There is also research going on on  genoeconomics , a  protoscience  that is based on the idea that a person's  financial  behavior could be traced to their  DNA  and that  genes  are related to  economic behavior . As of 2015 [update] , the results have been inconclusive. Some minor correlations have been identified. [ 91 ] [ 92 ]


Some studies show that our genes may affect some of our behaviors. For example, some genes may follow our state of stagnation, while others may be responsible for our bad habits. To give an example, the MAOA (Mono oxidase A) gene, the feature of this gene affects the release of hormones such as serotonin, epinephrine and dopamine and suppresses them. It prevents us from reacting in some situations and from stopping and making quick decisions in other situations, which can cause us to make wrong decisions in possible bad situations. As a result of some research, mood states such as aggression, feelings of compassion and irritability can be observed in people carrying this gene. Additionally, as a result of research conducted on people carrying the MAOA gene, this gene can be passed on genetically from parents, and mutations can also develop due to later epigenetic reasons. If we talk about epigenetic reasons, children of families growing up in bad environments begin to implement whatever they see from their parents. For this reason, those children begin to exhibit bad habits or behaviors such as irritability and aggression in the future. [ 93 ]


In December 2020, then- Director of National Intelligence   John Ratcliffe  said in an editorial for  The Wall Street Journal  that US intelligence shows China had conducted human testing on People's Liberation Army soldiers with the aim of creating "biologically enhanced" soldiers. [ 94 ] [ 95 ]


In 2022, the People's Liberation Army Academy of Military Sciences reported a notable experiment where military scientists inserted a  gene  from the  tardigrade  into human  embryonic stem cells . This experiment aimed to explore the potential enhancement of soldiers' resistance to acute  radiation syndrome , thereby increasing their ability to survive nuclear fallout. This development reflects the intersection of genetic engineering and  military  research, with a focus on bioenhancement for military personnel.
 [ 96 ]


CRISPR/Cas9  technologies have garnered attention for their potential applications in military contexts. Various projects are underway, including those focused on protecting soldiers from specific challenges. For instance, researchers are exploring the use of CRISPR/Cas9 to provide protection from  frostbite , reduce stress levels, alleviate  sleep deprivation , and enhance strength and endurance. The Defense Advanced Research Projects Agency ( DARPA ) is actively involved in researching and developing these technologies. One of their projects aims to engineer  human cells  to function as nutrient factories, potentially optimizing soldiers' performance and resilience in challenging environments.
 [ 97 ]


Additionally, military researchers are conducting animal trials to explore the prophylactic treatment for long-term protection against chemical weapons of mass destruction. This involves using non-pathogenic AAV8 vectors to deliver a candidate catalytic bioscavenger, PON1-IF11, into the  bloodstream  of  mice . These initiatives underscore the broader exploration of  genetic  and molecular interventions to enhance military capabilities and protect personnel from various threats. [ 98 ]


In the realm of bioenhancement, concerns have been raised about the use of dietary supplements and other  biomedical  enhancements by military personnel. A significant portion of American special operations forces reportedly use dietary supplements to enhance performance, but the extent of the use of other bioenhancement methods, such as steroids, human growth hormone, and erythropoietin, remains unclear. The lack of completed safety and efficacy testing for these bioenhancements raises ethical and regulatory questions. This concern is not new, as issues surrounding the off-label use of products like  pyridostigmine bromide  and botulinum toxoid vaccine during the  Gulf War , as well as the DoD's Anthrax Vaccine Immunization Program in 1998, have prompted discussions about the need for thorough  FDA approval  for specific military applications. [ 99 ]


The intersection of  genetic engineering ,  CRISPR/Cas9  technologies, and military research introduces complex ethical considerations regarding the potential augmentation of human capabilities for military purposes. Striking a balance between scientific advancements, ethical standards, and regulatory oversight over classified projects remain crucial as these technologies continue to evolve. [ 100 ]


George Church  has compiled a list of potential genetic modifications based on scientific studies for possibly advantageous traits such as less need  for sleep , cognition-related changes that protect against Alzheimer's disease, disease resistances, higher lean muscle mass and enhanced  learning  abilities along with some of the associated studies and potential negative effects. [ 101 ] [ 102 ]


RNA editing  (also  RNA modification ) is a molecular process through which some cells can make discrete changes to specific  nucleotide sequences  within an  RNA  molecule after it has been generated by  RNA polymerase . It occurs in all living organisms and is one of the most evolutionarily conserved properties of  RNAs . [ 1 ] [ 2 ] [ 3 ]  RNA editing may include the insertion, deletion, and base substitution of nucleotides within the RNA molecule. RNA editing is relatively rare, with common forms of RNA processing (e.g.  splicing , 5'- capping , and 3'- polyadenylation ) not usually considered as editing. It can affect the activity, localization as well as stability of RNAs, and has been linked with human diseases. [ 1 ] [ 2 ] [ 3 ] [ 4 ]


RNA editing has been observed in some  tRNA ,  rRNA ,  mRNA , or  miRNA  molecules of  eukaryotes  and their  viruses ,  archaea , and  prokaryotes . [ 5 ]  RNA editing occurs in the cell nucleus, as well as within  mitochondria  and  plastids . In vertebrates, editing is rare and usually consists of a small number of changes to the sequence of the affected molecules. In other organisms, such as  squids , [ 6 ]  extensive editing ( pan-editing ) can occur; in some cases the majority of nucleotides in an mRNA sequence may result from editing. More than 160 types of RNA modifications have been described so far. [ 7 ]


RNA-editing processes show great molecular diversity, and some appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing phenomena includes  nucleobase  modifications such as  cytidine  (C) to  uridine  (U) and  adenosine  (A) to  inosine  (I)  deaminations , as well as non-template nucleotide additions and insertions. RNA editing in mRNAs effectively alters the  amino acid sequence  of the encoded protein so that it differs from that predicted by the genomic DNA sequence. [ 8 ]


To identify diverse post-transcriptional modifications of RNA molecules and determine the transcriptome-wide landscape of RNA modifications by means of next generation RNA sequencing, recently many studies have developed conventional [ 9 ]  or specialised sequencing methods. [ 1 ] [ 2 ] [ 3 ]  Examples of specialised methods are  MeRIP-seq , [ 10 ]  m6A-seq, [ 11 ]  PA-m 5 C-seq  [ 12 ] ,  methylation-iCLIP, [ 13 ]  m6A-CLIP, [ 14 ]  Pseudo-seq, [ 15 ]  Ψ-seq, [ 16 ]  CeU-seq, [ 17 ]  Aza-IP [ 18 ]  and RiboMeth-seq [ 19 ] ). Many of these methods are based on specific capture of the RNA species containing the specific modification, for example through antibody binding coupled with sequencing of the captured reads. After the sequencing these reads are mapped against the whole transcriptome to see where they originate from. [ 20 ]  Generally with this kind of approach it is possible to see the location of the modifications together with possible identification of some consensus sequences that might help identification and mapping further on. One example of the specialize methods is PA-m 5 C-seq. This method was further developed from PA-m 6 A-seq method to identify m 5 C modifications on mRNA instead of the original target N6-methyladenosine. The easy switch between different modifications as target is made possible with a simple change of the capturing antibody form m6A specific to m 5 C specific. [ 12 ]  Application of these methods have identified various modifications (e.g. pseudouridine,  m 6 A , m5C, 2′-O-Me) within coding genes and non-coding genes (e.g. tRNA, lncRNAs, microRNAs) at single nucleotide or very high resolution. [ 4 ]


Mass spectrometry  is a way to quantify RNA modifications. [ 21 ]  More often than not, modifications cause an increase in mass for a given nucleoside. This gives a characteristic readout for the nucleoside and the modified counterpart. [ 21 ]  Moreover, mass spectrometry allows the investigation of modification dynamics by labelling RNA molecules with stable (non-radioactive) heavy isotopes  in vivo . Due to the defined mass increase of heavy isotope labeled nucleosides they can be distinguished from their respective unlabelled isotopomeres by mass spectrometry. This method, called  NAIL-MS  (nucleic acid isotope labelling coupled mass spectrometry), enables a variety of approaches to investigate RNA modification dynamics. [ 22 ] [ 23 ] [ 24 ]


Recently, functional experiments have revealed many novel functional roles of RNA modifications. Most of the RNA modifications are found on transfer-RNA and ribosomal-RNA, but also eukaryotic mRNA has been shown to be modified with multiple different modifications. 17 naturally occurring modifications on mRNA have been identified, from which the N6-methyladenosine is the most abundant and studied. [ 25 ]  mRNA modifications are linked to many functions in the cell. They ensure the correct maturation and function of the mRNA, but also at the same time act as part of cell's immune system. [ 26 ]  Certain modifications like 2’O-methylated nucleotides has been associated with cells ability to distinguish own mRNA from foreign RNA. [ 27 ]  For example, m 6 A has been predicted to affect protein translation and localization, [ 1 ] [ 2 ] [ 3 ]  mRNA stability, [ 28 ]  alternative polyA choice  [ 14 ]  and stem cell pluripotency. [ 29 ]   Pseudouridylation  of nonsense codons suppresses translation termination both  in vitro  and  in vivo , suggesting that RNA modification may provide a new way to expand the genetic code. [ 30 ]  5-methylcytosine on the other hand has been associated with mRNA transport from the nucleus to the cytoplasm and enhancement of translation. These functions of m 5 C are not fully known and proven but one strong argument towards these functions in the cell is the observed localization of m 5 C to translation initiation site. [ 31 ]  Importantly, many modification enzymes are dysregulated and genetically mutated in many disease types. [ 1 ]  For example, genetic mutations in pseudouridine synthases cause mitochondrial myopathy, sideroblastic anemia (MLASA)  [ 32 ]  and dyskeratosis congenital. [ 33 ]


Compared to the modifications identified from other RNA species like tRNA and rRNA, the amount of identified modifications on mRNA is very small. One of the biggest reasons why mRNA modifications are not so well known is missing research techniques. In addition to the lack of identified modifications, the knowledge of associated proteins is also behind other RNA species. Modifications are results of specific enzyme interactions with the RNA molecule. [ 25 ]  Considering mRNA modifications most of the known related enzymes are the writer enzymes that add the modification on the mRNA. The additional groups of enzymes readers and erasers are for most of the modifications either poorly known of not known at all. [ 34 ]  For these reasons there has been during the past decade huge interest in studying these modifications and their function. [ 20 ]


Transfer RNA  or tRNA is the most abundantly modified type of RNA. [ 35 ]  Modifications in tRNA play crucial roles in maintaining translation efficiency through supporting structure, anticodon-codon interactions, and interactions with enzymes. [ 36 ]


Anticodon modifications are important for proper decoding of mRNA. Since the genetic code is degenerate, anticodon modifications are necessary to properly decode mRNA. Particularly, the  wobble position  of the anticodon determines how the codons are read. For example, in eukaryotes an adenosine at position 34 of the anticodon can be converted to inosine. Inosine is a modification that is able to base-pair with cytosine, adenine, and uridine. [ 37 ]


Another commonly modified base in tRNA is the position adjacent to the anticodon. Position 37 is often hypermodified with bulky chemical modifications. These modifications prevent frameshifting and increase anticodon-codon binding stability through stacking interactions. [ 37 ]


Ribosomal RNA (rRNA) is essential to the makeup of ribosomes and peptide transfer during translation processes. [ 38 ]  Ribosomal RNA modifications are made throughout ribosome synthesis, and often occur during and/or after translation. Modifications primarily play a role in the structure of the rRNA in order to protect translational efficiency. [ 38 ]  Chemical modification in rRNA consists of  methylation  of  ribose sugars ,  isomerization  of uridines, and methylation and  acetylation  of individual bases. [ 39 ]


Methylation of rRNA upholds structural rigidity by blocking base pair stacking and surrounds the 2’-OH group to block hydrolysis. It occurs at specific parts of eukaryotic rRNA. The template for methylation consists of 10-21 nucleotides. [ 38 ]  2'-O-methylation of the ribose sugar is one of the most common rRNA modifications. [ 40 ]  Methylation is primarily introduced by small nucleolar RNA's, referred to as snoRNPs. There are two classes of snoRNPs that target methylation sites, and they are referred to box C/D and box H/ACA. [ 39 ] [ 40 ]  One type of methylation, 2′-O-methylation, contributes to helical stabilization. [ 38 ]


The isomerization of uridine to pseudouridine is the second most common rRNA modification. These pseudouridines are also introduced by the same classes of snoRNPs that participate in methylation. Pseudouridine synthases are the major participating enzymes in the reaction. [ 41 ]  The H/ACA box snoRNPs introduce guide sequences that are about 14-15 nucleotides long. [ 39 ]  Pseudouridylation is triggered in numerous places of rRNAs at once to preserve the thermal stability of RNA. [ 39 ]  Pseudouridine allows for increased hydrogen bonding and alters translation in rRNA and tRNA. [ 40 ] [ 41 ]  It alters translation by increasing the affinity of the ribosome subunit to specific mRNAs. [ 38 ]


Base Editing:


Base editing is the third major class of rRNA modification, specifically in eukaryotes. There are 8 categories of base edits that can occur at the gap between the small and large ribosomal subunits. [ 38 ]  RNA methyltransferases are the enzymes that introduce base methylation. [ 38 ]  Acetyltransferases are the enzymes responsible for acetylation of cytosine in rRNA. Base methylation plays a role in translation. These base modifications all work in conjunction with the two other main classes of modification to contribute to RNA structural stability. An example of this occurs in N7-methylation, which increases the nucleotide's charge to increase ionic interactions of proteins attaching to the RNA before translation.


RNA editing through the addition and deletion of uracil has been found in  kinetoplasts  from the mitochondria of  Trypanosoma brucei . [ 42 ]
Because this may involve a large fraction of the sites in a gene, it is sometimes called "pan-editing" to distinguish it from topical editing of one or a few sites.


Pan-editing starts with the base-pairing of the unedited primary transcript with a  guide RNA  (gRNA), which contains complementary sequences to the regions around the insertion/deletion points. The newly formed double-stranded region is then enveloped by an editosome, a large multi-protein complex that catalyzes the editing. [ 43 ] [ 44 ]  The editosome opens the transcript at the first mismatched nucleotide and starts inserting uridines. The inserted uridines will base-pair with the guide RNA, and insertion will continue as long as A or G is present in the guide RNA and will stop when a C or U is encountered. [ 45 ] [ 46 ]  The inserted nucleotides cause a  frameshift , and result in a translated protein that differs from its gene.


The mechanism of the  editosome  involves an  endonucleolytic  cut at the mismatch point between the guide RNA and the unedited transcript. The next step is catalyzed by one of the enzymes in the complex, a terminal U-transferase, which adds Us from UTP at the 3' end of the mRNA. [ 47 ]  The opened ends are held in place by other proteins in the complex. Another enzyme, a U-specific exoribonuclease, removes the unpaired Us. After editing has made mRNA complementary to gRNA, an RNA ligase rejoins the ends of the edited mRNA transcript. [ 48 ] [ 49 ]  As a consequence, the editosome can edit only in a 3' to 5' direction along the primary RNA transcript. The complex can act on only a single guide RNA at a time. Therefore, a RNA transcript requiring extensive editing will need more than one guide RNA and editosome complex.


The editing involves cytidine deaminase that deaminates a cytidine base into a uridine base. An example of C-to-U editing is with the  apolipoprotein B  gene in humans.  Apo B100  is expressed in the liver and apo B48 is expressed in the intestines. In the intestines, the mRNA has a CAA sequence edited to be UAA, a stop codon, thus producing the shorter B48 form.
C-to-U editing often occurs in the  mitochondrial RNA  of flowering plants. Different plants have different degrees of C-to-U editing; for example, eight editing events occur in mitochondria of the moss  Funaria hygrometrica , whereas over 1,700 editing events occur in the lycophytes  Isoetes engelmanii . [ 50 ]  C-to-U editing is performed by members of the pentatricopeptide repeat (PPR) protein family. Angiosperms have large PPR families, acting as  trans  -factors for  cis  -elements lacking a consensus sequence;  Arabidopsis  has around 450 members in its PPR family. There have been a number of discoveries of PPR proteins in both plastids and mitochondria. [ 51 ]


Adenosine-to-inosine (A-to-I) modifications contribute to nearly 90% of all editing events in RNA. The deamination of adenosine is catalyzed by the double-stranded RNA-specific adenosine deaminase ( ADAR ), which typically acts on pre-mRNAs. The deamination of adenosine to inosine disrupts and destabilizes the dsRNA base pairing, therefore rendering that particular dsRNA less able to produce  siRNA , which interferes with the  RNAi  pathway.


The  wobble base pairing  causes deaminated RNA to have a unique but different structure, which may be related to the inhibition of the initiation step of RNA translation. Studies have shown that I-RNA (RNA with many repeats of the I-U base pair) recruits methylases that are involved in the formation of  heterochromatin  and that this chemical modification heavily interferes with miRNA target sites. [ 52 ]  There is active research into the importance of A-to-I modifications and their purpose in the novel concept of  epitranscriptomics , in which modifications are made to RNA that alter their function. [ 53 ] [ 54 ]  A long established consequence of A-to-I in mRNA is the interpretation of I as a G, therefore leading to functional A-to-G substitution, e.g. in the interpretation of the genetic code by ribosomes. Newer studies, however, have weakened this correlation by showing that inosines can also be decoded by the ribosome (although in a lesser extent) as adenosines or uracils. Furthermore, it was shown that I's lead to the stalling of ribosomes on the I-rich mRNA. [ 55 ]


The development of high-throughput sequencing in recent years has allowed for the development of extensive databases for different modifications and edits of RNA. RADAR (Rigorously Annotated Database of A-to-I RNA editing) was developed in 2013 to catalog the vast variety of A-to-I sites and tissue-specific levels present in humans,  mice , and  flies . The addition of novel sites and overall edits to the database are ongoing. [ 56 ]  The level of editing for specific editing sites, e.g. in the filamin A transcript, is tissue-specific. [ 57 ]  The efficiency of mRNA-splicing is a major factor controlling the level of A-to-I RNA editing. [ 58 ] [ 59 ]  Interestingly, ADAR1 and ADAR2 also affect alternative splicing via both A-to-I editing ability and dsRNA binding ability. [ 60 ] [ 61 ]


Alternative U-to-C mRNA editing was first reported in  WT1  (Wilms Tumor-1) transcripts, [ 62 ]  and non-classic G-A mRNA changes were first observed in  HNRNPK  (heterogeneous nuclear ribonucleoprotein K) transcripts in both malignant and normal colorectal samples. [ 63 ]  The latter changes were also later seen alongside non-classic U-to-C alterations in brain cell  TPH2  (tryptophan hydroxylase 2) transcripts. [ 64 ]  Although the reverse amination might be the simplest explanation for U-to-C changes, transamination and transglycosylation mechanisms have been proposed for plant U-to-C editing events in mitochondrial transcripts. [ 65 ]  A recent study reported novel G-to-A mRNA changes in WT1 transcripts at two hotspots, proposing the  APOBEC3A  (apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3A) as the enzyme implicated in this class of alternative mRNA editing. [ 66 ]  It was also shown that alternative mRNA changes were associated with canonical WT1  splicing  variants, indicating their functional significance.


It has been shown in previous studies that the only types of RNA editing seen in the plants' mitochondria and plastids are conversion of C-to-U and U-to-C (very rare). [ 67 ] [ 68 ] [ 69 ] [ 70 ] [ 71 ] [ 72 ] [ 73 ] [ 74 ] [ 75 ] [ 76 ] [ 77 ] [ 78 ] [ 79 ]  RNA-editing sites are found mainly in the coding regions of mRNA,  introns , and other non-translated regions. [ 69 ]  In fact, RNA editing can restore the functionality of tRNA molecules. [ 71 ] [ 72 ]  The editing sites are found primarily upstream of mitochondrial or plastid RNAs. While the specific positions for C to U RNA editing events have been fairly well studied in both the mitochondrion and plastid, [ 80 ]  the identity and organization of all proteins comprising the editosome have yet to be established. Members of the expansive PPR protein family have been shown to function as  trans -acting factors for RNA sequence recognition. [ 81 ]  Specific members of the MORF (Multiple Organellar RNA editing Factor) family are also required for proper editing at several sites. As some of these MORF proteins have been shown to interact with members of the PPR family, it is possible MORF proteins are components of the editosome complex. [ 82 ]  An enzyme responsible for the trans- or deamination of the RNA transcript remains elusive, though it has been proposed that the PPR proteins may serve this function as well.


RNA editing is essential for the normal functioning of the plant's translation and respiration activity. Editing can restore the essential base-pairing sequences of tRNAs, restoring functionality. [ 83 ]  It has also been linked to the production of RNA-edited proteins that are incorporated into the polypeptide complexes of the respiration pathway. Therefore, it is highly probable that polypeptides synthesized from unedited RNAs would not function properly and hinder the activity of both mitochondria and plastids.


C-to-U RNA editing can create start and stop  codons , but it cannot destroy existing start and stop codons. A cryptic start codon is created when the codon ACG is edited to be AUG.


Viruses (i.e.,  measles ,  mumps , or  parainfluenza ), especially viruses that have an RNA genome, have been shown to have evolved to utilize RNA modifications in many ways when taking over the host cell. Viruses are known to utilize the RNA modifications in different parts of their infection cycle from immune evasion to protein translation enhancement. [ 27 ]  RNA editing is used for stability and generation of protein variants. [ 84 ] [ 85 ]  Viral RNAs are transcribed by a virus-encoded RNA-dependent  RNA polymerase , which is prone to pausing and "stuttering" at certain nucleotide combinations. In addition, up to several hundred non-templated A's are added by the polymerase at the 3' end of nascent mRNA. [ 86 ]  These As help stabilize the mRNA. Furthermore, the pausing and stuttering of the RNA polymerase allows the incorporation of one or two Gs or As upstream of the translational codon. [ 86 ]  The addition of the non-templated nucleotides shifts the reading frame, which generates a different protein.


Additionally, the RNA modifications are shown to have both positive and negative effects on the replication and translation efficiency depending on the virus.  For example, Courtney et al. [ 12 ]  showed that an RNA modification called 5-methylcytosine is added to the viral mRNA in infected host cells in order to enhance the protein translation of HIV-1 virus. The inhibition of the m 5 C modification on viral mRNA results in significant reduction in viral protein translation, but interestingly it has no effect on the expression of viral mRNAs in the cell. On the other hand, Lichinchi et al. [ 87 ]  showed that the N6-methyladenosine modification on ZIKV mRNA inhibits the viral replication.


The RNA-editing system seen in the animal may have evolved from mononucleotide deaminases, which have led to larger gene families that include the apobec-1 and adar genes. These genes share close identity with the bacterial deaminases involved in nucleotide metabolism. The adenosine deaminase of  E. coli  cannot deaminate a nucleoside in the RNA; the enzyme's reaction pocket is too small for the RNA strand to bind to. However, this active site is widened by amino acid changes in the corresponding human analog genes,  APOBEC1  and  ADAR , allowing deamination. [ 88 ] [ 89 ]
The gRNA-mediated pan-editing in  trypanosome  mitochondria, involving templated insertion of U residues, is an entirely different biochemical reaction. The enzymes involved have been shown in other studies to be recruited and adapted from different sources. [ 43 ] [ 90 ]  But the specificity of nucleotide insertion via the interaction between the gRNA and mRNA is similar to the tRNA editing processes in the animal and  Acanthamoeba  mitochondria. [ 91 ]  Eukaryotic ribose methylation of rRNAs by guide RNA molecules is a similar form of modification. [ 92 ]


Thus, RNA editing evolved more than once. Several adaptive rationales for editing have been suggested. [ 93 ]  Editing is often described as a mechanism of correction or repair to compensate for defects in gene sequences. However, in the case of gRNA-mediated editing, this explanation does not seem possible because if a defect happens first, there is no way to generate an error-free gRNA-encoding region, which presumably arises by duplication of the original gene region. A more plausible alternative for the evolutionary origins of this system is through  constructive neutral evolution , where the order of steps is reversed, with the gratuitous capacity for editing preceding the "defect". [ 94 ]


Directing edits to correct mutated sequences was first proposed and demonstrated in 1995. [ 95 ]   This initial work used synthetic RNA antisense oligonucleotides complementary to a pre-mature stop codon mutation in a dystrophin sequence to activate A-to-I editing of the stop codon to a read through codon in a model xenopus cell system. [ 95 ]   While this also led to nearby inadvertent A-to-I transitions, A to I (read as G) transitions can correct all three stop codons, but cannot create a stop codon.  Therefore, the changes led >25% correction of the targeted stop codon with read through to a downstream luciferase reporter sequence.  Follow on work by Rosenthal achieved editing of mutated mRNA sequence in mammalian cell culture by directing an oligonucleotide linked to a cytidine deaminase to correct a mutated cystic fibrosis sequence. [ 96 ]   More recently, CRISPR-Cas13 fused to deaminases has been employed to direct mRNA editing. [ 97 ]


In 2022, therapeutic RNA editing for Cas7-11 was reported. [ 98 ] [ 99 ]  It enables sufficiently targeted cuts and an early version of it was used for  in vitro  editing in 2021. [ 100 ]


Unlike DNA editing, which is permanent, the effects of RNA editing − including potential  off-target mutations  in RNA − are transient and are not inherited. RNA editing is therefore considered to be less risky. Furthermore, it may only require a guide RNA by using the ADAR protein already found in humans and many other eukaryotes' cells instead of needing to introduce a foreign protein into the body. [ 101 ]


2A peptides  are a class of 18–22  aa -long  peptides , which can induce ribosomal skipping during translation of a  protein  in a biological cell. [ 1 ] [ 2 ]  These peptides share a core  sequence motif  of  DxExNPGP , and are found in a wide range of  viral families . 2A peptides can be introduced artificially to help generate  polyproteins  from a single  ORF , by causing the  ribosome  to fail at making a peptide bond, and then resume translation. [ 3 ] [ 4 ]


The members of 2A peptides are named after the  virus  in which they have been first described. For example, F2A, the first described 2A peptide, is derived from  foot-and-mouth disease virus . The name "2A" itself comes from the gene numbering scheme of this virus. [ 1 ] [ 5 ]


These peptides are also known as  "self-cleaving"  peptides, which is a known misnomer, because the missing peptide bond is never synthesized by the ribosome, and is thus not cleaved.


Four members of 2A peptides family are frequently used in life science research. They are P2A, E2A, F2A, and T2A. F2A is derived from  foot-and-mouth disease virus  18; E2A is derived from   equine rhinitis A virus ; P2A is derived from  porcine teschovirus -1 2A; T2A is derived from   thosea asigna virus  2A. [ 1 ]


The following table shows the sequences of four members of 2A peptides. Adding the optional linker “ G S G ” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency. [ 6 ]


2A peptides trigger the ribosome to skip  peptide bond  formation between the  glycine  (G) and  proline  (P) near the C-terminus of the 2A peptide, resulting in the peptide located upstream of the 2A peptide having extra amino acids appended to its C-terminus while the protein downstream the 2A peptide will have an extra  proline  on its N-terminus. The exact molecular mechanism of 2A-peptide-mediated cleavage is still unknown. [ 7 ] [ 8 ]  However, it is believed to involve ribosomal "skipping" of glycyl-prolyl peptide bond formation rather than true proteolytic cleavage. [ 9 ] [ 10 ]


In molecular biology, 2A peptides are used to express two separate proteins from a single open-reading frame. 2A peptides can be used when direct protein fusion does not work or is undesirable.


Different 2A peptides have different peptide-bond-skipping efficiencies, with T2A and P2A being the most efficient and F2A the least efficient. [ 11 ] [ 12 ]  Therefore, up to 50% of F2A-linked proteins can in fact be produced as a  fusion protein , which might cause some unpredictable outcomes, including a gain of function. [ 13 ]  One study reported that 2A sites cause the ribosome to fall off approximately 60% of the time, and that, together with ribosome read-through of about 10% for P2A and T2A, this results in reducing expression of the downstream peptide chain by about 70%. [ 1 ]  However, the level of drop-off detected in this study varied widely depending on the exact construct used, with some constructs showing little evidence of drop-off; furthermore, within a tri-cistronic transcript it reported a higher level of ribosome drop-off after one 2A sequence than after two 2As combined, which is at odds with a linear model of translation.


variants:


Anti-CRISPR  (Anti-Clustered Regularly Interspaced Short Palindromic Repeats or Acr) is a group of proteins found in  phages , that inhibit the normal activity of  CRISPR -Cas, the immune system of certain  bacteria . [ 1 ]  CRISPR consists of  genomic  sequences that can be found in  prokaryotic organisms , that come from  bacteriophages  that infected the bacteria beforehand, and are used to defend the cell from further viral attacks. [ 2 ]  Anti-CRISPR results from an evolutionary process occurred in phages in order to avoid having their  genomes  destroyed by the  prokaryotic  cells that they will infect. [ 3 ]


Before the discovery of this type of family proteins, the acquisition of mutations was the only way known that phages could use to avoid  CRISPR-Cas  mediated shattering, by reducing the binding affinity of the phage and CRISPR. Nonetheless, bacteria have mechanisms to retarget the mutant bacteriophage, a process that it is called "priming adaptation". So, as far as researchers currently know, anti-CRISPR is the most effective way to ensure the survival of phages throughout the infection process of bacteria. [ 4 ]


Anti-CRISPR systems were first seen in  Pseudomonas aeruginosa  prophages, [ 5 ]  which disabled type I-F CRISPR–Cas system, characteristic of some strains of these bacteria. After analysing the genomic sequences of these phages, genes codifying five different Anti-CRISPR proteins (also named Acrs) were discovered. Such proteins were  AcrF1 ,  AcrF2 ,  AcrF3 ,  AcrF4  and  AcrF5 . Research found none of these proteins disrupted the expression of Cas genes nor the assembling of CRISPR molecules, so it was thought that those type I-F  proteins  directly affected the CRISPR–Cas interference. [ 6 ]


Further investigation confirmed this hypothesis with the discovery of 4 other proteins ( AcrE1 ,  AcrE2 ,  AcrE3  and  AcrE4 ), which were shown to impede  Pseudomonas aeruginosa ’s CRISPR-Cas system. [ 7 ]   Furthermore, the locus of the genes codifying these type I-E proteins was really close to the one responsible for the type I-F proteins expression in the same group of phages, leading to the conclusion that both types of proteins worked together. [ 8 ]  However, these first nine proteins shared no common  sequence motifs , which would have made easier the identification of new Anti-CRISPR protein families.


Later on, it was seen that phages that produced such proteins also encoded a putative  transcriptional regulator  named  Aca 1  (anti-CRISPR associated 1) which was genetically located really close to the anti-CRISPR genes. This regulatory protein is supposed to be the responsible for the anti-CRISPR gene expression during the infectious cycle of the phage,  therefore, both types of proteins (anti-CRISPR and Aca1) seem to work together as a single mechanism. [ 5 ]


After some studies, a similar  amino-acid sequence  to that of Aca1 was found, leading to the discovery of  Aca2 , a new family of Aca proteins. Aca2 also revealed the existence of five new groups of type I-F anti-CRISPR proteins due to their genomic proximity:  AcrF6 ,  AcrF7 ,  AcrF8 ,  AcrF9  and  AcrF10 . These proteins were not only present in  Pseudomonas aeruginosa ’s phages, as they also affected other cells of the  Pseudomonadota  (formerly  Proteobacteria ). [ 6 ]


Thanks to the use of bioinformatic tools, in 2016,  AcrIIC1 ,  AcrIIC2  and  AcrIIC3  protein families were discovered in  Neisseria meningitidis  (which had been infected by phages previously). Such proteins were the first inhibitors of type II CRISPR–Cas to be found (concretely, they impeded II-C CRISPR–Cas9, the type of mechanism used in the genetic edition of human cells). [ 9 ]  A year later, a study confirmed the presence of type II-A CRISPR–Cas9 inhibitors ( AcrIIA1 ,  AcrIIA2 ,  AcrIIA3  and  AcrIIA4 ) in  Listeria monocytogenes  (infected by bacteriophages which introduced the anti-CRISPR proteins). Two of those proteins (AcrIIA2 and AcrIIA4) were demonstrated to work properly against  Streptococcus pyogenes  type II-A defensive CRISPR system.


The result of all this research has been the discovery of 21 different Anti-CRISPR protein families, despite other inhibitors may exist due to the quick  mutational  process of phages. Thus, more research is needed to unravel the complexity of anti-CRISPR systems.


Anti-CRISPR genes can be found in different parts of the phage DNA: in the capsid, the tail and at the extreme end. Moreover, it has been found that many MGEs have two or even three Acr genes in a single  operon , which suggest that they could have been exchanged between MGEs. [ 10 ]


As all proteins, Acr family proteins are formed by the translation and transduction of the genes, and their classification is based on the type of CRISPR-Cas system they inhibit, due to the fact that each anti-CRISPR protein inhibits a specific CRISPR-Cas system. Although not many anti-CRISPR proteins have been discovered, these are the ones that have been found so far:


So far, genes encoding anti-CRISPR proteins have been found in  myophages ,  siphophages ,  putative conjugative elements  and  pathogenicity islands .


Attempts have been made to find common surrounding genetic features of anti-CRISPR genes, but without any success. Nevertheless, the presence of an  aca  gene just below anti-CRISPR genes has been observed. [ 10 ]


The first Acr protein families to be discovered were AcrF1, AcrF2, AcrF3, AcrF4 and AcrF5. [ 5 ]  These inhibitors are mainly found in  Pseudomonas  phages, which are capable of infecting  Pseudomonas aeruginosas  possessing a type I‑F CRISPR–Cas system. Then, in another study, AcrE1, AcrE2, AcrE3 and AcrE4 protein families were found to also inhibit the type I‑F CRISPR–Cas in  Pseudomonas aeruginosas. [ 7 ]


Later on, AcrF6, AcrF7, AcrF8, AcrF9 and AcrF10 protein families, which were also able to inhibit type I‑F CRISPR–Cas, were found to be very common in  Pseudomonadota   MGEs . [ 10 ]


The first inhibitors of a type II CRISPR–Cas system were then discovered: AcrIIC1, AcrIIC2 and AcrIIC3, that block the type II‑C CRISPR–Cas9 activity of  Neisseria meningitidis . [ 9 ]


Finally, AcrIIA1, AcrIIA2, AcrIIA3 and AcrIIA4 were found. These protein families have the ability to inhibit the type II‑A CRISPR–Cas system of  Listeria monocytogenes . [ 11 ]


As for the naming convention of Acr family proteins, it is established as follows: firstly, the type of system inhibited, then a numerical value referring to the protein family and finally the source of the specific anti-CRISPR protein. For example, AcrF9 Vpa  is active against the type I-F CRISPR–Cas system. It also was the ninth anti-CRISPR described for this system, and it is encoded in an integrated MGE in a  Vibrio parahaemolyticus  genome.


As exposed above, there is a wide spectrum of anti-CRISPR proteins, but few of these have been deeply studied. One of the most studied and well-defined Acrs is AcrIIA4, which inhibits Cas9, thus blocking the II-A CRISPR-Cas system of  Streptococcus pyogenes .


The protein was solved using  nuclear magnetic resonance  (NMR); it contains 87 residues and its molecular weight is 10.182 kDa. [ 13 ]  AcrIIA4 contains:


There is a good definition of the secondary structures, as the three α-helices are packed near the three β-strands. Strikingly, between β3 strand, α2 and α3 helices, there is a hydrophobic core, originated by a cluster of aromatic side chains which are attracted by non-covalent interactions, such as  pi stacking . Moreover, as it is an acidic protein, there is a high concentration of negatively charged residues in the loops between β3 and α2, between α2 and α3, and in the first part of α3, which may play an important role in the inhibition of Cas9, as negative charges might imitate  phosphates  of nucleic acids. [ 14 ]


On the other hand, there is another Acr, AcrF1, which may not have been as studied as the explained above, although there is a good description of its structure. It inhibits the I-F CRISPR-Cas system of  Pseudomonas aeruginosa . Maxwell et al. [ 15 ]  solved the 3D structure using NMR.


The protein contains 78 residues, [ 6 ]  between which interact to form secondary structures. The structure of AcrF1 is formed of two anti-parallel α-helices and a β-sheet, which contains four anti-parallel β-strands. This β-sheet is placed in the contrary side of the α-helical part, which creates a hydrophobic core formed of 13 amino acids. Turns can also be found in different parts of the protein, for instance, joining the β-strands. [ 15 ] [ 16 ]


There are surface residues which actively participate in the active site of AcrF1, two of which are  tyrosines  (Y6 and Y20) and the third amino acid is a  glutamic acid  (E31), as their mutation by an  alanine  causes a 100-fold decrease in the activity of the protein (with Y20A and E31A mutations), and a 10 7 -fold decrease when Y6 is mutated.


The different structures that form the protein create a strange combination, as Maxwell et al. conducted a DALI search in order to find similarities between other proteins, and they found no informative similarities. [ 15 ]


The principal function of anti-CRISPR proteins is to interact with specific components of CRISPR-Cas systems, such as the effector  nucleases , to avoid the destruction of the phage DNA (by binding or cleavage). [ 17 ]


A phage introduces its DNA into a prokaryotic cell, usually the cell detects a sequence known as "target", that activates CRISPR-Cas immune system, but the presence of an initial sequence (before the target) encoding the formation of Acr proteins, avoids phage destruction. Acr proteins are formed before the target sequence is read. This way, the CRISPR-Cas system is blocked before it can develop a response.


The procedure starts with the CRISPR  locus  being transcribed into crRNAs (CRISPR RNA). CrRNAs combine with Cas proteins forming a ribonucleoprotein complex called Cascade. This complex surveys the cell to find complementary sequences of the crRNA. When this sequence is found, the Cas3 nuclease is recruited to the Cascade, and the target DNA from the phage is cleaved. But, for instance, when AcrF1 and AcrF2 are found (anti-CRISPR proteins), these interact with Cas7f and Cas8f-Cas5f, respectively, not allowing the binding to the phage DNA. Moreover, the cleaving of the target is prevented by the union between AcrF3 and Cas3. [ 6 ]


The majority of Acr genes are located next to anti-CRISPR-associated (Aca) genes, which encode proteins with a helix-turn-helix DNA-binding motif. Aca genes are preserved, and researchers are using them to identify Acr genes, but the function of the proteins they encode is not totally clear. The Acr-associated promoter produces high levels of Acr transcription just after the phage DNA injection into the bacteria takes place and, afterward, Aca proteins repress the transcription. If this wasn't repressed, the constant transcription of the gene would be lethal to the phage. Therefore, Aca activity is essential to ensure its survival. [ 18 ]


Moreover, it has been verified that bacteria with CRISPR-Cas systems are still partially immune to Acr. Consequently, initial abortive phage infections may be unable to hamper CRISPR immunity, but phage-phage cooperation can increasingly boost Acr production and promote immunosuppression, which might produce an increase on the vulnerability of the host cell to reinfection, and finally allow a successful infection and spreading of a second phage. [ 17 ]  This cooperation creates an epidemiological tipping point, in which, depending on the initial density of Acr-phages and the strength of CRISPR/Acr binding, phages can either be eliminated or originate a phage epidemic (the number of bacteriophages is amplified). [ 19 ] [ 20 ]


If the starting levels of phages are high enough, the density of immunosuppressed hosts reaches a critical point where there are more successful infections than unsuccessful ones. Then, an epidemic begins. If this point is not reached, phage extinction occurs, and immunosuppressed hosts recover their initial state. [ 19 ] [ 20 ]


It has become clear that Acr proteins play an important role in allowing phage immune evasion, though it is still unclear how anti-CRISPR proteins synthesis can overcome the host’s CRISPR-Cas system, which can shatter the phage genome within minutes after the infection. [ 17 ]


Within all the Anti-CRISPR proteins that have been discovered so far, mechanisms have been described for only 15 of among them. These mechanisms can be divided into three different types: crRNA loading interference, DNA binding blockage and DNA  cleavage  prevention.


CrRNA (CRISPR RNA) loading interference mechanism has been mainly associated with the AcrIIC2 protein family. [ 22 ]  In order to block  Cas9  activity, it prevents the correct assembly of the crRNA-Cas9 complex.


AcrIIC2 has been shown not to be the only one capable of blocking DNA binding. There are 11 other Acr family proteins that can also carry it out. Some among those are AcrIF1, AcrIF2, and AcrIF10, which act on different subunits of the  Cascade effector  complex of the type I-F CRISPR-Cas system, preventing the DNA to bind to the complex. [ 23 ]


Furthermore, AcrIIC3 prevents DNA binding by promoting  dimerization  of Cas9  [ 22 ] [ 24 ]  and AcrIIA2 mimics DNA, thereby blocking the  PAM  recognition residues and consequently preventing dsDNA  (double-stranded DNA)  recognition and binding. [ 25 ] [ 26 ]


AcrE1, AcrIF3 and AcrIIC1 can prevent target DNA cleavage. Using  X-ray crystallography , AcrE1 was discovered to bind to the CRISPR associated Cas3. [ 27 ]  Likewise, biochemical and structural analysis of AcrIF3 showed its capacity of binding to Cas3 as a dimer so as to prevent the recruitment of Cas3 to the Cascade complex. [ 23 ] [ 28 ] [ 29 ]  Finally, thanks to biochemical and structural AcrIIC1 studies, it was found that it binds to the active site of the  HNH endonuclease  domain in Cas9, which prevents DNA from cleaving. Thus, it turns Cas9 into an inactive but DNA bound state. [ 24 ]


AcrIIA4 is one of the proteins responsible for the CRISPR-Cas9 system inhibition, the mechanism used in mammalian cells edition. Addition of AcrIIA4 in human cells avoids Cas9 interaction with the CRISPR system, reducing its ability to cut DNA. However, diverse studies have reached the conclusion that adding it in small proportions after the genome editing has been done, reduces the number of off-target cuts at the concrete sites in which Cas9 interacts, a thing that makes the whole system much more precise. [ 25 ]


One of the main objectives of using CRISPR-Cas9 technology is eradicating diseases, some of which are found in  disease vectors , such as mosquitoes. Anti-CRISPR proteins can impede  gene drive , which could create uncertain and catastrophic consequences in  ecosystems . [ 30 ]


In order to know whether a certain bacterium synthesises Cas9, and therefore uses CRISPR-Cas9, or to detect accidental or not allowed use of this system, AcrIIC1 can be used. As the aforementioned protein binds to Cas9, a centrifugal microfluidic platform has been designed to detect it and determine its catalytic activity. [ 30 ]


Antibiotic resistance  is a public health problem that is constantly increasing, because of the bad use of antibiotics.  Phage therapy  consists of the infection of bacteria using phages, which are much more specific and cause less side effects than antibiotics. Acrs could inhibit the CRISPR-Cas9 system of some bacteria and allow these phages to infect bacterial cells without being attacked by its immune system. [ 30 ]


ATryn  is the brand name of the  anticoagulant   antithrombin  manufactured by the  Massachusetts -based U.S. company  rEVO Biologics  (formerly known as GTC Biotherapeutics). It is made from the milk of goats that have been  genetically modified  to produce human antithrombin, a  plasma protein  with anticoagulant properties. [ 1 ]   Microinjection  was used to insert human antithrombin genes into the  cell nucleus  of their  embryos . [ 2 ]  ATryn is the first medicine produced using genetically engineered animals. [ 3 ]  GTC states that one genetically modified goat can produce the same amount of antithrombin in a year as 90,000 blood donations. [ 4 ]  GTC chose goats for the process because they reproduce more rapidly than cattle and produce more protein than rabbits or mice. [ 5 ]


On February 6, 2009, ATryn was approved by the  U.S. Food and Drug Administration  (FDA) for treatment of patients with hereditary  antithrombin deficiency  who are undergoing surgical or childbirth procedures. [ 1 ]  Along with the approval from the FDA's pharmaceutical regulatory board, the  Center for Veterinary Medicine  of the FDA also approved the genetic makeup of the goats that are used to manufacture ATryn. [ 3 ]  rEVO has the sole rights to sell ATryn in the United States, [ 3 ]  and the drug is available in the U.S. market. [ 1 ]  Earlier in 2006, the  European Medicines Agency  (EMA) initially rejected and, after an appeal from GTC, approved the drug for use in the  European Union  countries. [ 2 ] [ 6 ] [ 7 ]


According to Tom Newberry, the spokesperson for GTC, the company plans to acquire additional approval for treatment of those with non-hereditary antithrombin deficiency. [ 3 ]


The  Humane Society of the United States  has said of the process used to manufacture ATryn, "It is a mechanistic use of animals that seems to perpetuate the notion of their being merely tools for human use rather than sentient creatures." [ 4 ] [ 8 ]  However, the genetic changes have no known ill-effects on the host animal. [ citation needed ]


Ekkehard Karl Friedrich Bautz  is a molecular biologist and chair of the Institute of Molecular Genetics at the  University of Heidelberg .


He was born September 24, 1933, in  Konstanz , Germany. After studying chemistry at  Freiburg University  and the  University of Zürich , at the age of 26, he emigrated to the United States and later became a U.S. citizen. In 1961, he obtained a doctorate in molecular biology from the  University of Wisconsin . He did postdoctoral work at the  University of Illinois  with a fellowship awarded by the  Damon Runyon Memorial Fund for Cancer Research  and in 1962 became an assistant professor at the Institute of Microbiology at  Rutgers . In 1964, he participated in the  Evolving Genes and Proteins Symposium , a landmark event in the history of  molecular evolution  research. In 1966, he was promoted to associate  professor at the same institution. In 1970, he was appointed full professor there, but chose to return to Germany in the same year to become chair of the Institute of Molecular Genetics at the University of Heidelberg. [ 1 ]


Bautz's most important discovery is that of  sigma factor , the first known  transcription factor . [ 2 ]


He developed methods for the isolation of  messenger RNA [ 3 ]  and continued research on transcription. Later, he focused on novel selection methods, in particular  phage  display  [ 4 ]  and the generation of recombinant antibodies. [ 5 ]


In 1981, he founded the Center for Molecular Biology (ZMBH) [ 6 ]  in Heidelberg, where he served as chair of microbiology and acting director from 1983 to 1985. [ 7 ]


Bautz was on the editorial board of the  Journal of Virology [ 8 ]  from 1966 to 1970, and of  Molecular and General Genetics  from 1971 to 2000. He was chairman of the German Genetics Society  [ 9 ]  from 1979 to 1981, and a board member of the German Cancer Research Centre  [ 10 ]  from 1978 to 1983. In 1994, he was appointed a board member of the Zentralkommission für Biologische Sicherheit (ZKBS, engl.: Central Commission for Biological Safety), advising the German government on the biological safety of genetically engineered organisms. He retired from the commission in 2000.


In 1983, he founded Progen GmbH, a biotech startup, [ 11 ]  with  Werner Franke  and two other scientists from Heidelberg. He is also a cofounder of Peptide Specialty Laboratories (PSL)  [ 12 ]  and acted as general manager of Multimetrix GmbH from 2002 to 2007.


Benitec Biopharma Limited  is an Australian  biotechnology company  founded in 1997. [ 1 ]  It is engaged in the development of gene-silencing therapies for the treatment of chronic and life-threatening diseases using  DNA-directed RNA interference  (ddRNAi) technology. [ 2 ]


The  CSIRO  has researched RNAi extensively, developing the  small hairpin RNA  concept employed in ddRNAi. Benitec Biopharma has an exclusive license to this ddRNAi technology in human therapeutic uses and research. [ 3 ]


Benitec Biopharma is researching  ddRNAi  in the following fields:


Since the advent of  genetic engineering  in the 1970s, concerns have been raised about the dangers of the technology.  Laws, regulations, and treaties were created in the years following to  contain  genetically modified organisms  and prevent their  escape.   Nevertheless, there are several examples of failure to keep GM crops separate from conventional ones.


In the context of agriculture and food and feed production, co-existence means using cropping systems with and without  genetically modified crops  in parallel. In some countries, such as the United States, co-existence is not governed by any single law but instead is managed by regulatory agencies and  tort  law.  In other regions, such as Europe, regulations require that the separation and the identity of the respective food and feed products must be maintained at all stages of the production process.


Many consumers are critical of genetically modified plants and their products, while, conversely, most experts in charge of GMO approvals do not perceive concrete threats to health or the environment. The compromise chosen by some countries  - notably the European Union - has been to implement regulations specifically governing co-existence and  traceability . Traceability has become commonplace in the food and feed supply chains of most countries in the world, but the traceability of GMOs is made more challenging by the addition of very strict legal thresholds for unwanted mixing.  Within the  European Union , since 2001, conventional and organic food and feedstuffs can contain up to 0.9% of authorised GM material without being labelled GM [ 1 ]   (any trace of non-authorised GM products would cause shipments to be rejected [ 1 ] [ 2 ] ).


In the United States there is no legislation governing the co-existence of neighboring farms growing organic and GM crops; instead the US relies on a "complex but relaxed" combination of three federal agencies ( FDA ,  EPA , and  USDA / APHIS ) and the common law  tort  system, governed by state law, to manage risks of co-existence. [ 3 ] : 44


To limit mixing in the first stages of production, researchers and politicians are developing codes of  good agricultural practice  for GM crops. In addition to the thorough cleaning of machinery, recommended measures include the establishment of "isolation distances" and "pollen barriers". Isolation distances are the minimum distances required between GM and non-GM cultivations for most of the GM pollen to fall to the ground before reaching non-GM plants. Pollen barriers attempt actively catch pollen, and can consist of hedges and trees which physically hinder pollen movement. Pollen barriers consisting of conventional crops of the same species as the GM crop have a special advantage, as the conventional plants not only physically limit the GM pollen flow, but also produce competitive, conventional pollen. During harvest, the buffer strip of conventional crops is considered part of the GM crop yield. [ 4 ]


In addition to agricultural measures, there may be also biological tools to prevent the genetically modified crop from fertilising conventional fields. Researchers are investigating methods either to prevent GM crops from producing pollen at all (for example  male-sterile plants ), or to develop GM crops with pollen that nonetheless does not contain the additional, genetically engineered material. In an example of the latter,  transplastomic plants  can be generated in which the genetic modification has been integrated in the DNA of  chloroplasts . As the chloroplasts of plants are maternally inherited, the transgenes are not spread by pollen thus achieving biological containment.  In other words, the cell nucleus contains no transgenes, and the pollen contains no chloroplasts and thus no transgenes. [ 4 ]  Two important research projects on co-existence are and  Co-Extra . With the end of the de facto moratorium on genetically modified plants in Europe, several research programmes (e.g.  SIGMEA ,  Co-Extra , and  Transcontainer ) have begun investigating biological containment strategies for GMOs.


While SIGMEA was focused on co-existence at the farm level, Co-Extra studies co-existence along the whole production chain, and has a second focus on the traceability of GMOs, since co-existence cannot work without traceability. To be able to monitor and enforce compliance with co-existence regulations, authorities require the ability to trace,  detect and identify GMOs .


The development of a regulatory framework concerning genetic engineering began in 1975, at  Asilomar , California. The first use of  Recombinant DNA  (rDNA) technology had just been successfully accomplished by  Stanley Cohen  and  Herbert Boyer  two years previously and the scientific community recognized that as well as benefits this technology could also pose some risks. [ 5 ]  The  Asilomar meeting  recommended a set of guidelines regarding the cautious use of recombinant technology and any products resulting from that technology. [ 6 ]  The Asilomar recommendations were voluntary, but in 1976 the  US National Institute of Health  (NIH) formed a rDNA advisory committee. [ 7 ]  This was followed by other regulatory offices (the  United States Department of Agriculture  (USDA),  Environmental Protection Agency  (EPA) and  Food and Drug Administration  (FDA)), effectively making all rDNA research tightly regulated in the USA. [ 8 ]


In 1982 the  Organisation for Economic Co-operation and Development  (OECD) released a report into the potential hazards of releasing genetically modified organisms into the environment as the first transgenic plants were being developed. [ 9 ]  As the technology improved and genetically organisms moved from  model organisms  to potential commercial products the USA established a committee at the  Office of Science and Technology  (OSTP) to develop mechanisms to regulate the developing technology. [ 8 ]  In 1986 the OSTP assigned regulatory approval of genetically modified plants in the US to the USDA, FDA and EPA. [ 10 ]


The  Cartagena Protocol on Biosafety  was adopted on 29 January 2000 and entered into force on 11 September 2003. [ 11 ]  It is an international treaty that governs the transfer, handling, and use of genetically modified (GM) organisms. It is focussed on movement of GMOs between countries and has been called a de facto trade agreement. [ 12 ]  One hundred and fifty-seven countries are members of the Protocol and many use it as a reference point for their own regulations. [ 13 ]


In the face of continuing concerns about the economic losses that might be suffered by organic farmers by unintended intermixing, the  U.S. Secretary of Agriculture  convened an Advisory Committee on Biotechnology and 21st Century Agriculture (AC21) to study the issue and make recommendations as to whether to address these concerns and if so, how. economic losses to farmers caused by unintended presence of genetically engineered materials, as well as how such mechanisms might work.  The members  of AC21 included representatives of the biotechnology industry, the organic food industry, farming communities, the seed industry, food manufacturers, State government, consumer and community development groups, the medical profession, and academic researchers.  The AC21 recommended that a study should be conducted to answer the question of whether and to what extent there are any economic losses to US organic farmers; recommended that if the losses are serious, that a crop insurance program for organic farmers be put in place, and that an education program should be undertaken to ensure that organic farmers are putting appropriate contracts in place for their crops and that neighboring GM crop farmers are taking appropriate containment measures. Overall the report supported a diverse agriculture system in which many different farming systems could co-exist. [ 14 ] [ 15 ]


Since GM-free products yield higher prices in many countries, [ 16 ]  some governments have introduced limits for the mixing of both production systems, with compensation for non-GM farmers for economic losses in cases where mixing inadvertently occurred. [ 17 ]   One tool for compensation is a liability fund, to which all GM farmers, and sometimes GM seed producers, contribute. [ 17 ] : 88–91   After a notable GMO contamination event in  Western Australia  where a  certified organic  farm lost certification due to GMO  contamination , [ 18 ]  a Parliamentary Inquiry considered six proactive proposals for compensating farms contaminated by  GMOs , however the Inquiry did not recommend a particular mechanism of compensation. [ 19 ]


Mixing can occur already at the agricultural stage. Fundamentally, two reasons exist for the presence of GMOs in the harvest of a non-GM cultivation: first, that the seed has been contaminated already or, secondly, that the plants in the non-GM field have received  pollen  from neighbouring GM fields.  Mixing may also occur post-harvest, anywhere in the production chain. [ 20 ] [ 21 ]


In 1997,  Percy Schmeiser  discovered that  canola  growing on his farm was genetically modified to be resistant to  Roundup  although he had not planted GM seed. He had initially discovered that some canola growing by a roadside along one of his fields was Roundup resistant when he was killing weeds along the road; this led him to spray a 3- to 4‑acre section of his adjacent field and 60% of the canola survived. Schmeiser harvested the seed from the surviving, Roundup resistant plants, and planted the seed in 1998.  Monsanto  sued Schmeiser for patent infringement for the 1998 planting. Schmeiser claimed that because the 1997 plants grew from seed that was blown into his field from neighboring fields, that he owned the harvest and was entitled to do with it whatever he wished, including saving the seeds from the 1997 harvest and planting them in 1998. The case ( Monsanto Canada Inc v Schmeiser ) went to the  Supreme Court  which held for Monsanto by a 5‑4 vote in late May 2004. [ 22 ]   The case is widely cited or referenced by the anti-GM community in the context of a fear of a company claiming ownership of a farmer's crop based on the inadvertent presence of GM pollen grain or seed. [ 23 ] [ 24 ]  "The court record shows, however, that it was not just a few seeds from a passing truck, but that Mr Schmeiser was growing a crop of 95–98% pure Roundup Ready plants, a commercial level of purity far higher than one would expect from inadvertent or accidental presence. The judge could not account for how a few wayward seeds or pollen grains could come to dominate hundreds of acres without Mr Schmeiser's active participation, saying '. . .none of the suggested sources could reasonably explain the concentration or extent of Roundup Ready canola of a commercial quality evident from the results of tests on Schmeiser's crop'" – in other words, the original presence of Monsanto seed on his land in 1997 was indeed inadvertent, but the crop in 1998 was entirely purposeful. [ 25 ]


In 1999 scientists in Thailand claimed they discovered  glyphosate -resistant  genetically modified wheat  that was not yet approved for release in a  grain shipment  from the Pacific Northwest of the United States, even though transgenic wheat had never been approved for sale and was only ever grown in test plots. No one could explain how the transgenic wheat got into the food supply. [ 26 ]


In 2000,  Aventis   StarLink corn , which had been approved only as animal feed due to concerns about possible allergic reactions in humans, was found contaminating corn products in U.S. supermarkets and restaurants. This corn became the subject of a widely publicized  recall , when Taco Bell taco shells were found to contain the corn, eventually resulting in the recall of over 300 products. [ 27 ] [ 28 ]  It was the first-ever recall of a  genetically modified food .  The registration for the Starlink varieties was voluntarily withdrawn by Aventis in October 2000. [ 29 ]


In 2005, scientists at the UK  Centre for Ecology and Hydrology  reported the first evidence of  horizontal gene transfer  of  pesticide resistance  to weeds, in a few plants from a single season;  they found no evidence that any of the hybrids had survived in subsequent seasons. [ 30 ]


In 2006, American exports of rice to Europe were interrupted when the U.S. crop was contaminated with rice containing the  LibertyLink  modification, which had not been approved for release. [ 31 ]   An investigation by the USDA's Animal and Plant Health Inspection Service (APHIS) was unable to determine the cause of the contamination. [ 32 ]


In 2007, the U.S. Department of Agriculture fined Scotts Miracle-Gro $500,000 when modified genetic material from  creeping bentgrass , a new golf-course grass Scotts had been testing, was found within close relatives of the same genus ( Agrostis ) [ 33 ]  as well as in native grasses up to 21 km (13 mi) away from the test sites, released when freshly cut grass was blown by the wind. [ 34 ]


In 2009 the government of Mexico created a regulatory pathway for approval of  genetically modified maize , [ 35 ]  but because Mexico is the  center of diversity  for maize, concerns have been raised about the effect that genetically modified maize could have on local strains. [ 36 ] [ 37 ]  A 2001 report in  Nature  presented evidence that Bt maize was cross-breeding with unmodified maize in  Mexico , [ 38 ]  although the data in this paper was later described as originating from an artifact and  Nature  stated that "the evidence available is not sufficient to justify the publication of the original paper". [ 39 ]  A subsequent large-scale study, in 2005, failed to find any evidence of contamination in Oaxaca. [ 40 ]  However, other authors have stated that they also found evidence of cross-breeding between natural maize and  transgenic maize . [ 41 ]


A study published in 2010 by scientists at the  University of Arkansas ,  North Dakota State University ,  California State University  and the  US Environmental Protection Agency  showed that about 83 percent of wild or weedy canola tested contained genetically modified  herbicide resistance  genes. [ 42 ] [ 43 ] [ 44 ]   According to the researchers, the lack of reports in the US suggests inadequate oversight and monitoring protocols are in place in the US. [ 45 ]  The development of weeds resistant to glyphosate, the most commonly applied herbicide, could mean that farmers must return to more labour-intensive methods to control weeds, use more dangerous herbicides or  till  the soil (so increasing then risk of erosion). [ 46 ]  A 2010 report by the  National Academy of Sciences  stated that the advent of glyphosate-herbicide resistant weeds could cause the genetically engineered crops to lose their effectiveness unless farmers also use other established weed management strategies. [ 47 ] [ 48 ]  In  Australia , some of a 2010 planting of  Monsanto 's Roundup-Ready (RR) canola blew across a neighboring organic farm. [ 49 ]  The organic farm lost its organic certification and the organic farmer sued the GM farmer - so far without success. [ 49 ]  The certifier called it "contamination" and in the 2014 judgement the judge called it an "incursion" and rejected claims for  nuisance ,  negligence  and damages. [ 49 ]


In 2013,  glyphosate -resistant  genetically modified wheat  that was not yet approved for release, but which had been declared safe for consumption in the USA, [ 50 ]  was discovered in a farm in Oregon, growing as a weed or " volunteer plant ". The wheat had been created by  Monsanto , and was a strain that was field-tested from 1998 to 2005 and was in the American regulatory approval process before Monsanto withdrew it based on concern that importers would avoid the crop. The last field test in Oregon had occurred in 2001. Volunteer wheat from a field two miles away owned by the same farmer and planted with the same seed was tested and it was not found to be glyphosate-resistant. Monsanto was liable for fines of up to $1 million, if violations of the  Plant Protection Act  were found. [ 51 ] [ 52 ]  According to Monsanto it was "mystified" by its appearance, having destroyed all the material it held after completing trials in 2004 and because they did not think that seed left in the ground or pollen transfer could account for it. [ 50 ]  Later in the month, Monsanto suggested that the presence of the wheat was likely an act of "sabotage". [ 53 ]  The discovery could have threatened U.S. wheat exports, which totaled $8.1 billion in 2012; the US is the world's largest wheat exporter. [ 52 ] [ 54 ]   New Scientist  reported that the variety of wheat was rarely imported into Europe and doubted that the discovery of the wheat would affect Europe, but more likely destined for Asia. As a result of the discovery of the unapproved strain,  Japan  and  South Korea  halted wheat orders from the United States, leaving wheat growers in neighboring communities unable to decide what to plant next season. The crop growing when the genetically modified wheat was discovered had already been sold or insured. [ 55 ] [ 56 ]  On June 14, 2013, the  USDA  announced: "As of today, USDA has neither found nor been informed of anything that would indicate that this incident amounts to more than a single isolated incident in a single field on a single farm. All information collected so far shows no indication of the presence of GE [ sic ] wheat in commerce." [ 57 ]  As of August 30, while the source of the GM wheat remained unknown, Japan, South Korea and Taiwan had all resumed placing orders, and the export market resumed. The Oregon wheat commissioner, Blake Rowe, said that "the overall economic impact has been minimal". [ 58 ]


Bioreporters  are intact, living  microbial   cells  that have been  genetically engineered  to produce a measurable signal in response to a specific chemical or physical agent in their  environment . Bioreporters contain two essential genetic elements, a  promoter  gene and a  reporter gene . The promoter gene is turned on ( transcribed ) when the target agent is present in the cell’s environment. The promoter gene in a normal bacterial cell is linked to other genes that are then likewise transcribed and then translated into proteins that help the cell in either combating or adapting to the agent to which it has been exposed. In the case of a bioreporter, these genes, or portions thereof, have been removed and replaced with a reporter gene. As a result, turning on the promoter gene also turns on the reporter gene, leading to the production of reporter  proteins  that output a detectable signal. The presence of a signal indicates that the bioreporter has sensed a particular agent in its environment.


Originally developed for fundamental analysis of factors affecting  gene expression , bioreporters were early on applied for the detection of environmental contaminants [ 1 ]  and have since evolved into fields as diverse as  medical diagnostics ,  precision agriculture ,  food safety  assurance, process monitoring and control, and bio- microelectronic  computing. Their versatility stems from the fact that there exist a large number of  reporter gene  systems that are capable of generating a variety of signals. Additionally, reporter genes can be genetically inserted into  bacterial ,  yeast ,  plant , and  mammalian  cells, thereby providing considerable functionality over a wide range of host vectors.


Several types of reporter genes are available for use in the construction of bioreporter organisms, and the signals they generate can usually be categorized as either  colorimetric ,  fluorescent ,  luminescent ,  chemiluminescent  or  electrochemical . Although each functions differently, their end product always remains the same – a measurable signal that is proportional to the concentration of the unique chemical or physical agent to which they have been exposed. In some instances, the signal only occurs when a secondary  substrate  is added to the  bioassay  ( luxAB , Luc, and aequorin). For other bioreporters, the signal must be activated by an external light source (GFP and UMT), and for a select few bioreporters, the signal is completely self-induced, with no exogenous substrate or external activation being required ( luxCDABE ). The following sections outline in brief some of the reporter gene systems available and their existing applications.


Luciferase  is a generic name for an  enzyme  that  catalyzes  a light-emitting reaction. Luciferases can be found in bacteria, algae, fungi, jellyfish, insects, shrimp, and squid, and the resulting light that these organisms produce is termed bioluminescence. In bacteria, the genes responsible for the light-emitting reaction (the  lux  genes) have been isolated and used extensively in the construction of bioreporters that emit a blue-green light with a maximum intensity at 490 nm. [ 2 ]  Three variants of  lux  are available, one that functions at < 30°C, another at < 37°C, and a third at < 45°C. The  lux  genetic system consists of five genes,  luxA ,  luxB ,  luxC ,  luxD , and  luxE . Depending on the combination of these genes used, several different types of  bioluminescent  bioreporters can be constructed.


luxAB  bioreporters contain only the  luxA  and  luxB  genes, which together are responsible for generating the light signal. However, to fully complete the light-emitting reaction, a  substrate  must be supplied to the cell. Typically, this occurs through the addition of the chemical  decanal  at some point during the bioassay procedure. Numerous  luxAB  bioreporters have been constructed within bacterial, yeast, insect, nematode, plant, and mammalian cell systems.


Instead of containing only the  luxA  and  luxB  genes, bioreporters can contain all five  genes  of the  lux  cassette, thereby allowing for a completely independent light generating system that requires no extraneous additions of substrate nor any  excitation  by an external light source. So in this bioassay, the bioreporter is simply exposed to a target  analyte  and a  quantitative  increase in bioluminescence results, often within less than one hour. Due to their rapidity and ease of use, along with the ability to perform the bioassay repetitively in  real time  and on-line, makes  luxCDABE  bioreporters extremely attractive. Consequently, they have been incorporated into a diverse array of detection methodologies ranging from the sensing of environmental contaminants to the real-time monitoring of pathogen infections in living mice.


Nonspecific  lux  bioreporters are typically used for the detection of chemical toxins. They are usually designed to continuously bioluminesce. Upon exposure to a chemical toxin, either the cell dies or its metabolic activity is retarded, leading to a decrease in  bioluminescent  light levels. Their most familiar application is in the Microtox assay where, following a short exposure to several concentrations of the sample, the decreased  bioluminescence  can be correlated to relative levels of  toxicity . [ 3 ] [ 4 ]


Firefly luciferase  catalyzes a reaction that produces visible light in the 550 to 575 nm range. A click-beetle luciferase is also available that produces light at a peak closer to 595 nm. Both luciferases require the addition of an exogenous substrate (luciferin) for the light reaction to occur. Numerous  luc -based bioreporters have been constructed for the detection of a wide array of  inorganic  and  organic compounds  of environmental concern. The most promising applications, however, probably rely on introducing the genetic code of the firefly luciferase into other eukaryotic cells and tissues. [ 5 ]


Insertion of the  luc  genes into a human cervical carcinoma cell line ( HeLa ) illustrated that tumor-cell clearance could be visualized within a living mouse by simply scanning with a  charge-coupled device  camera, allowing for  chemotherapy  treatment to rapidly be monitored on-line and in real-time. [ 6 ]  In another example, the  luc  genes were inserted into human breast cancer cell lines to develop a bioassay for the detection and measurement of substances with potential  estrogenic  and antiestrogenic activity. [ 7 ]


Particular  promoters  can be placed upstream of the  luc  gene, that is, the  luc  sequence can be fused to the promoter sequence at DNA level. If such construct is not too large in size, it can simply be introduced into eukaryotic cells using  plasmids . This approach is widely used to study the activity of a given promoter in a given cell/tissue type, since the amount of light produced by the luciferase is directly proportional to the promoter activity. [ 8 ]  In addition to studying the promoters, firefly luciferase assays offer the option of studying  transcriptional activators : in these experiments typically the  GAL4/UAS_system  is used and its Gal4 upstream activating DNA sequence (UAS) is placed upstream the  luc  gene while the different activators or the different variants/fragments of the same activator are fused to the GAL4 DNA binding module at protein level. This way the transcriptional activity of the different GAL4 fusion proteins can be directly compared using light as a readout. [ 9 ]


Aequorin  is a photoprotein isolated from the bioluminescent jellyfish  Aequorea victoria . Upon addition of  calcium  ions (Ca2+) and coelenterazine, a reaction occurs whose result is the generation of blue light in the 460 to 470 nm range. Aequorin has been incorporated into human  B cell  lines for the detection of  pathogenic  bacteria and viruses in what is referred to as the  Cell CANARY  assay (Cellular Analysis and Notification of Antigen Risks and Yields). [ 10 ]  The B cells are genetically engineered to produce aequorin. Upon exposure to antigens of different pathogens, the recombinant B cells emit light as a result of activation of an intracellular signaling cascade that releases calcium ions inside the cell.


Green fluorescent protein  (GFP) is also a photoprotein isolated and cloned from the jellyfish  Aequorea victoria . [ 11 ]  Variants have also been isolated from the sea pansy  Renilla reniformis . GFP, like aequorin, produces a blue fluorescent signal, but without the required addition of an exogenous substrate. All that is required is an  ultraviolet light  source to activate the fluorescent properties of the photoprotein. This ability to autofluoresce makes GFP highly desirable in biosensing assays since it can be used on-line and in to monitor intact, living cells. Additionally, the ability to alter GFP to produce light emissions besides blue (i.e., cyan, red, and yellow) allows it to be used as a multianalyte detector. Consequently, GFP has been used extensively in bioreporter constructs within bacterial, yeast, nematode, plant, and mammalian hosts.


Uroporphyrinogen (urogen) III methyltransferase  (UMT) catalyzes a reaction that yields two  fluorescent  products which produce a red-orange fluorescence in the 590 to 770 nm range when illuminated with  ultraviolet light . [ 12 ]  So as with GFP, no addition of exogenous substrates is required. UMT has been used as a bioreporter for the selection of recombinant  plasmids , as a marker for gene  transcription  in bacterial, yeast, and mammalian cells, and for the detection of toxic salts such as  arsenite  and  antimonite .


Bioretrosynthesis  is a technique for synthesizing  organic chemicals  from inexpensive precursors and evolved  enzymes . [ 1 ]  The technique builds on the retro-evolution hypothesis proposed in 1945 by  geneticist   Norman Horowitz . [ 2 ]


The technique works backwards from the target to identify a precursor molecule and an enzyme that converts it into the target, and then a second precursor that can produce the first and so on until a simple, inexpensive molecule becomes the beginning of the series. [ 1 ]  For each precursor, the enzyme is evolved using induced mutations and  natural selection  to produce a more productive version. The evolutionary process can be repeated over multiple generations until acceptable productivity is achieved. [ 1 ]  The process does not require high temperature, high pressure, the use of exotic  catalysts  or other elements that can increase costs. [ 1 ]  The enzyme "optimizations" that increase the production of one precursor from another are cumulative in that the same precursor productivity improvements can potentially be leveraged across multiple target molecules. [ 1 ]


In 2014 the technique was used to produce the  HIV  drug  didanosine : [ 2 ]  a simpler molecule was identified that can be converted into didanosine when subjected to a specific chemical transformation in the presence of a specific enzyme. [ 2 ]  The  gene  that creates the enzyme was then "copied", adding random  mutations  to each copy using  ribokinase  engineering. [ 2 ]  The mutant genes were inserted into  Escherichia coli   bacteria  and used to produce (now-mutant) enzymes. The enzymes were then mixed with the precursor and the mutant enzymes that produced the greatest amount of didanosine were retained and replicated. One mutant stimulated a 50x increase in didanosine production. [ 2 ]  The first step was repeated, using the first precursor in place of didanosine, finding a yet simpler precursor and an enzyme to produce it. One mutated enzyme produced a 9,500x increase in nucleoside production. [ 2 ]  A third retrogression allowed them to start with the simple and inexpensive sugar named  dideoxyribose  and produce didanosine in a three-step sequence. [ 2 ]


Biosafety  is the prevention of large-scale loss of  biological integrity , focusing both on  ecology  and  human health . [ 1 ]
These prevention mechanisms include the conduction of regular reviews of biosafety in laboratory settings, as well as strict guidelines to follow. Biosafety is used to protect from harmful incidents. Many laboratories handling biohazards employ an ongoing risk management assessment and enforcement process for biosafety.  Failures to follow such protocols can lead to increased risk of exposure to biohazards or pathogens.  Human error  and poor technique contribute to unnecessary exposure and compromise the best safeguards set into place for protection.


The international  Cartagena Protocol on Biosafety  deals primarily with the agricultural definition but many advocacy groups seek to expand it to include post-genetic threats: new molecules, artificial life forms, and even robots which may compete directly in the natural food chain. [ citation needed ]


Biosafety in agriculture, chemistry, medicine, exobiology and beyond will likely require the application of the  precautionary principle , and a new definition focused on the biological nature of the threatened organism rather than the nature of the threat.


When  biological warfare  or new, currently hypothetical, threats (i.e., robots, new artificial bacteria) are considered, biosafety precautions are generally not sufficient. The new field of  biosecurity  addresses these complex threats.


Biosafety level  refers to the stringency of  biocontainment  precautions deemed necessary by the  Centers for Disease Control and Prevention  (CDC) for laboratory work with infectious materials.


Typically, institutions that experiment with or create potentially harmful biological material will have a committee or board of supervisors that is in charge of the institution's biosafety. They create and monitor the biosafety standards that must be met by labs in order to prevent the accidental release of potentially destructive biological material. (note that in the US, several groups are involved, and efforts are being made to improve processes for government run labs, but there is no unifying regulatory authority for all labs.


Biosafety is related to several fields:


Chemical hazards  typically found in laboratory settings include  carcinogens ,  toxins ,  irritants ,  corrosives , and  sensitizers .   Biological hazards  include viruses, bacteria, fungi,  prions , and biologically derived toxins, which may be present in body fluids and tissue,  cell culture  specimens, and  laboratory animals .  Routes of exposure for chemical and biological hazards include  inhalation ,  ingestion , skin contact, and eye contact. [ 2 ]


Physical hazards  include  ergonomic hazards ,  ionizing  and  non-ionizing radiation , and  noise hazards .  Additional safety hazards include  burns  and  cuts  from  autoclaves , injuries from  centrifuges ,  compressed gas  leaks,  cold burns  from  cryogens ,  electrical hazards ,  fires ,  injuries from machinery , and  falls . [ 2 ]


A complete understanding of experimental risks associated with synthetic biology is helping to enforce the knowledge and effectiveness of biosafety. [ 3 ]
With the potential future creation of man-made unicellular organisms, some are beginning to consider the effect that these organisms will have on biomass already present. Scientists estimate that within the next few decades, organism design will be sophisticated enough to accomplish tasks such as creating biofuels and lowering the levels of harmful substances in the atmosphere. [ 4 ]  Scientist that favor the development of synthetic biology claim that the use of biosafety mechanisms such as  suicide genes  and nutrient dependencies will ensure the organisms cannot survive outside of the lab setting in which they were originally created. [ 5 ]   Organizations like the  ETC Group  argue that regulations should control the creation of organisms that could potentially harm existing life. They also argue that the development of these organisms will simply shift the consumption of petroleum to the utilization of biomass in order to create energy. [ 6 ]  These organisms can harm existing life by affecting the prey/predator food chain, reproduction between species, as well as competition against other species (species at risk, or act as an invasive species).
 Synthetic vaccines  are now being produced in the lab. These have caused a lot of excitement in the  pharmaceutical industry  as they will be cheaper to produce, allow quicker production, as well as enhance the knowledge of  virology  and  immunology .


Biosafety, in medicine and health care settings, specifically refers to proper handling of organs or tissues from biological origin, or genetic therapy products, viruses with respect to the environment, [ 7 ]  to ensure the safety of health care workers, researchers, lab staff, patients, and the general public. Laboratories are assigned a biosafety level numbered 1 through 4 based on their potential biohazard risk level. [ 8 ]  The employing authority, through the laboratory director, is responsible for ensuring that there is adequate surveillance of the health of laboratory personnel. [ 9 ]  The objective of such surveillance is to monitor for occupationally acquired diseases. [ 10 ]  The World Health Organization attributes human error and poor technique [ 10 ]  as the primary cause of mishandling of biohazardous materials.


Biosafety is also becoming a global concern and requires multilevel resources and international collaboration to monitor, prevent and correct accidents from unintended and malicious release and also to prevent that bioterrorists get their hands-on biologics sample to create biologic weapons of mass destruction.  Even people outside of the health sector needs to be involved as in the case of the Ebola outbreak the impact that it had on businesses and travel required that private sectors, international banks together pledged more than $2 billion to combat the epidemic. [ 11 ]  The bureau of international Security and nonproliferation (ISN) is responsible for managing a broad range of U.S. nonproliferation policies, programs, agreements, and initiatives, and biological weapon is one their concerns
Biosafety has its risks and benefits. All stakeholders must try to find a balance between cost-effectiveness of safety measures and use evidence-based safety practices and recommendations, measure the outcomes and consistently reevaluate the potential benefits that biosafety represents for human health.
Biosafety level designations are based on a composite of the design features, construction, containment facilities, equipment, practices and operational procedures required for working with agents from the various risk groups. [ 10 ]


Classification of biohazardous materials is subjective and the risk assessment is determined by the individuals most familiar with the specific characteristics of the organism. [ 10 ]  There are several factors taken into account when assessing an organism and the classification process.


See World Health Organization Biosafety Laboratory Guidelines (4th edition, 2020):  World Health Organization Biosafety Laboratory Guidelines


Investigations have shown that there are hundreds of unreported biosafety accidents, with laboratories self-policing the handling of biohazardous materials and lack of reporting. [ 13 ]  Poor record keeping, improper disposal, and mishandling biohazardous materials result in increased risks of biochemical contamination for both the public and environment. [ 14 ]


Along with the precautions taken during the handling process of biohazardous materials, the World Health Organization recommends:
Staff training should always include information on safe methods for highly hazardous procedures that are commonly encountered by all laboratory personnel, and which involve: [ 10 ]


First of all the  laboratory  director, who holds immediate responsibility for the laboratory, is tasked with ensuring the development and adoption of a biosafety management plan as well as a safety or operations manual. Secondly, the laboratory supervisor, who reports to the laboratory director, is responsible for organizing regular training sessions on laboratory safety. [ 9 ]


The third point, the personnel must be informed about any special  hazards  and be required to review the safety or operations manual and adhere to established practices and procedures. The laboratory supervisor is responsible for ensuring that all personnel have a clear understanding of these guidelines, and a copy of the safety or operations manual should be readily available within the laboratory. Finally, adequate medical assessment, monitoring, and treatment must be made available to all personnel when needed, and comprehensive medical records should be maintained. [ 9 ]


In June 2009, the Trans-Federal Task Force on Optimizing Biosafety and Biocontainment Oversight recommended the formation of an agency to coordinate high safety risk level labs (3 and 4), and voluntary, non-punitive measures for incident reporting. [ 15 ]  However, it is unclear as to what changes may or may not have been implemented following their recommendations.


The United States  Code of Federal Regulations  is the  codification (law) , or collection of laws specific to a specific to a  jurisdiction  that represent broad areas subject to federal regulation. [ 16 ]   Title 42 of the Code of Federal Regulations  addresses laws concerning Public Health issues including biosafety which can be found under the citation 42 CFR 73 to 42 CFR 73.21 by accessing the US Code of Federal Regulations (CFR) website. [ 17 ]


Title 42 Section 73 of the CFR addresses specific aspects of biosafety including  occupational safety and health , transportation of biohazardous materials and safety plans for laboratories using potential biohazards. While  biocontainment , as defined in the Biosafety in Microbiological and Biomedical Laboratories [ 18 ]  and Primary Containment for Biohazards: Selection, Installation and Use of Biosafety Cabinets [ 18 ]  manuals available at the  Centers for Disease Control and Prevention  website much of the design, implementation and monitoring of protocols are left up to state and local authorities. [ 17 ]


The United States CFR states  "An individual or entity required to register [as a user of biological agents] must develop and implement a written biosafety plan that is commensurate with the risk of the select agent or toxin" [ 17 ]  which is followed by three recommended sources for laboratory reference:


While clearly the needs of  biocontainment  and biosafety measures vary across government, academic and private industry laboratories,  biological agents  pose similar risks independent of their locale. [ 19 ]   Laws relating to biosafety are not easily accessible and there are few federal regulations that are readily available for a potential trainee to reference outside of the publications recommended in 42 CFR 73.12. [ 17 ] [ 18 ]  Therefore, training is the responsibility of lab employers [ 17 ]  and is not consistent across various laboratory types thereby increasing the risk of accidental release of  biological hazards  that pose serious health threats to the humans, animals and the  ecosystem  as a whole.


Many government agencies have made guidelines and recommendations in an effort to increase biosafety measures across laboratories in the United States.  Agencies involved in producing policies surrounding biosafety within a hospital, pharmacy or clinical research laboratory include: the CDC,  FDA ,  USDA ,  DHHS , DoT,  EPA  and potentially other local organizations including public health departments.  The federal government does set some standards and recommendations for States to meet their standards, most of which fall under the  Occupational Safety and Health Act  of 1970. [ 20 ]  but currently, there is no single federal regulating agency directly responsible for ensuring the safety of biohazardous handling, storage, identification, clean-up and disposal.  In addition to the CDC, the  Environmental Protection Agency  has some of the most accessible information on ecological impacts of biohazards, how to handle spills, reporting guidelines and proper disposal of agents dangerous to the environment. [ 21 ]  Many of these agencies have their own manuals and guidance documents relating to training and certain aspects of biosafety directly tied to their agency's scope, including transportation, storage and handling of blood borne pathogens (OSHA, [ 22 ]   IATA ).  The American Biological Safety Association (ABSA) has a list of such agencies and links to their websites, [ 23 ]  along with links to publications and guidance documents to assist in risk assessment, lab design and adherence to laboratory exposure control plans. Many of these agencies were members of the 2009 Task Force on BioSafety. [ 24 ]  There was also a formation of a  Blue Ribbon Study Panel on Biodefense , but this is more concerned with national defense programs and biosecurity.


Ultimately states and local governments, as well as private industry labs, are left to make the final determinants for their own biosafety programs, which vary widely in scope and enforcement across the United States. [ 25 ]  Not all state programs address biosafety from all necessary perspectives, which should not just include personal safety, but also emphasize an full understanding among laboratory personnel of quality control and assurance, exposure potential impacts on the environment, and general public safety. [ 26 ]


Toby Ord  puts into question whether the current international conventions regarding biotechnology research and development regulation, and self-regulation by biotechnology companies and the scientific community are adequate. [ 27 ]


State occupational safety plans are often focused on transportation, disposal, and risk assessment, allowing caveats for safety audits, but ultimately leaves the training in the hands of the employer. [ 28 ]   22 states have approved Occupational Safety plans by OSHA that are audited annually for effectiveness. [ 20 ]  These plans apply to private and public sector workers, and not necessarily state/ government workers, and not all specifically have a comprehensive program for all aspects of biohazard management from start to finish.  Sometimes biohazard management plans are limited only to workers in transportation specific job titles.  The enforcement and training on such regulations can vary from lab to lab based on the State's plans for occupational health and safety.  With the exception of DoD lab personnel, CDC lab personnel, First responders, and DoT employees, enforcement of training is inconsistent, and while training is required to be done, specifics on the breadth and frequency of refresher training does not seem consistent from state to state; penalties may never be assessed without larger regulating bodies being aware of non-compliance, and enforcement is limited. [ 29 ]


Medical waste  management was identified as an issue in the 1980s, with the  Medical Waste Tracking Act  of 1988 [ 30 ]  becoming the new standard in biohazard  waste disposal .


Although the Federal Government, EPA & DOT provide some oversight of regulated medical waste storage, transportation, and disposal the majority of biohazard medical waste is regulated at the state level. [ 30 ]  Each state is responsible for regulation and management of their own biohazardous waste with each state varying in their regulatory process. Record keeping of biohazardous waste also varies between states.


Medical healthcare centers, hospitals veterinary clinics, clinical laboratories and other facilities generate over one million tons of waste each year. [ 30 ]  Although the majority of this waste is as harmless as common household waste, as much as 15 percent of this waste poses a potential infection hazard, according to the Environmental Protection Agency (EPA). [ 30 ]  Medical waste is required to be rendered non-infectious before it can be disposed of. [ 30 ]  There are several different methods to treat and dispose of biohazardous waste. In the United States, the primary methods for treatment and disposal of biohazard, medical and sharps waste may include: [ 30 ]


Different forms of biohazardous wasted required different treatments for their proper waste management. This is determined largely be each states regulations.