell's  nuclear envelope  directly into the  nucleus . [ 26 ]   Superovulated  fertilised eggs are collected at the single cell stage and cultured  in vitro . When the  pronuclei  from the sperm head and egg are visible through the  protoplasm  the genetic material is injected into one of them. The  oocyte  is then implanted in the  oviduct  of a  pseudopregnant  animal. [ 41 ]  Another method is Embryonic Stem Cell-Mediated Gene Transfer. The gene is transfected into  embryonic stem cells  and then they are inserted into mouse  blastocysts  that are then implanted into foster mothers. The resulting offspring are  chimeric , and further mating can produce mice fully transgenic with the gene of interest. [ 42 ]


Transduction is the process by which foreign  DNA  is introduced into a cell by a  virus  or  viral vector . [ 43 ]  Genetically modified viruses can be used as  viral vectors  to transfer target genes to another organism in  gene therapy . [ 44 ]  First the virulent genes are removed from the virus and the target genes are inserted instead. The sequences that allow the virus to insert the genes into the host organism must be left intact. Popular virus vectors are developed from  retroviruses  or  adenoviruses . Other viruses used as vectors include,  lentiviruses ,  pox viruses  and  herpes viruses . The type of virus used will depend on the cells targeted and whether the DNA is to be altered permanently or temporarily.


As often only a single cell is transformed with genetic material, the organism must be  regenerated  from that single cell. In plants this is accomplished through the use of  tissue culture . [ 45 ] [ 46 ]  Each plant species has different requirements for successful regeneration. If successful, the technique produces an adult plant that contains the  transgene  in every cell. [ 47 ]  In animals it is necessary to ensure that the inserted DNA is present in the  embryonic stem cells . [ 27 ]  Offspring can be screened for the gene. All offspring from the first generation are  heterozygous  for the inserted gene and must be inbred to produce a  homozygous  specimen. [ citation needed ]  Bacteria consist of a single cell and reproduce clonally so regeneration is not necessary.  Selectable markers  are used to easily differentiate transformed from untransformed cells.


Cells that have been successfully transformed with the DNA contain the marker gene, while those not transformed will not. By growing the cells in the presence of an antibiotic or chemical that  selects  or marks the cells expressing that gene, it is possible to separate modified from unmodified cells. Another screening method involves a  DNA probe  that sticks only to the inserted gene. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant. [ 48 ]


Finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that they will be appropriately expressed in the intended tissues. Further testing using PCR,  Southern hybridization , and  DNA sequencing  is conducted to confirm that an organism contains the new gene. [ 49 ]  These tests can also confirm the chromosomal location and copy number of the inserted gene. Once confirmed methods that look for and measure the gene products (RNA and protein) are also used to assess gene expression, transcription, RNA processing patterns and expression and localization of protein product(s). These include  northern hybridisation , quantitative  RT-PCR ,  Western blot ,  immunofluorescence ,  ELISA  and phenotypic analysis. [ 50 ]  When appropriate, the organism's offspring are studied to confirm that the transgene and associated  phenotype  are stably inherited.


Traditional methods of genetic engineering generally insert the new genetic material randomly within the host genome.  This can impair or alter other genes within the organism. Methods were developed that inserted the new genetic material into  specific sites  within an organism genome. Early methods that targeted genes at certain sites within a genome relied on  homologous recombination  (HR). [ 51 ]  By creating DNA constructs that contain a template that matches the targeted genome sequence, it is possible that the HR processes within the cell will insert the construct at the desired location. Using this method on  embryonic stem cells  led to the development of  transgenic mice  with targeted  knocked out . It has also been possible to  knock in  genes or alter  gene expression  patterns. [ 52 ]


If a vital gene is knocked out it can prove lethal to the organism. In order to study the function of these genes,  site specific recombinases  (SSR) were used. The two most common types are the  Cre-LoxP  and  Flp-FRT  systems.  Cre recombinase  is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites. The Flip-FRT system operates in a similar way, with the Flip recombinase recognizing FRT sequences. By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that expresses the SSR under control of  tissue specific promoters , it is possible to knock out or switch on genes only in certain cells. This has also been used to remove marker genes from transgenic animals. Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired times or  stages of development . [ 52 ]


Genome editing uses artificially engineered  nucleases  that create specific  double-stranded breaks   at desired locations in the genome. The breaks are subject to cellular  DNA repair  processes that can be exploited for targeted gene knock-out, correction or insertion at high frequencies. If a donor DNA containing the appropriate sequence (homologies) is present, then new genetic material containing the transgene will be integrated at the targeted site with high efficiency by  homologous recombination . [ 53 ]  There are four families of engineered nucleases:  meganucleases , [ 54 ] [ 55 ]   ZFNs , [ 56 ] [ 57 ]   transcription activator-like effector nucleases  (TALEN), [ 58 ] [ 59 ]  t he CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPRassociated protein (e.g. CRISPR/Cas9) . [ 60 ] [ 61 ]  Among the four types, TALEN and CRISPR/Cas are the two most commonly used. [ 62 ]  Recent advances have looked at combining multiple systems to  exploit the best features of both (e.g. megaTAL that are a fusion of a TALE DNA binding domain and a meganuclease). [ 63 ]  Recent research has also focused on developing strategies to create gene knock-out or corrections without creating double stranded breaks (base editors). [ 62 ]


Meganucleases were first used in 1988 in mammalian cells. [ 64 ]  Meganucleases are  endodeoxyribonucleases  that function as restriction enzymes with long recognition sites, making them more specific to their target site than other  restriction enzymes . This increases their specificity and reduces their toxicity as they will not target as many sites within a genome. The most studied meganucleases are the  LAGLIDADG family . While meganucleases are still quite susceptible to off-target binding, which makes them less attractive than other gene editing tools, their smaller size still makes them attractive particularly for viral vectorization perspectives. [ 65 ] [ 53 ]


Zinc-finger nucleases (ZFNs), used for the first time in 1996, are typically created through the fusion of Zinc-finger domains and the  Fok I nuclease domain. ZFNs have thus the ability to cleave DNA at target sites. [ 53 ]  By engineering the zinc finger domain to target a specific site within the genome, it is possible to edit the genomic sequence at the desired  location . [ 65 ] [ 66 ] [ 53 ]  ZFNs have a greater specificity, but still hold the potential to bind to non-specific sequences.. While a certain amount of off-target cleavage is acceptable for creating transgenic model organisms, they might not be optimal for all human gene therapy treatments. [ 65 ]


Access to the code governing the DNA recognition by transcription activator-like effectors (TALE) in 2009 opened the way to the development of a new class of efficient TAL-based gene editing tools. TALE, proteins secreted by the Xanthomonas plant pathogen, bind with great specificity to genes within the plant host and initiate  transcription   of the genes helping infection. Engineering TALE by fusing the DNA binding core to the  Fok I nuclease  catalytic domain allowed creation of a new tool of designer nucleases, the TALE nuclease (TALEN). [ 67 ]  They have one of the greatest specificities of all the current engineered nucleases. Due to the presence of repeat sequences, they are difficult to construct through standard molecular biology procedure and rely on more complicated method of such as  Golden gate cloning . [ 62 ]


In 2011, another major breakthrough technology was developed based on CRISPR/Cas (clustered regularly interspaced short palindromic repeat / CRISPR associated protein) systems that function as an adaptive immune system in bacteria and  archaea .  The CRISPR/Cas system allows bacteria and archaea to fight against invading viruses by cleaving viral DNA and inserting pieces of that DNA into their own genome. The organism then transcribes this DNA into  RNA  and combines this RNA with  Cas9  proteins to make double-stranded breaks in the invading viral DNA. The RNA serves as a guide RNA to direct the Cas9 enzyme to the correct spot in the virus DNA. By pairing Cas proteins with a designed guide RNA CRISPR/Cas9 can be used to induce double-stranded breaks at specific points within DNA sequences. The break gets repaired by cellular DNA repair enzymes, creating a small insertion/deletion type mutation in most cases. Targeted DNA repair is possible by providing a donor DNA template that represents the desired change and that is (sometimes) used for double-strand break repair by homologous recombination. It was later demonstrated that CRISPR/Cas9 can edit human cells in a dish. Although the early generation lacks the specificity of TALEN, the major advantage of this technology is the simplicity of the design. It also allows multiple sites to be targeted simultaneously, allowing the editing of multiple genes at once.  CRISPR/Cpf1  is a more recently discovered system that requires a different guide RNA to create particular double-stranded breaks (leaves overhangs when cleaving the DNA) when compared to CRISPR/Cas9. [ 62 ]


CRISPR/Cas9 is efficient at gene disruption. The creation of HIV-resistant babies by Chinese researcher He Jiankui is perhaps the most famous example of gene disruption using this method. [ 68 ]  It is far less effective at gene correction. Methods of base editing are under development in which a “nuclease-dead” Cas 9 endonuclease or a related enzyme is used for gene targeting while a linked deaminase enzyme makes a targeted base change in the DNA. [ 69 ]  The most recent refinement of CRISPR-Cas9 is called Prime Editing. This method links a reverse transcriptase to an RNA-guided engineered nuclease that only makes single-strand cuts but no double-strand breaks. It replaces the portion of DNA next to the cut by the successive action of nuclease and reverse transcriptase, introducing the desired change from an RNA template. [ 70 ]


Genetic erosion  (also known as  genetic depletion ) is a process where the limited  gene pool  of an  endangered species  diminishes even more when reproductive individuals die off before reproducing with others in their endangered  low population . The term is sometimes used in a narrow sense, such as when describing the loss of particular  alleles  or genes, as well as being used more broadly, as when referring to the loss of a phenotype or whole species.


Genetic erosion occurs because each individual organism has many unique genes which get lost when it dies without getting a chance to breed. Low genetic diversity in a population of wild animals and plants leads to a further diminishing gene pool – inbreeding and a weakening immune system can then "fast-track" that species towards eventual  extinction .


By definition, endangered species suffer varying degrees of genetic erosion. Many species benefit from a human-assisted breeding program to keep their population viable, [ citation needed ]  thereby avoiding extinction over long time-frames. Small populations are more susceptible to genetic erosion than larger populations.


Genetic erosion gets compounded and accelerated by  habitat loss  and  habitat fragmentation  – many endangered species are threatened by habitat loss and (fragmentation)  habitat . Fragmented habitat create barriers in gene flow between populations.


The  gene pool  of a  species  or a  population  is the complete set of unique alleles that would be found by inspecting the genetic material of every living member of that species or population. A large gene pool indicates extensive  genetic diversity , which is associated with robust populations that can survive bouts of intense  selection . Meanwhile, low genetic diversity (see  inbreeding  and  population bottlenecks ) can cause reduced  biological fitness  and increase the chance of  extinction  of that species or population.


Population bottlenecks create shrinking gene pools, which leave fewer and fewer fertile mating partners. The genetic implications can be illustrated by considering the analogy of a high-stakes poker game with a crooked dealer. Consider that the game begins with a 52-card deck (representing high genetic diversity). Reduction of the number of breeding pairs with unique genes resembles the situation where the dealer deals only the same five cards over and over, producing only a few limited "hands".


As specimens begin to inbreed, both physical and reproductive congenital effects and defects appear more often. Abnormal sperm increases, infertility rises, and birthrates decline. "Most perilous are the effects on the immune defense systems, which become weakened and less and less able to fight off an increasing number of bacterial, viral, fungal, parasitic, and other disease-producing threats. Thus, even if an endangered species in a bottleneck can withstand whatever human development may be eating away at its habitat, it still faces the threat of an epidemic that could be fatal to the entire population." [ 1 ]


Genetic erosion in  agricultural  and  livestock  is the loss of biological genetic diversity – including the loss of individual genes, and the loss of particular  recombinants  of genes (or gene complexes) – such as those manifested in locally adapted  landraces  of  domesticated  animals or plants that have become adapted to the natural environment in which they originated.


The major driving forces behind genetic erosion in crops are variety replacement, land clearing,  overexploitation  of species,  population pressure ,  environmental degradation ,  overgrazing , governmental policy, and changing agricultural systems. The main factor, however, is the replacement of local varieties of domestic plants and animals by other varieties or species that are non-local. A large number of varieties can also often be dramatically reduced when commercial varieties are introduced into traditional farming systems. Many researchers believe that the main problem related to agro-ecosystem management is the general tendency towards genetic and ecological uniformity imposed by the development of modern agriculture.


In the case of  Animal Genetic Resources for Food and Agriculture , major causes of genetic erosion are reported to include indiscriminate cross-breeding, increased use of exotic breeds, weak policies and institutions in animal  genetic resources  management, neglect of certain breeds because of a lack of profitability or competitiveness, the intensification of production systems, the effects of diseases and disease management, loss of pastures or other elements of the production environment, and poor control of inbreeding. [ 2 ]


With advances in modern bioscience, several techniques and safeguards have emerged to check the relentless advance of genetic erosion and the resulting acceleration of endangered species towards eventual extinction. However, many of these techniques and safeguards are too expensive yet to be practical, and so the best way to protect species is to protect their  habitat  and to let them live in it as naturally as possible. Complicating matters, the conservation of substantial amounts of genetic diversity often requires the maintenance of multiple independent populations across a species distribution. [ 3 ]  For example, to conserve at least 90% of the genetic diversity of the northern quoll requires the conservation of at least eight populations across the continent of Australia. [ 3 ]


Wildlife sanctuaries  and  national parks  have been created to preserve entire  ecosystems  with all the web of species native to the area.  Wildlife corridors  are created to join fragmented habitats (see  Habitat fragmentation ) to enable endangered species to travel, meet, and breed with others of their kind. Scientific  conservation  and modern  wildlife management  techniques, with the expertise of scientifically trained staff, help manage these protected ecosystems and the wildlife found in them. Wild animals are also  translocated  and  reintroduced  to other locations physically when fragmented wildlife habitats are too far and isolated to be able to link together via a wildlife corridor, or when local extinctions have already occurred.


Modern policies of  zoo associations  and  zoos  around the world have begun putting dramatically increased emphasis on keeping and breeding wild-sourced  species  and  subspecies  of animals in their registered endangered species breeding programs. These specimens are intended to have a chance to be reintroduced and survive back in the wild. The main objectives of zoos today have changed, and greater resources are being invested in breeding species and subspecies for then ultimate purpose of assisting conservation efforts in the wild. Zoos do this by maintaining extremely detailed scientific breeding records ( i.e.   studbooks )) and by loaning their wild animals to other zoos around the country (and often globally) for breeding, to safeguard against  inbreeding  by attempting to maximize genetic diversity however possible.


Costly (and sometimes controversial)  ex-situ conservation  techniques aim to increase the genetic  biodiversity  on our planet, as well as the diversity in local  gene pools . by guarding against genetic erosion. Modern concepts like  seedbanks ,  sperm banks , and  tissue banks  have become much more commonplace and valuable.  Sperm ,  eggs , and  embryos  can now be frozen and kept in banks, which are sometimes called "Modern Noah's Arks" or " Frozen Zoos ".  Cryopreservation  techniques are used to freeze these living materials and keep them alive in perpetuity by storing them submerged in  liquid nitrogen  tanks at very low temperatures. Thus, preserved materials can then be used for  artificial insemination ,  in vitro  fertilization ,  embryo transfer , and  cloning  methodologies to protect diversity in the gene pool of critically endangered species.


It can be possible to save an  endangered species  from  extinction  by preserving only  parts  of specimens, such as tissues, sperm, eggs, etc. – even after the death of a critically endangered animal, or collected from one found freshly dead, in  captivity  or from the  wild . A new specimen can then be "resurrected" with the help of cloning, so as to give it another chance to breed its genes into the living population of the respective threatened species. Resurrection of dead critically endangered wildlife specimens with the help of cloning is still being perfected, and is still too expensive to be practical, but with time and further advancements in science and methodology it may well become a routine procedure not too far into the future.


Genetic pollution  is a term for uncontrolled [ 1 ] [ 2 ]   gene flow  into wild populations. It is defined as "the dispersal of contaminated altered genes from genetically engineered organisms to natural organisms, esp. by cross-pollination", [ 3 ]  but has come to be used in some broader ways.  It is related to the  population genetics  concept of gene flow, and  genetic rescue , which is genetic material intentionally introduced to increase the fitness of a population. [ 4 ]   It is called genetic pollution when it negatively impacts the fitness of a population, such as through  outbreeding depression  and the introduction of unwanted phenotypes which can lead to extinction.


Conservation biologists and conservationists have used the term to describe gene flow from domestic, feral, and non-native species into  wild   indigenous species , which they consider undesirable. They promote awareness of the effects of introduced  invasive species  that may " hybridize with native species, causing genetic pollution ". In the fields of  agriculture ,  agroforestry  and  animal husbandry ,  genetic pollution  is used to describe gene flows between genetically engineered species and wild relatives. The use of the word "pollution" is meant to convey the idea that mixing genetic information is bad for the environment, but because the mixing of genetic information can lead to a variety of outcomes, "pollution" may not always be the most accurate descriptor.


Some  conservation biologists  and  conservationists  have used genetic pollution for a number of years as a term to describe gene flow from a  non-native ,  invasive subspecies ,  domestic , or  genetically-engineered  population to a  wild   indigenous  population. [ 1 ] [ 5 ] [ 6 ]


The introduction of genetic material into the gene pool of a population by human intervention can have both positive and negative effects on populations. When genetic material is intentionally introduced to increase the fitness of a population, this is called  genetic rescue . When genetic material is unintentionally introduced to a population, this is called genetic pollution and can negatively affect the fitness of a population (primarily through  outbreeding depression ), introduce other unwanted phenotypes, or theoretically lead to extinction.


An introduced species is one that is not native to a given population that is either intentionally or accidentally brought into a given ecosystem. Effects of introduction are highly variable, but if an introduced species has a major negative impact on its new environment, it can be considered an invasive species. One such example is the introduction of the Asian Longhorned beetle in North America, which was first detected in 1996 in Brooklyn, New York. It is believed that these beetles were introduced through cargo at trade ports. The beetles are highly damaging to the environment, and are estimated to cause risk to 35% of urban trees, excluding natural forests. [ 7 ]  These beetles cause severe damage to the wood of trees by larval funneling. Their presence in the ecosystem destabilizes community structure, having a negative influence on many species in the system.


Introduced species are not always disruptive to an environment, however. Tomás Carlo and Jason Gleditch of Penn State University found that the number of "invasive" honeysuckle plants in the area correlated with the number and diversity of the birds in the Happy Valley Region of Pennsylvania, suggesting introduced honeysuckle plants and birds formed a mutually beneficial relationship. [ 8 ]  Presence of introduced honeysuckle was associated with higher diversity of the bird populations in that area, demonstrating that introduced species are not always detrimental to a given environment and it is completely context dependent.


Conservation biologists and conservationists have, for a number of years, used the term to describe gene flow from domestic, feral, and non-native species into wild  indigenous species , which they consider undesirable. [ 1 ] [ 5 ] [ 6 ]   For example,  TRAFFIC  is the international wildlife trade monitoring network that works to limit  trade  in wild plants and animals so that it is not a threat to conservationist goals. They promote awareness of the effects of introduced invasive species that may " hybridize with native species, causing genetic pollution ". [ 9 ]  Furthermore, the  Joint Nature Conservation Committee , the statutory adviser to the  UK government , has stated that invasive species "will alter the genetic pool (a process called  genetic pollution ), which is an irreversible change." [ 10 ]


Invasive species can invade both large and small native populations and have a profound effect. Upon invasion, invasive species interbreed with native species to form sterile or more evolutionarily fit hybrids that can outcompete the native populations. Invasive species can cause extinctions of small populations on islands that are particularly vulnerable due to their smaller amounts of genetic diversity. In these populations, local adaptations can be disrupted by the introduction of new genes that may not be as suitable for the small island environments. For example, the  Cercocarpus traskiae  of the Catalina Island off the coast of California has faced near extinction with only a single population remaining due to the hybridization of its offspring with  Cercocarpus betuloides . [ 11 ]


Increased contact between wild and  domesticated  populations of organisms can lead to reproductive interactions that are detrimental to the wild population's ability to survive. A wild population is one that lives in natural areas and is not regularly looked after by humans. This contrasts with domesticated populations that live in human controlled areas and are regularly, and historically, in contact with humans.  Genes  from domesticated populations are added to wild populations as a result of reproduction. In many crop populations this can be the result of pollen traveling from farmed crops to neighboring wild plants of the same species. For farmed animals, this reproduction may happen as the result of escaped or released animals.


A popular example of this phenomenon is the gene flow between wolves and domesticated dogs.  The New York Times  cites, from the words of biologist Luigi Boitani, "Although wolves and dogs have always lived in close contact in Italy and have presumably mated in the past, the newly worrisome element, in Dr. Boitani's opinion, is the increasing disparity in numbers, which suggests that interbreeding will become fairly common. As a result, 'genetic pollution of the wolf gene pool might reach irreversible levels', he warned. 'By hybridization, dogs can easily absorb the wolf genes and destroy the wolf, as it is,' he said. The wolf might survive as a more doglike animal, better adapted to living close to people, he said, but it would not be 'what we today call a wolf.'" [ 1 ]


Aquaculture  is the practice of farming aquatic animals or plants for the purpose of consumption. This practice is becoming increasingly common for the production of  salmon . This is specifically termed  aquaculture of salmonoids . One of the dangers of this practice is the possibility of domesticated salmon breaking free from their containment. The occurrence of escaping incidents is becoming increasingly common as aquaculture gains popularity. [ 12 ] [ 13 ] [ 14 ]  Farming structures may be ineffective at holding the vast number of fast growing animals they house. [ 15 ]  Natural disasters, high tides, and other environmental occurrences can also trigger aquatic animal escapes. [ 16 ] [ 17 ]  The reason these escapes are considered dangers is the impact they pose for the wild population they reproduce with after escaping. In many instances the wild population experiences a decreased likelihood of survival after reproducing with domesticated populations of salmon. [ 18 ] [ 19 ]


The  Washington Department of Fish and Wildlife  cites that "commonly expressed concerns surrounding escaped Atlantic salmon include competition with native salmon, predation, disease transfer, hybridization, and colonization." [ 20 ]  A report done by that organization in 1999 did not find that escaped salmon posed a significant risk to wild populations. [ 21 ]


Crops  refer to groups of plants grown for consumption. Despite domestication over many years, these plants are not so far removed from their wild relatives that they couldn't reproduce if brought together. Many crops are still grown in the areas they originated and gene flow between crops and wild relatives impacts the evolution of wild populations. [ 22 ]  Farmers can avoid reproduction between the different populations by timing their planting of crops so that crops are not flowering when wild relatives would be. Domesticated crops have been changed through artificial selection and genetic engineering. The genetic make-ups of many crops is different from those of their wild relatives, [ 23 ]  but the closer they grow to one another the more likely they are to share genes through pollen. Gene flow persists between crops and wild counterparts.


Genetically engineered organisms are genetically modified in a laboratory, and therefore distinct from those that were bred through artificial selection. In the fields of  agriculture ,  agroforestry  and  animal husbandry ,  genetic pollution  is being used to describe gene flows between GE species and wild relatives. [ 24 ]
An early use of the term " genetic pollution"  in this later sense appears in a wide-ranging review of the potential ecological effects of genetic engineering in  The Ecologist magazine in July 1989 . It was also popularized by environmentalist  Jeremy Rifkin  in his 1998 book  The Biotech Century . [ 25 ]   While intentional crossbreeding between two genetically distinct varieties is described as  hybridization  with the subsequent  introgression  of genes, Rifkin, who had played a leading role in the ethical debate for over a decade before, used genetic pollution to describe what he considered to be problems that might occur due to the unintentional process of (modernly)  genetically modified organisms  (GMOs) dispersing their genes into the natural environment by breeding with wild plants or animals. [ 24 ] [ 26 ] [ 27 ]


Concerns about negative consequences from gene flow between genetically engineered organisms and wild populations are valid. Most corn and soybean crops grown in the midwestern USA are genetically modified. There are corn and soybean varieties that are resistant to herbicides like glyphosate [ 28 ]  and corn that produces  neonicotinoid  pesticide within all of its tissues. [ 29 ]   These genetic modifications are meant to increase yields of crops but there is little evidence that yields actually increase. [ 29 ]  While scientists are concerned genetically engineered organisms can have negative effects on surrounding plant and animal communities, the risk of gene flow between genetically engineered organisms and wild populations is yet another concern. Many farmed crops may be weed resistant and reproduce with wild relatives. [ 30 ]  More research is necessary to understand how much gene flow between genetically engineered crops and wild populations occurs, and the impacts of genetic mixing.


Mutations within organisms can be executed through the process of exposing the organism to chemicals or radiation in order to generate mutations.  This has been done in plants in order to create mutants that have a desired trait.  These mutants can then be bred with other mutants or individuals that are not mutated in order to maintain the mutant trait.  However, similar to the risks associated with introducing individuals to a certain environment, the variation created by mutated individuals could have a negative impact on native populations as well.


Since 2005 there has existed a  GM Contamination Register , launched for  GeneWatch  UK and  Greenpeace  International that records all incidents of intentional or accidental [ 31 ] [ 32 ]  release of organisms genetically modified using modern techniques. [ 33 ]


Genetic use restriction technologies  (GURTs) were developed for the purpose of property protection, but could be beneficial in preventing the dispersal of transgenes. GeneSafe technologies introduced a method that became known as "Terminator." This method is based on seeds that produce sterile plants. This would prevent movement of transgenes into wild populations as hybridization would not be possible. [ 34 ]  However, this technology has never been deployed as it disproportionately negatively affects farmers in developing countries, who save seeds to use each year (whereas in developed countries, farmers generally buy seeds from seed production companies). [ 34 ]


Physical containment has also been utilized to prevent the escape of transgenes. Physical containment includes barriers such as filters in labs, screens in greenhouses, and isolation distances in the field. Isolation distances have not always been successful, such as transgene escape from an isolated field into the wild in  herbicide-resistant  bentgrass  Agrostis stolonifera . [ 35 ]


Another suggested method that applies specifically to protection traits (e.g. pathogen resistance) is mitigation. Mitigation involves linking the positive trait (beneficial to fitness) to a trait that is negative (harmful to fitness) to wild but not domesticated individuals. [ 35 ]  In this case, if the protection trait was introduced to a weed, the negative trait would also be introduced in order to decrease overall fitness of the weed and decrease possibility of the individual’s reproduction and thus propagation of the transgene.


Not all genetically engineered organisms cause genetic pollution.  Genetic engineering has a variety of uses and is specifically defined as a direct manipulation of the genome of an organism.  Genetic pollution can occur in response to the introduction of a species that is not native to a particular environment, and genetically engineered organisms are examples of individuals that could cause genetic pollution following introduction.  Due to these risks, studies have been done in order to assess the risks of genetic pollution associated with organisms that have been genetically engineered:


Not only are there risks in terms of genetic engineering, but there are risks that emerge from species hybridization. In Czechoslovakia, ibex were introduced from Turkey and Sinai to help promote the ibex population there, which caused hybrids that produced offspring too early, which caused the overall population to disappear completely. [ 4 ]  The genes of each population of the ibex in Turkey and Sinai were locally adapted to their environments so when placed in a new environmental context did not flourish. Additionally, the environmental toll that may arise from the introduction of a new species may be so disruptive that the ecosystem is no longer able to sustain certain populations.


The use of the word "pollution" in the term genetic pollution has a deliberate negative connotation and is meant to convey the idea that mixing genetic information is bad for the environment. However, because the mixing of genetic information can lead to a variety of outcomes, "pollution" may not be the most accurate descriptor. Gene flow is undesirable according to some  environmentalists  and  conservationists , including groups such as  Greenpeace ,  TRAFFIC , and  GeneWatch UK . [ 44 ] [ 31 ] [ 33 ] [ 45 ] [ 5 ] [ 9 ] [ 46 ]


" Invasive species have been a major cause of  extinction  throughout the world in the past few hundred years. Some of them prey on native wildlife, compete with it for resources, or spread disease, while others may hybridize with native species, causing " genetic pollution ". In these ways, invasive species are as big a threat to the balance of nature as the direct  overexploitation  by humans of some species. " [ 47 ]


It can also be considered undesirable if it leads to a loss of  fitness  in the wild populations. [ 48 ]  The term can be associated with the gene flow from a  mutation bred ,  synthetic organism  or  genetically engineered  organism to a non GE organism, [ 24 ]  by those who consider such gene flow detrimental. [ 44 ]  These environmentalist groups stand in complete opposition to the development and production of genetically engineered organisms.


From a governmental perspective, genetic pollution is defined as follows by the  Food and Agriculture Organization of the United Nations :


"Uncontrolled spread of genetic information (frequently referring to transgenes) into the genomes of organisms in which such genes are not present in nature." [ 49 ]


Use of the term 'genetic pollution' and similar phrases such as  genetic deterioration ,  genetic swamping ,  genetic takeover , and  genetic aggression , are being debated by scientists as many do not find it scientifically appropriate. Rhymer and  Simberloff  argue that these types of terms:


...imply either that hybrids are less fit than the parentals, which need not be the case, or that there is an inherent value in "pure" gene pools. [ 47 ]


They recommend that gene flow from invasive species be termed  genetic mixing  since:


"Mixing" need not be value-laden, and we use it here to denote mixing of gene pools whether or not associated with a decline in fitness. [ 47 ]